Editors Leslie C. Grammer M.D. Professor of Medicine Department of Medicine; Vice Chief, Division of Allergy-Immunology; Director, Ernest S. B a zl e y A s t h m a a n d A l l e r g i c D i s e a s e s C e n t e r , N o r t h w e s t e r n U n i v e r s i t y M e d i c a l S c h o o l ; Attending Physician, Northwestern Memorial Hospital, Chicago, Illinois P a u l A. G r e e n b e r g e r M . D . Professor of Medicine Department of Medicine; Associate Chief, Education and Clinical Affairs, Division of AllergyImmunology, Northwestern University Medical School; Attending Physician, Northwestern Memorial Hospital, Chicago, Illinois
Contributing Authors H o w a r d L . Al t M . D . Assistant Professor of Clinical Medicine Department of Psychiatry, Northwestern University Medical School, Chicago, Illinois A n d r e a J . Ap t e r M . D . , M . S c . Chief; Associate Professor Department of Allergy and Immunology, Division of Pulmonary, Allergy, and Critical Care, Hospital of the University of Pennsylvania, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania Banani Banerjee Ph.D. Instructor of Pediatrics (Allergy/Immunology) and Medicine Medical College of Wisconsin, Milwaukee, Wisconsin Melvin Berger M.D. Ph.D. Professor Departments of Pediatrics and Pathology, Case Western Reserve University; Chief, Department of Pediatrics, Rainbow, Babies and Children's Hospital, University Hospitals Health System, Cleveland, Ohio David I. Bernstein M.D. Professor of Medicine Department of Immunology, University of Cincinnati College of Medicine, Cincinnati, Ohio J o n a t h a n A. B e r n s t e i n M . D . Associate Professor Department of Internal Medicine, University of Cincinnati, Cincinnati, Ohio Michael S. Blaiss M.D. Clinical Professor Departments of Pediatrics and Medicine, University of Tennessee, Memphis, Department of Pediatrics, Le Bonheur Children's Medical Center, Memphis, Tennessee Bernard H. Booth III M.D. Clinical Professor Department of Medicine, University of Mississippi Medical Center, Jackson, Mississippi G. Daniel Brooks M.D. Department of Medicine, University of Wisconsin, Department of Medicine, University of Wisconsin Hospitals and Clinics, Madison, Wisconsin Robert K. Bush M.D. Professor Department of Medicine, University of Wisconsin-Madison; Chief, Department of Allergy, William. S. Middleton V.A. Hospital, Madison, Wisconsin Rakesh K. Chandra M.D. Chief Resident Department of Otolaryngology Head & Neck Northwestern University, Chicago, Illinois David B. Conle y M.D. Assistant Professor Department of Otolaryngology School, Chicago, Illinois
Head
&
Neck
Surgery,
Northwestern
Memorial
Surgery,
Northwestern
University
Hospital,
Medical
Thomas Corbridge M.D., F.C.C.P. Associate Professor Department of Medicine, Northwestern University Medical School; Director, Medical Intensive Care Unit, Northwestern Memorial Hospital, Chicago, Illinois An n e M. Ditto M.D. Assistant Professor Department of Medicine, Division of Allergy-Immunology, Northwestern University Medical School, Chicago, Illinois Jordan N. Fink M.D. Professor and Chief Department of Medicine, Division Complex, Milwaukee, Wisconsin
of
Allergy-Immunology,
Milwaukee
County
Medical
Leslie C. Grammer M.D. Professor of Medicine Department of Medicine; Vice Chief, Division of Allergy-Immunology; Director, Ernest S. B a zl e y A s t h m a a n d A l l e r g i c D i s e a s e s C e n t e r , N o r t h w e s t e r n U n i v e r s i t y M e d i c a l S c h o o l , Chicago, Illinois Thomas H. Grant D.O. Associate Professor
Department of Radiology, Northwestern Memorial Hospital, Northwestern University Medical School, Chicago, Illinois P a u l A. G r e e n b e r g e r M . D . Professor of Medicine Department of Medicine; Associate Chief, Education and Clinical Affairs, Division of AllergyImmunology, Northwestern University Medical School, Chicago, Illinois Kathleen E. Harris B.S. Senior Life Sciences Researcher Department of Medicine, Division of Allergy-Immunology, Northwestern University Medical School, Chicago, Illinois Mary Beth Hogan M.D. Assistant Professor Department of Pediatrics, West Virginia University School of Medicine, Morgantown, West Virginia Carla Irani M.D. Section of Allergy and Immunology, Division of Pulmonary, Allergy, Critical Care Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania Kevin J. Kelly M.D. Professor and Chief Department of Allergy/Immunology, Medical College of Wisconsin; Chief, Department of Medicine, Division of Allergy/Immunology, Children's Hospital of Wisconsin, Milwaukee, Wisconsin R o b e r t C . K e r n M . D . , M . S . , F . A. C . S . Chairman Division of Otolaryngology, Cook County Hospital; Associate Professor, Department of Otolaryngology-Head & Neck Surgery, Northwestern University Medical School, Chicago, Illinois Theodore M. Lee M.D. Peachtree Allergy and Asthma Clinic, PC, Atlanta, Georgia Donald Y M Leung M.D., Ph.D. Professor Department of Pediatrics, University of Colorado Health Sciences Center; Head, Department of Pediatric Allergy-Immunology, National Jewish Medical and Research Center, Denver, Colorado Phil Lieberman M.D. Clinical Professor Department of Internal Medicine and Pediatrics, University of Tennessee College of Medicine , Cordova, Tennessee Kris G. McGrath M.D. Associate Professor Department of Medicine, Northwestern University Medical School; Chief, Department of Allergy-Immunology, Saint Joseph Hospital, Chicago, Illinois Roger W. Melvold Ph.D. Professor and Department Chair Department of Microbiology & Immunology, School University of North Dakota, Grand Forks, North Dakota
of
Medicine
and
Health
Sciences,
W. James Metzger M.D. Professor and Section Head Department of Allergy, Asthma and Immunology, East Carolina University School of Medicine, Greenville, North Carolina Babak Mokhlesi M.D. Assistant Professor Department of Medicine, Division of Pulmonary and Critical Care, Rush Medical College/Cook County Hospital, Chicago, Illinois Michelle J. Naidich M.D. Department of Radiology, Northwestern Memorial Hospital, Northwestern University Medical School, Chicago, Illinois Sai R. Nimmagadda M.D. Assistant Professor Department of Pediatrics, Northwestern University Medical Pediatrics/Allergy, Children's Memorial Hospital, Chicago, Illinois
School,
Department
of
Peck Y. Ong M.D. Fellow Department of Pediatrics, Division of Allergy and Immunology, National Jewish Medical and Research Center, Denver, Colorado Roy Patterson M.D. Ernest S. Bazley Professor of Medicine Department of Medicine; Chief, Division of Allergy-Immunology, Northwestern University Medical School, Chicago, Illinois Neill T. Peters M.D. Clinical Instructor Department of Dermatology, Mercy Hospital and Medical Center, Chicago, Illinois J a c q u e l i n e A. P o n g r a c i c M . D . Assistant Professor Department of Pediatrics and Medicine, Northwestern University Medical School; Acting Division Head, Department of Allergy, Children's Memorial Hospital, Chicago, Illinois Jacob J. Pruzansky Ph.D.
Emeritus Professor of Microbiology Department of Medicine, Division of Allergy-Immunology, Northwestern University Medical School, Chicago, Illinois Robert E. Reisman M.D. Clinical Professor Departments of Medicine and Pediatrics, State University of New York at Buffalo, Department of Medicine (Allergy/Immunology), Buffalo General Hospital, Buffalo, New York An t hon y J. Ricketti M.D. Associate Professor of Medicine Seton Hall University, Graduate School of Medicine, Chairman, Department of Medicine, St. Francis Medical Center, Trenton, New Jersey E r i c J . R u s s e l l M . D . , F . A. C . R . Professor Departments of Radiology, Neurosurgery, and Otolaryngology, Northwestern University, Chief of Neuroradiology, Department of Radiology, Northwestern Memorial Hospital, Chicago, Illinois C a r o l A. S a l t o u n M . D . Clinical Instructor Department of Medicine, Division of Allergy-Immunology, Northwestern University Medical School, Chicago, Illinois An d rew Scheman M.D. Associate Professor of Clinical Dermatology and Department of Dermatology Northwestern University Medical Center, Chicago, Illinois William R. Solomon M.D. Professor Emeritus Department of Internal Medicine (Allergy), University of Michigan Medical School and, University of Michigan Medical Center, Ann Arbor, Michigan Abba I. Terr M.D. Associate Professor Department of Medicine, University of California-San Francisco, School of Medicine, San Francisco, California An j u Tripathi M.D. Assistant Professor Departments of Medicine and Allergy, Northwestern University Medical School , Chicago, Illinois Stephen I. Wasserman M.D. The Helen M. Ranney Professor Department of Medicine, University of California-San Diego, La Jolla, California; Professor and Chief of Allergic Diseases, Department of Medicine, University of California-San Diego Medical Center, San Diego, California C a r o l An n W i g g i n s M . D . Department of Allergy and Immunology, Emory University School of Medicine, Department of Allergy and Immunology, Piedmont Hospital, Atlanta, Georgia Nevin W. Wilson M.D. Associate Professor Department of Pediatrics, West Virginia University, School of Medicine, Morgantown, West Virginia Lisa F. Wolfe M.D. Assistant Professor of Medicine Clinical Instructor, Division of Pulmonary & Critical Care Medicine and the Center for Sleep and Circadian Biology, Northwestern University Medical School, Chicago, Illinois Michael C. Zacharisen M.D. Assistant Professor Departments of Pediatrics and Medicine, Medical College of Wisconsin, Children's Hospital of Wisconsin, Milwaukee, Wisconsin C . R a ym o n d Z e i s s M . D . Emeritus Professor of Medicine Department of Medicine, Division of Allergy-Immunology, Northwestern University Medical School, VA Chicago Health Care System–Lakeside Division, Chicago, Illinois
In Memoriam In Memoriam Jacob J. Pruzansky, Ph.D. June 20, 1921–April 5, 2001 J a c k P r u za n s k y s e r v e d a s a n a u t h o r i n f i v e e d i t i o n s o f A l l e r g i c D i s e a s e s . H e s p e n t m a n y hours mentoring fellows in the Allergy-Immunology Division during his 35-year tenure at Northwestern University. After his retirement, he still functioned as a consultant and provided scientific expertise to the division. Jack was an expert on in vitro basophil histamine release and discovered how dilute hydrochloric acid would allow for removal of IgE from basophils. This discovery allowed him to study passive transfer experimentally and led to studies of histamine releasing factors. His intellect and advice helped fellows and faculty stay out of “dark alleys” as he would say. He will be missed.
In Memoriam Martha A. Shaughnessy, B.S. December 3, 1943– September 9, 1997 Martha Shaughnessy was a chapter author in the last two editions of Allergic Diseases. She had a terrific sense of humor and quick wit. Her contributions to the division included
performing immunologic research, writing research papers and grants, teaching fellows, and administration of the Allergy-Immunology Division. She coauthored 71 peer-reviewed papers, 66 of which were in collaboration with other members of the Allergy-Immunology Division. She also coauthored ten book chapters, primarily in areas of allergen immunotherapy and occupational immunologic lung disease, her two major research interests. She was admired and respected by her co-investigators and all who worked with her. We, her colleagues and friends, now honor her.
In Memory of Ernest S. Bazley T h e E r n e s t S . B a zl e y G r a n t t o N o r t h w e s t e r n M e m o r i a l H o s p i t a l a n d N o r t h w e s t e r n U n i v e r s i t y has provided continuing research support that has been invaluable to the Allergy-Immunology Division of Northwestern University.
In Memoriam W. James Metzger, Jr., M.D. October 30, 1945– November 17, 2000 Jim was a fellow at Northwestern University from 1974 to 1976 and spent his career in academic medicine at the University of Iowa and East Carolina Medical School where he was Division Chief of Allergy Asthma and Immunology and Vice-Chair of the Department of Medicine. He moved to Denver to the National Jewish Hospital in the months before he became ill. He was an author in three editions of Allergic Diseases. Jim had many accomplishments in Allergy-Immunology including the early discovery that allergen vaccine therapy inhibited some of the late airway response to allergen. Jim was liked by everyone and is deeply missed. It was stated, “As in so many things, Jim was the catalyst that altered and enlarged an experience, while managing to stay almost invisible himself.”
Foreword A Major Honor I was informed by the publisher that the name Roy Patterson would be on all future editions of Allergic Diseases: Diagnosis and Management. I consider this a great honor and at this time I would like to acknowledge several colleagues for their contributions to my personal achievements. As of this writing, I am 75 years old (as of 4/26/01) and am the Ernest S. Bazley Professor of Medicine of Northwestern University Medical School and the Chief of the Allergy-Immunology Division of the Department of Medicine. I owe a debt of gratitude to the following for all their support during my academic career. I wish to thank: The Ernest S. Bazley Trustees: Catherine Ryan of the Bank of America, Illinois, the late Ernest S. Bazley, Jr., and the late Gunnard Swanson. A major effort of the Northwestern Allergy-Immunology program has been the diagnosis and management of all forms of asthma. Research funding from the Bazley Trust has made many of our accomplishments in the area possible. The National Institute of Allergy and Infectious Diseases: Richard Krause, M.D., Anthony Fauci, M.D., Sheldon Cohen, M.D., and Dorothy Sogn, M.D. Full time facult y: p a rt ic ul ar l y t he l at e Jac ob J. Pruz ans k y, Ph. D . V o l u n t a r y f a c u l t y: p a r t i c u l a r l y R i c h a r d S . D e S w a r t e , M . D . Technical and support staff: Mary Roberts, R.N., Kathleen E. Harris, B.S., Margaret A. Mateja-Wieckert, and the late Martha A. Shaughnessy, B.S. F i n a l l y , t h e G r a d u a t e s o f t h e Al l e r g y- I m m u n o l o g y t r a i n i n g p r o g r a m . Roy Patterson M.D.
Preface The sixth edition covers allergic and immunologic problems that are encountered in the ambulatory practice or in hospitalized patients. The quantity and complexity of general and specific knowledge in the field of Allergy-Immunology continues to expand at an exciting but daunting pace. Further, the practice of Allergy-Immunology requires familiarity with many specific details. The 42 chapters contain useful, applicable, and up-to-date information on how to diagnose and manage nearly all of the conditions in Allergy-Immunology. The history of Allergic Diseases: Diagnosis and Management through six editions includes being a useful textbook where important, crucial information can be found and applied. Edition six has 15 new chapters, and the classic Drug Allergy chapter originated by Richard DeSwarte has been divided into three parts. The new chapters covering radiologic findings of the sinuses and lungs, role of rhinoscopy and surgery for chronic sinusitis, and work-up for immunodeficiency reflect the broadened base of knowledge required for the practice of Allergy-Immunology. In that asthma is now recognized as of one the most complicated disorders a physicians must treat, five new chapters were prepared to cover medications for asthma, inhaler devices and delivery systems, and novel approaches to treatment. There are shortages of specialists in Allergy-Immunology in practice and even more so in academic medical centers. We hope this textbook assists physicians to provide better care for their patients, inspires medical students and residents to pursue training in AllergyImmunology, and assists investigators in advancing our knowledge. We will always have much more to learn. We are grateful to the authors for their superb chapters, each of which has been approved by us, should there be any oversights. We could not have completed this textbook without the love and support from our families who give us the time to continue in the Northwestern University Allergy-Immunology tradition that is now over 40 years old! Special appreciation goes to our support staff, especially Kathleen E. Harris and Margaret Mateja Wieckert, our trainees and graduates, and our patients, from whom we can always learn. Leslie C. Grammer M.D.
Paul A. Greenberger M.D.
1 Review of Immunology Roger W. Melvold Department of Microbiology and Immunology, School of Medicine and Health Sciences, University of North Dakota, Grand Forks, North Dakota
Contents • • • • • • • •
ANTIGENS MOLECULES OF THE IMMUNE SYSTEM MOLECULES FOR ADHESION, RECIRCULATION, AND HOMING CELLS OF THE IMMUNE SYSTEM PRIMARY ORGANS: BONE MARROW AND THYMUS INTERACTIONS IN IMMUNE RESPONSES THE IMMUNE SYSTEM: A DOUBLE-EDGED SWORD TOLERANCE
Although immunology is a relative newcomer among the sciences, its phenomena have long been recognized and manipulated. Ancient peoples understood that survivors of particular diseases were protected from those diseases for the remainder of their lives, and the ancient Chinese and Egyptians even practiced forms of immunization. Surgeons have also long understood that tissues and organs would not survive when exchanged between different individuals (e.g., from cadaver donors) but could succeed when transplanted from one site to another within the same individual. However, only during the past century have the mechanisms of the immune system been illuminated, at least in part. Keep in mind that the immune system, as we usually think of it, is the body's second line of defense. The first line of defense consists of a number of barriers, including the skin and mucous membranes, the fatty acids of the skin, the high pH of the stomach, resident microbial populations, and cells that act nonspecifically against infectious organisms (1). Like the nervous and endocrine systems, the immune system is adaptive, specific, and communicative. It recognizes and responds to changes in the environment, and it displays memory by adapting or altering its response to stimuli that it has encountered previously. It can detect the presence of millions of different substances (antigens) and has an exquisite ability to discriminate among closely related molecules. Communication and interaction, involving both direct contact and soluble mediators, must occur among a variety of lymphoid and other cells for optimal function. The complexity of the immune system is extended by genetic differences among individuals. This is because the “repertoire” of immune responses varies among unrelated individuals in an outbred, genetically heterogeneous species such as our own. Furthermore, each of us, in a sense, is “immunologically incomplete” because none of us is able to recognize and respond to all of the possible antigens that exist. Several factors contribute to this: (a) genetic or environmentally induced conditions that nonspecifically diminish immune functions, (b) variation among individuals in the genes encoding the antigen receptors of lymphocytes, (c) genetically encoded differences among individuals (often determined by the highly polymorphic genes of the human leukocyte antigen [HLA] complex) that dictate whether and how the individual will respond to specific antigens, and (d) the fact that each individual's immune system must differentiate between self (those substances that are a normal part of the body) and foreign, or nonself, in order to avoid autoimmunity. However, because self differs from one individual to the next, what is foreign also differs among individuals.
ANTIGENS Part of "1 - Review of Immunology" Antigens were initially defined as substances identified and bound by antibodies (immunog-lobulins) produced by B lymphocytes. However, because the specific antigen receptors of P.2 T lymphocytes are not immunoglobulins, the definition must be broadened to include substances that can be specifically recognized by the receptors of T or B lymphocytes or both. It is estimated that the immune system can specifically recognize at least 106 to 107 different antigens. These include both substances that are foreign to the body (nonself) and substances that are normal constituents of the body (self). The immune system must distinguish between nonself and self antigens so that, under normal conditions, it can attack the former but not the latter. Thus, the immune system should be tolerant of self but intolerant of nonself. Autoimmune diseases arise when such distinctions are lost and the immune system attacks self antigens, a phenomenon originally described by Paul Erlich as horror autotoxicus. Well-known examples include rheumatoid arthritis, psoriasis, systemic lupus erythematosus, and some forms of diabetes. Antigens can be divided into three general types—immunogens, haptens, and tolerogens—depending on the way in which they stimulate and interact with the immune system (2,3 and 4). An immunogen can, by itself, both stimulate an immune response and subsequently serve as a target of that response. The terms immunogen and antigen are often, but inappropriately, used interchangeably. A
hapten cannot, by itself, stimulate an immune response. However, if a hapten is attached to a larger immunogenic molecule (a “carrier”), responses can be stimulated against both the carrier and the hapten, and the hapten itself can subsequently serve as the target of a response so invoked. A tolerogen is a substance that, after an initial exposure to the immune system, inhibits future responses against itself. Because of the genetic diversity among individuals, a substance that is an immunogen for one person may be a tolerogen for another and may be ignored completely by the immune system of still others. Also, a substance that acts as an immunogen when administered by one route (e.g., intramuscularly) may act as a tolerogen when applied by a different route (e.g., intragastrically). Antigens are usually protein or carbohydrate in nature and may be found as free single molecules or as parts of larger structures (e.g., expressed on the surface of an infectious agent). Although some antigens are very small and simple, others are large and complex, containing many different sites that can be individually identified by lymphocyte receptors or free immunoglobulins. Each such individual part of an antigen that can be distinctly identified by the immune system is called an epitope or determinant (i.e., the smallest identifiable antigenic unit). Thus, a single large antigen may contain many different epitopes. In general, the more complex the molecule and the greater the number of epitopes it displays, the more potent it is as an immunogen. Adjuvants are substances that, when administered together with an immunogen (or a hapten coupled to an immunogen), enhance the response against it (5). For example, immunogens may be suspended in mixtures (e.g., colloidal suspensions of mycobacterial proteins and oil) that induce localized inflammations and aid in arousal of the immune system.
MOLECULES OF THE IMMUNE SYSTEM Part of "1 - Review of Immunology"
Immunoglobulin B lymphocytes synthesize receptors (immunog-lobulins) able to recognize and bind specific structures (antigens, determinants, epitopes). All immunoglobulins produced by a single B cell, or by a clonally derived set of B cells, have the same specificity and are able to recognize and bind only a single antigen or epitope (2,3 and 4). Immunoglobulin exists either as a surface membrane-bound molecule or in a secreted form by B cells that have been appropriately stimulated and matured. The immunoglobulin molecule is a glycoprotein composed of two identical light chains and two identical heavy chains (Fig. 1.1) linked by disulfide bonds (6). Enzymatic cleavage of the immunoglobulin molecule creates defined fragments. Papain produces two antigen-binding fragments (Fab) and one crystallizable fragment (Fc). Pepsin produces only a divalent antigen-binding fragment termed F(ab′)2, and the remainder of the molecule tends to be degraded and lost.
FIG. 1.1. The immunoglobulin molecule. P.3 Each chain (heavy and light) contains one or more constant regions (CH or CL) and a variable region (VH or VL). Together, the variable regions of the light and heavy chains contribute to the antigenbinding sites (Fab) of the immunoglobulin molecule. The constant regions of the heavy chain (particularly in the Fc portion) determine what subsequent interactions may occur between the bound immunoglobulin and other cells or molecules of the immune system. When the antigen-binding sites are filled, a signal is transmitted through the immunoglobulin molecule, which results in conformational changes in the Fc portion of the heavy chain. These conformational changes permit the Fc portion to then interact with other molecules and cells. The conformationally altered Fc may
be recognized by receptors (Fc receptors [FcR]) on macrophages and other cells, which allow them to distinguish bound from unbound immunoglobulin molecules (7,8), increasing their efficiency of phagocytosis. Other conformational changes in the Fc portion of bound immunoglobulin permit the binding of complement component C1q to initiate the classic pathway of complement activation. The Fab and F(ab′)2 fragments are useful experimental and therapeutic tools that can bind antigens without the ensuing consequences resulting from the presence of the Fc region (9). Immunoglobulin light chains contain one of two types of constant regions, κ or λ. The constant regions of the heavy chains exist in five major forms (Table 1.1), each associated with a particular immunoglobulin isotype or class: Cα (immunoglobulin A [IgA]), Cδ (IgD), Cε (IgE), Cγ (IgG), and Cµ (IgM). Some of these can be subdivided into subclasses (e.g., IgG1, IgG2, IgG3, and IgG4). Each normal individual can generate all of the isotypes. Within a single immunoglobulin molecule, both light chains are identical and of the same type (both κ or both λ), and the two heavy chains are likewise identical and of the same isotype. IgD, IgG, and IgE exist only as monomeric basic immunoglobulin units (two heavy chains and two light chains), but serum IgM exists as a pentamer of five basic units united by a J (joining) chain. IgA can be found in a variety of forms (monomers, dimers, trimers, tetramers) but is most commonly seen as a monomer (in serum) or as a dimer (in external body fluids, such as mucus, tears, and saliva). P.4 The dimeric form contains two basic units, bound together by a J chain. In passing through specialized epithelial cells to external fluids, it also adds a “secretory piece,” which increases its resistance to degradation by external enzymes (10).
T AB L E 1 . 1. Immunoglobulin isotypes In addition to antigen-binding specificity, variability among immunoglobulin molecules derives from three further sources: allotypes, isotypes, and idiotypes. Allotypes are dictated by minor amino acid sequence differences in the constant regions of heavy or light chains, which result from slight polymorphisms in the genes encoding these molecules. Allotypic differences typically do not affect the function of P.5 the molecule and segregate within families like typical mendelian traits. Isotypes, as already discussed, are determined by more substantial differences in the heavy chain constant regions affecting the functional properties of the immunoglobulins (11) (Table 1.1). Finally, many antigenic determinants may be bound in more than one way, and thus there may be multiple, structurally distinct, immunoglobulins with the same antigenic specificity. These differences within the antigenbinding domains of immunoglobulins that bind the same antigenic determinants are termed idiotypes.
Generation of Antigen Binding Diversity among Immunoglobulins Each immunoglobulin chain, light and heavy, is encoded not by a single gene but by a series of genes occurring in clusters along the chromosome (11). In humans, the series of genes encoding κ light chains, the series encoding λ light chains, and the series encoding heavy chains are all located on separate chromosomes. Within each series, the genes are found in clusters, each containing a set
of similar, but not identical, genes. All of the genes are present in embryonic and germ cells and in cells other than B lymphocytes. When a cell becomes committed to the B-lymphocyte lineage, it rearranges the DNA encoding its light and heavy chains (11,12) by clipping out and degrading some of the DNA sequences. Each differentiating B cell chooses either the κ series or the λ series (but not both). In addition, although both the maternally and paternally derived chromosomes carry these sets of genes, each B cell uses only one of them (either paternal or maternal) to produce a functional chain, a phenomenon termed allelic exclusion. For the light chains, there are three distinct clusters of genes that contribute to the synthesis of the entire polypeptide: variable genes (VL), joining genes (JL), and constant genes (CL) (Fig. 1.2). In addition, each V gene is preceded by a leader sequence encoding a portion of the polypeptide that is important during the synthetic process but is removed when the molecule becomes functional. The VL and DL genes are used to produce the variable domain of the light chain. This is accomplished by the random selection of a single VL gene and a single JL gene to be united (VL–JL) by splicing out and discarding the intervening DNA. Henceforth, that cell and all of its clonal descendants are committed to that particular VL–JL combination. Messenger RNA for the light chain is transcribed to include the VL–JL genes, the CL gene or genes, and the intervening DNA between them. Before translation, the messenger RNA (mRNA) is spliced to unite the VL–JL genes with a CL gene so that a single continuous polypeptide can be produced from three genes that were originally separated on the chromosome.
FIG. 1.2. Synthesis of immunoglobulin light chains. For heavy chains, there are four distinct clusters of genes involved (Fig. 1.3): variable genes (VH), diversity genes (DH), joining genes (JH), and a series of distinct constant genes (Cµ, Cδ, Cγ, Cε, and Cα). As with the light chain genes, each V gene is preceded by a leader sequence (L) that plays a role during synthesis but is subsequently lost. One VH gene, one DH gene, and one JH gene are randomly selected, and the intervening DNA segments are excised and discarded to bring these genes together (VH–DH–JH). Messenger RNA is then transcribed to include both the VH–DH–JH and constant genes, but unlike for the light chains, the processes involving constant genes are distinctly different in stimulated and unstimulated B lymphocytes.
FIG. 1.3. Synthesis of immunoglobulin heavy chains. Unstimulated B cells transcribe heavy chain mRNA from VH–DH–JH through the Cµ and Cδ genes. This transcript does not contain the information from the Cγ, Cε, or Cα genes. The mRNA is then spliced to bring VH–DH–JH adjacent to either Cµ or Cδ, which permits the translation of a single continuous polypeptide with a variable domain (from VH–DH–JH) and a constant domain (from either Cµ or Cδ). Thus, the surface immunoglobulin of naïve unstimulated B cells includes only the IgM and IgD isotypes. After antigenic stimulation, B cells can undergo an isotype switch in which splicing of DNA, rather than RNA, brings the united VH–DH–JH genes adjacent to a constant region gene (13,14). This transition is controlled by cytokines secreted by T lymphocytes. Depending on the amount of DNA excised, the VH–DH–JH genes P.6 P.7 may be joined to any of the different CH genes (Fig. 1.4). As a result of the isotype switch, B-cell “subclones” are generated that produce an array of immunoglobulins that have identical antigenbinding specificity but different isotypes.
FIG. 1.4. The isotype switch. Two additional sources of diversity in the variable (antigen-binding) regions of light and heavy immunoglobulin chains occur. First, junctional diversity may result from imprecision in the precise placement of the cutting and splicing that bring V, D, and J genes together; second, somatic mutations may occur and accumulate in successive generations of clonally derived B lymphocytes when they undergo restimulation through later exposures to the same antigenic epitopes (15,16).
T-cell Receptor T lymphocytes (T cells) do not use immunoglobulins as antigen receptors but rather use a distinct set of genes encoding four polypeptide chains (α, β, γ, and δ), each with variable and constant domains, used to form T-cell receptors (TCRs) (17,18 and 19). The TCR is a heterodimer, either an α β or a γ δ chain combination, which recognizes and binds antigen (Fig. 1.5). This heterodimer, which is not covalently linked together, is complexed with several other molecules (e.g., CD3, CD4, and CD8), that provide stability and auxiliary functions for the receptor (20,21 and 22). Unlike immunoglobulin, which can bind to free antigen alone, TCRs bind only to specific combinations of antigen and certain self cell surface molecules. They are therefore restricted to recognition and binding of antigen on cell surfaces and are unable to bind free antigen. In humans, the self molecules are encoded by the polymorphic genes of the HLA complex (23): class I (encoded by the HLA-A, -B, and -C loci) and class II (encoded by the -DP, -DQ, and -DR loci within the D/DR region). TCRs of T cells in which CD8 is part of the TCR complex can recognize and bind antigen P.8 only when that antigen is associated with (or presented by) class I molecules, whereas those T cells in which CD4 is part of the TCR complex can recognize and bind antigen only when the antigen is presented by class II molecules.
FIG. 1.5. The T-cell receptor. Like immunoglobulin, the TCR chains contain variable and constant domains. The variable domains are encoded by a series of V, J, and sometimes D (β and δ chains only) gene clusters that undergo DNA rearrangement, and the constant regions are encoded by constant genes. TCRs do not undergo any changes equivalent to the isotype switch. Junctional diversity provides an additional source of variation for the variable domains of α and β chains but not for the γ and δ chains. Somatic mutation, so important in the diversity of immunoglobulins, does not occur in TCRs and is apparently “forbidden.”
Cell Determinant Molecules Several cell surface molecules, the cell determinant (CD) molecules, indicate the functional capacities of lymphocytes and other cells (24). The most commonly used are those distinguishing Tlymphocyte subsets.
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CD3 is a complex of several molecules associated with the T-cell antigen receptor (21,22). It provides support for the TCR and is involved in transmembrane signaling when the TCR is filled. It is found on all T cells.
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CD4 is found on T lymphocytes of the helper T-cell (TH) and delayed hypersensitivity (Tdh) subsets (25,26 and 27). CD4 molecules are found in association with the TCR and recognize class II major histocompatibility complex (MHC) molecules on antigen-presenting cells (APCs). The TCR of CD4+ cells are thus restricted to recognizing combinations of antigen and class II MHC.
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CD8 is found on T cells of the cytotoxic T-lymphocyte (CTL) and suppressor T-cell (Ts) subsets (25,27). CD8 molecules are found in association with the TCR and recognize class I MHC molecules on APCs. The TCR of CD8+ cells are thus restricted to recognizing combinations of antigen and class I MHC.
All αβ T cells express either CD4 or CD8 (and, during an early developmental stage, both). The γδ T cells, on the other hand, often express neither CD4 nor CD8 (19).
Human Leukocyte Antigen Molecules The HLA is the MHC of humans (23). It is a small region of chromosome 6 containing several (10 to 20) individual genes encoding proteins of three P.9 different types, called class I, II, and III MHC molecules (Fig. 1.6A).
F I G . 1 . 6 . A: T h e H L A c o m p l e x . B : T h e D / D R r e g i o n .
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Class I molecules are membrane-bound glycoproteins found on all nucleated cells (28). They are a single large polypeptide (about 350 amino acids) associated with a smaller molecule (β2-microglobulin). The HLA complex includes three distinct class I loci (HLA-A, -B, and -C), each having scores of alleles.
•
Class II molecules are heterodimers (Fig. 1.6B) consisting of two membrane-bound, noncovalently linked chains (α and β) and show a much more limited cellular distribution than class I molecules (29). They are encoded by the DR, DP, and DQ regions of the HLA complex. They are expressed constitutively on B lymphocytes, macrophages, monocytes, and similar cells in various tissues (Kupffer cells, astrocytes, Langerhans cells of the skin). Some other cells (e.g., vascular epithelium) are able to express class II molecules transiently under particular conditions.
•
Class III molecules are those complement molecules (e.g., C2, C4, Bf) encoded within the HLA complex (30).
Cytokines and Ligands Cytokines are short-range acting, soluble products that are important in the cellular communication necessary for the generation of immune responses (31,32,33,34,35,36 and 37). Those produced predominantly by lymphocytes or monocytes are often referred to as lymphokines or monokines, but because so many are produced by multiple cell types, the term cytokine has gained favor. A large number of cytokines have been identified, although the roles of many of them are not yet well understood. Many of the cytokines are crucial in regulating lymphocyte development and the types of immune responses evoked by specific responses (37,38,39 and 40). Those most basically involved in common immune responses are listed in Table 1.2.
T AB L E 1 . 2. Cy tokines P.10 Ligands are cell surface molecules that bind molecules on the surface of other cells in order to transmit or receive signals critical to development or activation. Among those important for immune function are B7/CD28 and CD40/CD40 ligand. B7 on APCs binds CD28 or CTLA-4 (or both) on T lymphocytes to provide signals for activation and inhibition, respectively (41). CD40 ligand (CD40L) on activated T lymphocytes binds CD40 on B lymphocytes and macrophages to provide activation signals to those cells (42,43).
Complement Complement is the composite term for a number of serum proteins (complement components) that can interact with one another, as well as with P.11 antibodies under some circ*mstances, to produce several different chemical signals and destructive responses (44). The complement components (C1 through C9 plus B, D, and P) act on one another sequentially (the complement “cascade”) (Fig. 1.7). The cascade begins with the binding of either component C1 to an antigen–antibody complex or of component C3 to a bacterial or other membrane surface (without the assistance of antibody). The binding of C1 initiates what is termed the classic pathway (involving the subsequent binding of components C4, C2, and C3), whereas the direct binding of C3 initiates the alternative pathway (involving the additional binding of components D, B, and P). A third pathway for complement activation, the lectin pathway, begins with the binding of mannose-binding protein (MBP) to mannose on bacterial cell surfaces. All three pathways eventually
lead to the activation and binding of component C5, followed by components C6, C7, C8, and C9. The completion of this combination of C5 through C9 is termed the membrane attack complex and results in the rupture of the cell surface to which it is attached (45).
FIG. 1.7. The complement cascade. As complement components interact with one another, each is cleaved into fragments. Some become enzymatically active to continue the cascade. The smaller fragments gain hormone-like functions and are important in stimulating various inflammatory reactions (46). C5a (a fragment of C5) attracts neutrophils and macrophages to the site of interest. C3a (a fragment of C3) causes smooth muscle contraction and stimulates basophils, mast cells, and platelets to release histamine and other chemicals contributing to inflammation. C3b (another fragment of C3) stimulates the ingestion (opsonization) of the cells onto which the C3b is bound by monocytes and other phagocytic cells. C4a (fragment of C4) has activity similar to C5a, although less effective.
Antigen–Antibody Complexes Binding of antigen with antibody is noncovalent and reversible. The strength of the interaction is termed affinity and determines the relative concentrations of bound versus free antigen and antibody. The formation of antigen–antibody complexes results into lattice-like aggregates of soluble antigen and antibody, and the efficiency of such binding is affected by the relative concentrations of antigen and antibody (2,3 and 4,47). This is best illustrated by the quantitative precipitin reaction (Fig. 1.8). When there is an excess of either antibody or antigen, the antigen–antibody complexes tend to remain small and in solution. The optimal binding, producing large aggregates P.12 that fall out of solution, occurs when the concentrations of antibody and antigen are in equivalence. The quantitative precipitin curve provides the basis of laboratory methods for determining the amount of antigen or antibody in, for example, a patient's serum.
FIG. 1.8. The precipitin reactions.
MOLECULES HOMING
FOR
ADHESION,
RECIRCULATION,
AND
Part of "1 - Review of Immunology" A number of surface molecules (adhesins, integrins, selectins) are used by various elements of the immune system to stabilize binding between cells to facilitate binding of antigen-specific receptors, to facilitate attachment of leukocytes to endothelial surfaces in order to leave the blood vessels and enter into the surrounding tissues, to identify and accumulate at sites of inflammation, and to identify organ- or tissue-specific sites (e.g., lymph nodes, intestinal mucosa) into which they must enter in order to undergo developmental processes or carry out other immunologic functions (48,49 and 50).
CELLS OF THE IMMUNE SYSTEM Part of "1 - Review of Immunology"
Lymphocytes (General) The ability of the immune system to recognize specifically a diverse range of antigens resides with the lymphocytes (2,3 and 4). The lymphocytic lineage, derived from stem cells residing within the bone marrow, includes the B lymphocytes, T lymphocytes, and null cells. B lymphocytes mature in the bone marrow, and those destined to become T lymphocytes migrate to the thymus, where they mature. The bone marrow and thymus thus constitute the primary lymphoid organs of the immune system, as opposed to the secondary organs (e.g., spleen, lymph nodes, Peyer patches), where cells later periodically congregate as they circulate throughout the body. The ability of the immune system to identify so many different antigens is based on a division of labor—each lymphocyte (or clone of lymphocytes) is able to identify only one epitope or determinant. During its development and differentiation, each cell that is committed to becoming a B or T lymphocyte rearranges the DNA encoding its receptors (as previously described) to construct a unique antigen receptor. Thereafter, that cell and all of its clonal descendants express receptors with the same antigenic specificity. Other surface molecules and secreted products serve to define functional subsets of lymphocytes (Table 1.3). The specificity of an immune response lies in the fact that the entry of a foreign antigen into the body stimulates only those lymphocytes whose receptors recognize and bind the determinants expressed on the antigen. As a result of this specific binding and subsequent intercellular communication, a response is initiated that includes the following distinct phases:
T AB L E 1 . 3. Cells of the human immune sy ste m: markers and fu nc tions
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Recognition of antigen by binding to the receptors of lymphoid cells—often manifested by clonal proliferation of the stimulated cells P.13
• • •
Differentiation and maturation of the stimulated cells to mature functional capacity Response against the antigen, cell, or organism by any of several methods Establishment of immunologic memory
Memory resides in a portion of the stimulated lymphocytes that do not carry out effector functions (51,52). Instead, they remain quiescent in the system, providing an enlarged pool of activated cells specific for the original stimulating epitope. As a result, subsequent exposures to that same epitope can produce faster and higher (secondary or anamnestic) responses than were seen in the initial (primary) response. Memory can persist for long periods of time and is primarily maintained by T lymphocytes.
B Lymphocytes
Immunoglobulins recognize and bind specific antigens and determinants. Each B cell, or clonally derived set of B cells, expresses only a single “species” of immunoglobulin and is capable of P.14 recognizing and binding to only a single epitope. Immunoglobulin can be either membrane bound or secreted, and these forms serve two different purposes:
•
When membrane-bound on a B-cell surface, immunoglobulin detects the antigen or epitope for which that particular B cell is specific. The binding of antigen to the surface immunoglobulin, together with “help” from T lymphocytes (proliferative and maturation factors), induces the B cell to proliferate and mature into a plasma cell that secretes large amounts of immunoglobulin or becomes a memory B cell (53).
•
When secreted by plasma cells, immunoglobulin binds to the antigen of interest, “tagging” it for removal or for subsequent interaction with other cells and molecules (e.g., complement or phagocytic cells). The binding specificity of the membrane-bound and secreted immunoglobulins from a single B cell or clonal set of B cells and plasma cells are essentially identical. However, as mentioned previously, mutations can occur and accumulate in the immunoglobulin-encoding genes of B lymphocytes undergoing proliferation after restimulation with antigen. Where the mutated immunoglobulins are capable of binding more tightly to the antigen, the cells producing those immunoglobulins are stimulated to proliferate more rapidly. In this way, an ongoing antibody response can generate new immunoglobulin varieties with higher affinity for the antigen in question, a process known as affinity maturation.
T Lymphocytes T lymphocytes (T cells) also bear antigen-specific surface receptors. The TCR of most T cells is an α-β heterodimer, complexed with other molecules (e.g., CD3, CD4, and CD8) providing auxiliary functions. As described earlier, the TCRs bind not to antigen alone, but rather to specific combinations of antigen and class I or II MHC molecules (54,55). T cells include several different functional groups:
•
Helper T cells initiate responses by proliferating and providing help to B cells and to other T cells (e.g., cytotoxic T lymphocytes) and participate in inflammatory responses. T-cell help consists of a variety of cytokines that are required for activation, proliferation, and differentiation of cells involved in the immune response, including the helper T cells themselves. Helper T cells, in turn, comprise two broad categories: TH1 and TH2 (56,57,58 and 59), which secrete different sets of cytokines. These two particular subsets have been best characterized in mice, and comparable subsets are being identified in humans. All helper T cells, both TH1 and TH2, bear the CD4 marker and receptors that recognize combinations of antigen and class II HLA molecules.
•
TH1 cells help other effector T cells (e.g., cytotoxic T lymphocytes) to carry out cellmediated responses (57,58). In mice, they also help B cells producing immunoglobulins of the IgG2a isotype. TH1 cells are characterized by the production of interleukin-2 (IL-2), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) (Table 1.2). They participate in delayed-typed hypersensitivity (DTH) responses, but it is unclear whether the cells doing so are a distinct subset of TH1. In addition to its helper functions, IFN-γ also diminishes the activity of TH2 cells.
• •
TH2 cells cells provide help for most B cells (with exceptions such as IgG2a in mice) and are characterized by the production of IL-4, IL-5, IL-6, and IL-10 (Table 1.2). In addition to their helper functions, IL-4 and IL-10 diminish the activity of TH1 cells. CTLs can lyse other cells, which they identify as altered by infection or transformation, through direct contact (60,61,62 and 63) and using a short-range acting cytolysin, which does not damage the membrane of the CTL itself. These cells, which require help from TH1 cells to proliferate and differentiate, bear CD8 molecules and TCRs recognizing antigen and class I HLA molecule combinations on the surface of antigen-producing cells (where they are first stimulated) and later on the surface of cells that they subsequently identify as targets for destruction. In order for a CTL to attack and P.15
lyse a potential target cell, it must see (on that target) the same combination of antigen and class I HLA molecule that provided its initial stimulation.
•
DTH T cells (a subset of TH1) mediate an effector mechanism whereby the Tdh, bearing CD4 molecules and triggered by specific combinations of antigen and class II HLA molecules, produce cytokines that attract and activate macrophages (57,58). The activated macrophages, which themselves have no specificity for antigen, then produce a localized inflammatory response arising 24 to 72 hours after antigenic challenge.
•
Ts cells provide negative regulation to the immune system—the counterweight to TH cells (64). These cells, classically defined as bearing CD8 markers and recognizing combinations of antigen and class I MHC molecules, are involved in keeping immune responses within acceptable levels of intensity, depressing them as the antigenic stimulation declines, and preventing aberrant immune responses against self antigens. The mechanisms by which Ts cells carry out these functions is currently a topic of intense debate, and some investigators question their existence altogether. More recently, CD4+ T cells have also been implicated in
some types of suppression, the mutual negative regulation of TH1 and TH2 cells providing one such example. Although T lymphocytes with αβ TCR also express either the CD4 or CD8 markers, those with γδ TCR usually express neither. The ontogeny, distribution, and functional roles of γδ T lymphocytes are still not as well understood as those of αβ T lymphocytes (65). The TCRs of a lineage of T cells do not accumulate mutations and, under affinity, maturation, as do immunoglobulins.
Macrophages and Other Antigen-presenting Cells TCRs do not usually recognize antigen alone in its natural form, but rather bind to antigen that has been processed and presented on the surface of appropriate APCs (66,67 and 68). APCs internalize antigen, enzymatically degrade it into fragments (processing), and put the fragments back onto their surface in association with class I and II MHC molecules (presentation) (7). APCs (Table 1.3) include monocytes, macrophages, and other related tissue-specific cells that express class II MHC molecules (e.g., astrocytes in the central nervous system, Langerhans cells in the skin, Kupffer cells in the liver, and so forth). In addition, B lymphocytes (which normally express class II) can efficiently process and present antigen (69,70). There are some other cells that are capable of transient expression of class II (e.g., vascular endothelium). In addition, a variety of other molecules on APCs and T cells serve to stabilize the contact between the TCR and combination of antigen and MHC molecule.
Null Cells In addition to T and B cells, the lymphoid lineage includes a subset of cells lacking both of the classic lymphoid antigen receptors (immunoglobulin and TCR). This subset includes killer (K) and natural killer (NK) cells, and probably other cells, such as lymphokine-activated killer cells and large granular lymphocytes, which may represent differentially activated forms of K and NK cells (71,72). K cells bear receptors capable of recognizing the Fc portion of bound immunoglobulins. If the antigen is on the surface of a cell, the K cell uses the bound immunoglobulin to make contact with that cell and lyse it by direct contact, a process termed antibody-dependent cellular cytotoxicity (ADCC). The K cell has no specificity for the antigen that is bound to the antibody, only for the Fc portion of the bound antibody. NK cells appear to distinguish between altered (by malignant transformation or viral infection) cells and comparable normal cells and to bind preferentially and lyse the former. The means by which they make this distinction is unknown, but their activity is heightened by IFN-γ and IL-2. NK cells are able to recognize decreases in the levels of class I MHC molecules or other molecules on the surface of infected or malignant cells (71,72). Recent evidence suggests that K cells may be a subset of NK cells and that the distinction between them may simply reflect P.16 distinct stages of differentiation, or even simply the use of different assay systems.
Mast Cells and Granulocytes A variety of other cells are involved in some immune responses, particularly those involving inflammation (Table 1.3). Mast cells and basophils bear receptors (FcεRI) for the Fc portion of unbound IgE, permitting them to use IgE on their own surface as an antigen detector (73). When antigen binds simultaneously to two or more such IgE molecules on the same mast cell (called bridging), a signal is transmitted into the cell, leading to degranulation and release of a variety of mediators, including histamine, resulting in immediate hypersensitivity (allergic) responses (74). Neutrophils are drawn to sites of inflammation by cytokines, where their phagocytic activity and production of enzymes and other soluble mediators contribute to the inflammation. Eosinophils (75,76) are involved in immune responses against large parasites, such as roundworms, and are apparently capable of killing them by direct contact.
PRIMARY ORGANS: BONE MARROW AND THYMUS Part of "1 - Review of Immunology" The primordial stem cells that ultimately produce the human immune system (and other elements of the hematopoietic system) originate in the yolk sac, about 60 days after fertilization. These cells migrate to the fetal liver and then (beginning about 80 days after fertilization) to the bone marrow, where they remain for life. These primordial hematopoietic stem cells give rise to more specialized stem cells, which lead to the erythrocytic, granulocytic, thrombocytic (platelet), myelocytic (e.g., macrophages and monocytes), and lymphocytic lineages. Primary lymphoid organs consist of the bone marrow and thymus, where B and T lymphocytes, respectively, mature. B cells undergo their development, including generation of immunoglobulin receptors, while in the bone marrow. Cells of the T-lymphocyte lineage, however, migrate from the bone marrow to the thymus, where they undergo development and generation of TCRs (77,78). More than 95% of the cells that migrate into the thymus perish there, failing to survive a rigorous selection process to promote the development of those relevant to the individual's MHC genotype and to eliminate potentially self-reactive cells. It is in the thymus, under the influence of thymic stroma, nurse cells, and thymic APCs, that T cells receive an initial “thymic education” with regard to what should be recognized as self (79,80).
Secondary Organs: Spleen and Lymph Nodes The secondary organs (e.g., spleen, lymph nodes, Peyer patches) provide sites where recirculating lymphocytes and APCs enter after passage through diverse parts of the body, “mingle” in close proximity for a period of time, and then leave again to recirculate. This intimate contact between recirculating cells facilitates the close interactions needed to initiate immune responses and generate appropriately sensitized cells, whose activities may then be expressed throughout the body (2,3 and 4). Thus, most immune responses are actually initiated in the secondary organs.
INTERACTIONS IN IMMUNE RESPONSES Part of "1 - Review of Immunology"
Antibody Responses More than 99% of antibody responses are against T-dependent antigens, which require the involvement of T lymphocytes in generation of the responses. The relatively few T-independent (TI) antigens, which can provoke antibody production in the absence of T-cell involvement, fall into two general categories: TI-1 and TI-2. TI-1 antigens (e.g., a variety of lectins) are mitogenic, inducing proliferation and differentiation through binding of B-cell surface molecules other than immunoglobulins, whereas TI-2 antigens have regular repeating structures (e.g., dextran, with repetitive carbohydrate moieties) and are capable of cross-linking multiple immunoglobulin molecules on the surface of the same B cell. Antibody responses to most antigens are T dependent and require interactions between APCs P.17 (e.g., macrophages), T lymphocytes, and B lymphocytes (53,81,82,83 and 84), as illustrated in Fig. 1.9. B lymphocytes responding to T-dependent antigens require two signals for proliferation and differentiation: (a) the binding of their surface immunoglobulin by appropriate specific antigen, and (b) the binding of cytokines (e.g., IL-4 and other helper factors) produced by activated helper T cells (85,86). The help provided by T cells acts only over a short range; thus, the T and B cells must be in fairly intimate contact for these interactions to occur successfully. The involvement of APCs, such as macrophages or even B cells themselves, is essential for the activation of helper T cells and provides a means of bringing T and B cells into proximity.
FIG. 1.9. Interactions in antibody production.
Cellular Responses
The mixed lymphocyte response (MLR) is an in vitro measure of T-cell proliferation (primarily of CD4+ T cells) that is often used as a measure of the initial phase (recognition and proliferation) of the cellular response. Splenic or lymph node T cells (or both) from the individual in question (responder) are mixed with lymphocytes from another individual (sensitizer) against whom the response is to be evaluated. The sensitizing cells are usually treated (e.g., with mitomycin or irradiation) to prevent them from proliferating. The two cell populations are incubated together for 4 to 5 days, after which time tritiated thymidine is added to the culture for a few hours. If the responder cells actively proliferate as a result of the recognition of foreign antigens on the sensitizing cells, significant increases of thymidine incorporation (over control levels) can be measured. The strongest MLR responses typically occur when the sensitizing cells bear different class II MHC molecules than the responding cells, although primary significant MLR responses can also often be observed for class I MHC differences only, and even for some non-MHC gene differences, such as the Mls gene in the mouse (87). If the responder was sufficiently sensitized in vivo before the MLR, significant responses to other non-MHC alloantigens can often be seen as well. The MLR is a special subset of Tproliferative assays, one that is directed at genetically encoded alloantigens between two populations of lymphocytes. The same principle can, however, be used to assess the proliferation of T cells against antigen in other forms, such as soluble antigen on the surface of APCs. Cell-mediated lysis is the response function of cytotoxic T lymphocytes. After appropriate stimulation (by antigen in conjunction with class I P.18 MHC molecules on the surface of APC, together with help from TH1 cells), CTLs proliferate and differentiate to become capable of binding and destroying target cells through direct cell–cell contact (Fig. 1.10). Clonally derived CTLs can lyse only those cells that bear the same combination of antigen and class I MHC molecules originally recognized by the originally stimulated CTL from which the clone was generated. Death of the target cell can be induced through the action of perforins secreted by the CTL or through apoptosis induced by binding of target cell receptors by ligands on the surface of CTLS or by cytokines secreted by the CTL.
FIG. 1.10. Cytotoxic T lymphocytes. DTH is an in vivo response by inflammatory TH1 (or Tdh) cells (Fig. 1.11). Individuals presensitized against a particular antigen, then later challenged intradermally with a small amount of the same antigen, display local inflammatory responses 24 to 72 hours later at the site of challenge. Perhaps the best known example is a positive tuberculin skin test (Mantoux test). The response is mediated by CD4+ TH1 cells, previously sensitized against a particular combination of antigen and class II MHC molecules. Upon subsequent exposure to the same combination of antigen and class II MHC molecules, the TH1 cells respond by secreting a series of cytokines (Table 1.2) that attract macrophages to the site of interest and activate them. The activated macrophages exhibit an increased size and activity, enabling them to destroy and phagocytize the antigenic stimulus. However, because macrophages are not antigen specific, they may also destroy normal cells and tissues in the local area, referred to as innocent bystander destruction.
FIG. 1.11. Delayed-type hypersensitivity.
THE IMMUNE SYSTEM: A DOUBLE-EDGED SWORD Part of "1 - Review of Immunology"
The immune system evolved to protect the body from a variety of external (infectious agents or harmful molecules) and internal (malignant cells) threats. In this regard, the immune system provides the body with a means for minimizing or preventing disease. This is most clearly illustrated by individuals who have defects in immune function (immunodeficiency disease) resulting from genetic, developmental, infective, or therapeutic causes. Because of its destructive potential, however, the immune system is also capable of causing disease when confronted with inappropriate antigenic stimulation or loss of regulatory control (88). P.19
Transplantation Transplantation involves the ability to replace damaged or diseased body parts by transplanting organs from one individual to another. Unfortunately, the immune system is exquisitely adept at recognizing nonself and rejecting transplanted organs from donors differing genetically from the recipient (89). The genetically encoded molecules that trigger the rejection response are termed histocompatibility antigens and are divided into two primary categories: major (encoded by class I and II MHC genes) and minor (scores, possibly hundreds, of antigens encoded by widely diverse genes scattered across the chromosomes). Because a genetically perfect match between host and donor in humans exists only between identical twins, transplantation surgeons are forced to minimize or eliminate the recipient immune response against the transplanted organ. Some of these responses can be minimized by using the closest possible genetic match between donor and recipient by tissue typing, but in humans, this is possible only for the HLA system. The alternative is the use of drugs to reduce immune responsiveness. Ideally, only the ability of the immune system to react to the antigens on the transplanted organ would be diminished (i.e., induction of antigen-specific immunologic tolerance), leaving the rest of the immune system intact. However, we currently must rely on drugs that depress the immune system in a relatively nonspecific fashion, thus leaving the patient susceptible to potentially fatal opportunistic infections. Recently, some agents (i.e., cyclosporine and FK506) have been found to diminish immune responses in a somewhat more specific fashion, but their long-term use may have secondary adverse effects on organs. Bone marrow transplantation represents a special case in which the graft itself comprises immunocompetent tissue and the host is either immunodeficient or immunosuppressed. Thus, there is the possibility of the graft mounting an immune response against foreign host cells and tissues, leading to graft-versus-host disease (90,91)
Autoimmunity Autoimmune diseases involve the development of antibody or cell-mediated immune responses directed against self antigens (88,92). In many autoimmune diseases, an individual's risk is P.20 affected by his or her HLA genes (2,3 and 4,93,94). There are several possible scenarios under which such undesirable responses might be initiated. Autoimmune responses may arise when antigens that have been normally sequestered from the immune system (e.g., in immunologically privileged sites) are exposed as a result of trauma. Having never been detected previously by the immune system as it developed its sense of self versus nonself, such antigens are now seen as foreign. Second, the interaction of self molecules with small reactive chemicals (e.g., haptens) or with infectious agents may produce alterations in self molecules (altered antigens or neoantigens), resulting in their detection as nonself. Third, immune responses against determinants on infectious agents may generate clones of lymphocytes with receptors capable of cross-reacting with self antigens (cross-reactive antigens). A classic example is rheumatic fever, which results from immune responses against streptococcal antigens that are cross-reactive with molecules found on cardiac tissue. Finally, some autoimmune responses, especially those that tend to develop in later life, may result from senescence of inhibitory mechanisms, such as suppressor T lymphocytes, that keep autoimmune responses under control. For example, the onset of systemic lupus erythematosus is associated with age and an accompanying decline in suppressor Tcell function.
Immune Complex Diseases The humoral immune response is generally efficient in eliminating antigen–antibody complexes through the phagocytic cells of the reticuloendothelial system. There are, however, situations in which antigen–antibody complexes (involving IgG and IgM antibodies) reach such high concentrations that they precipitate out of solution and accumulate in tissues, often unrelated to the source of the antigen. This may lead to systemic or localized inflammation as the complexes bind and activate serum complement components, attract phagocytic cells, and induce the release of proteolytic enzymes and other mediators of inflammation. Attempts to clear depositions of antigen–antibody complexes often damage the tissues and organs involved. Such situations most often arise as a secondary effect of situations in which there is a persistence of antigen (e.g., chronic infection, cancer, autoimmunity, or frequent repeated administration of an external reagent), leading to continual stimulation of the immune system and production of high levels of antibodies against the persisting antigen. Among the most commonly damaged sites are the kidneys, of which the filtration apparatus tends to accumulate deposited complexes (glomerulonephritis); the synovial joint membranes (rheumatoid arthritis); the skin (rashes); and the endothelial walls of blood vessels (arteritis).
Contact Dermatitis Contact dermatitis is an example of a normally protective T-cell–mediated immune response that becomes harmful under certain circ*mstances. Contact dermatitis is a DTH response, usually caused by the presence of small, chemically reactive antigens (e.g., heavy metals or, as in the case of poison ivy, plant lipids such as catechol) that bind to self proteins (e.g., class II MHC molecules) on the skin and produce neoantigens.
Allergies and Anaphylaxis (Immediate Hypersensitivity) Allergies and anaphylaxis represent antigen-specific immunologic reactions involving IgE antibodies bound (by their Fc domain) to the membranes of mast cells and basophils (95). When antigen is bound, resulting in cross-linking of the IgE molecules, the mast cells are stimulated to degranulate and release histamine, serotonin, platelet-activating factors, and other mediators of immediate
hypersensitivity. The result is the rapid onset of an inflammatory response. Immediate hypersensitivity may develop against a wide array of environmental substances and may be localized (e.g., itching, tearing) or systemic (e.g., involving the circulatory system). The latter may be lifethreatening if severe. Treatment P.21 involves the prompt administration of pharmaceutical agents (e.g., epinephrine, antihistamines).
TOLERANCE Part of "1 - Review of Immunology" In many cases, it is desirable to diminish or eliminate immune responses, thus inducing tolerance to some particular antigen. For example, autoimmune responses, asthmatic and allergic responses, and the host responses against transplanted tissues or organs all represent situations in which such tolerance would be desirable. There are two approaches: nonspecific and specific. Immunosuppression is the elimination of all immune responses, regardless of the specificity of those responses. This may occur naturally, as in the case of individuals who are deficient in immune function for genetic reasons (e.g., severe combined immunodeficiency disease) or as the result of infection (e.g., acquired immunodeficiency syndrome). Alternatively, it may be intentionally imposed by the application of radiation, drugs, or other therapeutic reagents (e.g., antilymphocyte sera). Such procedures, however, impose a new set of risks because their nonspecificity leaves the patient (or experimental animal) open to infections by opportunistic pathogens. Attempts to diminish these consequences involve the development of reagents with narrower effects, including drugs such as cyclosporine and FK506, or the application of antibodies specific for only particular subsets of lymphocytes (96). Immunologic tolerance is the specific acquired inability of individuals to respond to a specific immunogenic determinant toward which they would otherwise normally respond. Tolerance is more desirable than immunosuppression because it eliminates or inactivates only those lymphocytes involved in the responses of concern, leaving the remainder of the immune system intact to deal with opportunistic infections. The natural induction of tolerance during the development of the immune system prevents immune responses against self antigens (self tolerance), thus preventing autoimmunity (97,98). Experimentally, tolerance can be induced in immunocompetent adult animals by manipulating a variety of factors, including age, the physical nature and dose of antigen, and the route of administration (99,100). Tolerance may be induced in both T and B lymphocytes, although tolerance of T cells generally requires lower doses of antigen and is effective for a longer period of time. In addition, because B lymphocytes require T-cell help, the induction of tolerance in T cells often also diminishes corresponding antibody responses. The means by which specific tolerance is induced and maintained involve three general mechanisms, all of which probably occur in various situations. Clonal deletion or abortion is the actual elimination of those clones of lymphocytes that encounter the specific antigen under particular conditions. Clonal anergy is the functional inactivation of those clones of lymphocytes that encounter the specific antigen in a tolerogenic form. This may be reversible. Antigen-specific suppression relies on the presence of cells that inhibit the antigen-specific induction or expression of immune responses by other T or B lymphocytes. It is known that TH1 cells (promoting cellular and inflammatory responses) and TH2 cells (promoting antibody responses) directed against the same antigen may inhibit one another through the cytokines they secrete. Thus, a response against a given antigen may be dominated by cellular responses in one case and by antibody responses in another. And, for example, attempts to alleviate cellular inflammatory responses directed against a given antigen may involve the promotion of antibody responses against the same antigen. The association of autoimmune disorders with advancing age is often attributed to age-related declines in suppressor T cells. The immune system is an amazing biologic system. Precise interactions must occur, in appropriate sequences and quantities, between a bewildering array of cells and molecules. Moreover, these highly specific cells and molecules must find one another, after patrolling throughout the entire body, in order to coordinate their activities. It is so complex that it seems incredible P.22 at times not that it all usually works so well, but that it works at all. It can malfunction, however, with potentially harmful consequences, and we still have far to go in learning how to correct and alleviate these occasions.
2 Evaluation and Management of Immune Deficiency in Allergy Practice Melvin Berger Departments of Pediatrics and Pathology, Case Western Reserve University; Department of Pediatrics, Rainbow Babies' and Childrens' Hospital, University Hospitals Health System, Cleveland, Ohio
Contents • • • • • •
INDICATIONS FOR AN IMMUNOLOGIC WORKUP DOCUMENTING THE HISTORY OF INFECTION THE PHYSICAL EXAMINATION IN CASES OF SUSPECTED IMMUNE DEFICIENCY GENERAL LABORATORY SCREENING TESTS IMMUNOLOGIC SCREENING TESTS DETAILED IMMUNOLOGIC LABORATORY EVALUATION
• • •
MOLECULAR DIAGNOSIS AND OTHER ADVANCED TESTING EARLY MANAGEMENT OF CELLULAR AND SEVERE COMBINED IMMUNE DEFICIENCY MANAGEMENT OF ANTIBODY DEFICIENCY SYNDROMES
Because there is considerable overlap between the manifestations of allergy and infection (i.e., rhinorrhea, sneezing, cough, wheezing) and because allergy may be a predisposing factor in sinusitis, otitis, and other respiratory infections, the allergist is frequently called on to evaluate patients with symptoms that have been attributed to recurrent infections and in which the competence of the patient's immune system has been or should be questioned. Because half or more of all patients with primary immune defects have antibody deficiencies (1,2) and most of them have problems with recurrent sinopulmonary infections, this is not an uncommon problem in clinical practice. The intent of this chapter is to provide a practical approach to the diagnosis and management of such patients, not to provide a comprehensive review of immune deficiency disorders or their molecular bases. Readers who wish a more in-depth analysis of immune deficiency disorders should consult comprehensive texts devoted to those disorders, such as the multiauthor works edited by Ochs, Smith, and Puck (3) or Stiehm (4).
INDICATIONS FOR AN IMMUNOLOGIC WORKUP Part of "2 - Evaluation and Management of Immune Deficiency in Allergy Practice" Although many immune-deficient patients present with a clear history of distinct episodes of infection, the allergist is frequently called on to see patients with less severe, nonspecific symptoms such as nasal stuffiness, chronic and recurrent rhinorrhea, or cough, which may be due to infection, allergy, or other factors. The first step in sorting out such complaints is to try to distinguish whether the symptoms are, in fact, due to infection. Inciting factors, such as seasonality, and clearly identifiable trigger factors may suggest allergic etiologies, but changes in the weather and changes in seasons are frequently accompanied by changes in exposure to infectious diseases. Exposure to other people with similar symptoms and characteristics, such as the presence or absence of fever, description of excessive secretions (clear and watery versus thick and purulent), and the response to antibiotics, may help to distinguish between infectious and noninfectious etiologies. After an estimate of the real incidence of infection is obtained, this can be compared with benchmarks such as the “10 warning signs of immune deficiency” (Fig. 2.1). The incidence of infection should be compared with the incidence for that age group in the community, but the exposure history also needs to be taken into consideration. For example, a 40-year-old who lives alone and sits in front of a computer screen all day would be expected to have a different degree of exposure to infectious agents than a kindergarten teacher, day care worker, or pediatric office nurse. College students moving from home to the dormitory for the first time and military recruits P.26 P.27 often have sharp increases in infectious disease exposure. Similarly, a first-born baby at home often has a very different degree of exposure than a similar-aged child in day care or with many siblings. Generally, the frequency of respiratory infection among school-aged children in the United States is about six to eight upper respiratory infections per year, but as many as one a month while school is in session is not unusual. About half of these are primary bacterial infections or secondary bacterial sequelae, such as otitis media, sinusitis, pneumonia, or bronchitis.
FIG. 2.1. The 10 warning signs of primary immune deficiency. (Presented as a public service by The Jeffrey Modell Foundation and American Red Cross. These warnings signs were developed by The Jeffrey Modell Foundation Medical Advisory Board.) Patients with clear histories of more than 10 distinct episodes of infection per year, more than two documented episodes of pneumonia per year, or more than one life-threatening infection should be evaluated for possible immune deficiency or other underlying abnormality that may be contributing to this increased incidence of infection. However, the specialist must be careful in interpreting the history from the patient or parent. Frequently, antibiotics are given when the patient does not have clear evidence for bacterial infection, but the conclusion is drawn that antibiotics do not work, suggesting that there is something “wrong” with the patient's immune system. Frequent upper respiratory symptoms may represent individual viral upper respiratory infections; on the other hand, there may be prolonged symptoms from conditions, such as chronic sinusitis, that have not been adequately treated despite multiple short courses of oral antibiotics. The demonstration of densities on chest radiograph may represent atelectasis due to asthma rather than infiltrates and should not necessarily be taken as indicating recurrent pneumonia unless there is documentation of concomitant fever, elevated white blood cell count, or positive sputum Gram stain or culture.
Patients with unusually severe infections, such as those requiring parenteral antibiotics, prolonged or multiple courses of antibiotics for a single infection, or surgical intervention such as incision and drainage of abscesses or removal of seriously infected tissue (e.g., a segment of lung or infected bone), should probably also undergo at least screening (see later) to exclude immune deficiency. Patients with unusual or opportunistic infections should also be evaluated, as should those with unusual responses, such as prostration or excessive fever, to seemingly common organisms. Although many patients with primary immune deficiencies present with recurrent and chronic respiratory infections (2,5,6), gastrointestinal disorders are also common in these patients. The combination of recurrent respiratory infections with recurrent gastrointestinal symptoms may prompt immunologic screening even when the involvement of either organ system itself is not severe. Infection with Giardia lamblia (7,8) and bacterial overgrowth in the small intestine are not infrequent in patients with antibody deficiencies and may present with symptoms such as cramps or diarrhea after eating, leading to suspicion of food allergy on the part of the patient, parent (if a child), or referring physician, despite the absence of other manifestations of true allergy. In some immune deficient patients, there may be organized lymphonodular hyperplasia in the intestine or infiltration of the submucosa with scattered aggregates of lymphocytes (7); and patients with gastrointestinal workups or biopsy results not typical for recognized patterns of inflammatory bowel disease should also undergo evaluation for immune deficiencies. The presence of nonimmunologic findings on physical examination may also provide indications for evaluation to exclude immune deficiency (Table 2.1). These may include malabsorption with failure to thrive or excessive weight loss; eczema or thrombocytopenia in Wiskott-Aldrich syndrome (9); and facial, cardiac, or skeletal features suggestive of a recognizable pattern of malformation such as that seen in DiGeorge syndrome, short-limbed dwarfism, or cartilage-hair hypoplasia (10,11). Characteristic facial and skeletal abnormalities or eczematoid dermatitis may suggest the hyperimmunoglobulin E (hyper-IgE) or Job syndrome (12), and rib or other skeletal abnormalities may be present in severe combined immune deficiency (SCID) due to adenosine deaminase (ADA) deficiency (10,13). Alopecia or endocrinopathies occur with increased frequency in chronic mucocutaneous candidiasis (14). Nystagmus, clumsiness, and other neurologic abnormalities may occur before P.28 observable telangiectasias and can suggest the diagnosis of ataxia-telangiectasia (15), and neurologic disorders are also common in purine nucleoside phosphorylase deficiency (16). Although delayed separation of the umbilical cord stump is widely recognized as an indicator of leukocyte adherence protein deficiency, in fact, there is a wide variation in the time at which the stump separates, and this should not be overemphasized in an otherwise well infant (17). Of course, patients with positive screening tests for human immunodeficiency virus (HIV) would also be candidates for immunologic evaluation.
T AB L E 2 . 1. Physical findings not due to infectious disease associated with immune deficiency syndromes Several immune deficiencies can clearly be hereditary. For many of these, the patterns of inheritance and the precise molecular defects have been defined (18) (Table 2.2). Family members suspected of having these disorders, perhaps because an older sibling has already been diagnosed, should undergo assessment of their immune status. When available, tests for the specific molecular lesion should be included so that treatment aimed at correcting or compensating for the basic defect can be instituted early enough to prevent or minimize end-organ damage. Prenatal diagnosis and screening for the carrier state is now available for many of these disorders and can be used both in counseling and in ensuring that prompt and appropriate therapy is offered to affected newborns.
T AB L E 2 . 2. Inherited immune deficiencies
DOCUMENTING THE HISTORY OF INFECTION Part of "2 - Evaluation and Management of Immune Deficiency in Allergy Practice" A major goal in questioning the patient and reviewing the medical records is to develop a firm impression of the types of infections that the patient has suffered so that subsequent laboratory tests can be targeted to analyze specifically those components of the immune system whose defects P.29 would most likely explain the patient's symptoms. This will be best served by keeping in mind general patterns of infection that might be caused by defects in specific immunologic defense mechanisms. Thus, infections with encapsulated extracellular bacterial pathogens, particularly of the respiratory tract, are suggestive of defects in antibody production (19,20), which constitute the majority of all immune deficiencies (1). Superficial mucosal infections may particularly suggest isolated IgA deficiency (21). Infections with opportunistic pathogens, including protozoans and fungi, and recurrent episodes of chickenpox or chronic herpetic lesions, may suggest problems in cell-mediated immunity (20). Failure to clear bacteria promptly from the blood stream, resulting in bacteremia, sepsis, or hematogenously disseminated infections such as osteomyelitis, may be seen in
deficiencies of C3 or early-acting components of the complement system (22), but may also indicate asplenia or poor reticuloendothelial system function, as in sickle cell disease. Problems with recurrent or disseminated neisserial infections may suggest deficiency of the later-acting complement components that form the membrane attack complex (22). Abscesses and infections with unusual bacteria or fungi may suggest neutropenia or defects in neutrophil function (19,20,23,24). Enteroviral meningoencephalitis may suggest X-linked agammaglobulinemia. The number and types of infections and their individual and cumulative morbidity should be assessed. It is necessary to exclude carefully other causes of nonspecific symptoms; for example, is sniffling or congestion due to recurrent P.30 upper respiratory infection, allergy, or other types of rhinitis? If cough is a major complaint, it is important to determine whether this is due to sputum production, irritation, or other causes. Could it represent cough-equivalent asthma? If failure to thrive and cough are both present, could the patient have cystic fibrosis? Inflammatory bowel disease may mimic hypogammaglobulinemia in children with poor weight gain who also have recurrent rhinitis due to multiple mucosal viral infections, which by themselves would not be considered significant. Isolation and identification of responsible organisms is clearly the gold standard for rigorous diagnosis of infection. Documentation of fever, white blood count with differential, and sensitive but nonspecific measures such as the erythrocyte sedimentation rate and C-reactive protein, can help distinguish between chronic, recurrent sinusitis and headaches due to other causes and can help with the differential diagnosis of recurrent cough or other chest symptoms. The importance of culture and examination of smears of nasal secretions for bacteria and neutrophils versus eosinophils cannot be overemphasized in distinguishing infectious from allergic and other noninfectious etiologies, particularly in small children. In some cases, the most appropriate step in the workup is to send the patient back to the primary care physician with instructions to have appropriate cultures and those simple laboratory tests performed every time an infection is suspected or the symptoms recur. Sometimes, the culture result points to the diagnosis, as in the case of Pseudomonas aeruginosa suggesting cystic fibrosis, invasive aspergilli suggesting neutropenia or chronic granulomatous d i s e a s e ( C G D ) ( 2 4 ) o r S t r e p t o c o c c u s p n e u m o n i a e o r H a e m o p h i l u s i n f l u e n za e s u g g e s t i n g a n a n t i b o d y deficiency (19,20). Clues to the severity and overall morbidity resulting from infection may be obtained by asking whether hospitalization or intravenous antibiotics have been required to treat infections or whether oral antibiotics have generally been sufficient. The response to therapy should be evaluated carefully. Continued high fever or other symptoms suggesting a lack of response of culture-confirmed bacterial infection to antibiotics is more likely indicative of a significant immune deficiency than is the frequently seen pattern in which the fever and symptoms resolve promptly when antibiotic therapy is started (e.g., for otitis media) only to recur again shortly after the prescribed course of therapy is concluded. In many situations, the latter may actually represent a distinct new infection. This pattern is quite commonly seen in children in day care and in adults with frequent exposure to small children. Similarly, it is also important to distinguish inadequate or inappropriate therapy from failure to respond, and it is important to differentiate chronic infections from recurrent episodes. Absence from school or work should be quantitated if possible, and any long-term sequelae or disability should be documented. The family history should include questions about siblings and preceding generations. Family trees with premature deaths of male infants should raise suspicion of X-linked immune deficiencies (Table 2.2). Questions should also be asked about the family history of asthma and allergy as well as other genetic diseases that may present with recurrent infection such as cystic fibrosis. In evaluating a child, it may be important to determine whether the parents have died prematurely or have known risk factors for HIV infection. The age at onset of infections of unusual frequency or severity may yield important insights into possible underlying immune deficiencies. It must be kept in mind that term newborns have IgG levels equivalent to those of their mothers, from whom most of their IgG has been transferred across the placenta (25). Thus, babies who have problems with infections before the age of 6 months may have T-cell or phagocyte problems but are unlikely to have agammaglobulinemia or other isolated problems in antibody production. In contrast, disorders of antibody production are more likely to present after the age of 6 months. The history of exposure must be carefully considered in evaluating this issue because the frequency of common types of infections often increases after a child's exposure to infectious agents is increased after attending day care or preschool, particularly if there are no siblings in the home. Although patients with P.31 severe antibody deficiency such as that seen in Bruton agammaglobulinemia generally present between 6 months and 2 years of age (6,26), the diagnosis of the X-linked hyper-IgM syndrome is frequently delayed until later in childhood (27,28), and those with common variable immunodeficiency disease (CVID) may present at any age (5,29). It may not be clear whether this represents an earlyonset deficiency that has not been previously recognized or a newly acquired problem. Just as some infants may have delayed development of the full range of immune responses (30), it seems likely that some adults may undergo premature senescence of immune responsiveness (31) and may present with recurrent bacterial infections in their 40s or 50s.
THE PHYSICAL EXAMINATION IN CASES OF SUSPECTED IMMUNE DEFICIENCY Part of "2 - Evaluation and Management of Immune Deficiency in Allergy Practice" The physical examination often provides important evidence for or against immune deficiency and may also allow the physician to assess critically the cumulative morbidity due to infection. Most importantly, the presence or absence of lymphoid tissue should be carefully documented. The absence of visible tonsils in patients who have not had them surgically removed and the absence of palpable cervical or inguinal lymph nodes should promote a strong suspicion of a significant antibody deficiency because the bulk of these tissues is composed of B-lineage lymphoid cells involved in antibody synthesis. Conversely, the presence of palpable lymph nodes and easily visible tonsils
essentially excludes Bruton agammaglobulinemia and may suggest the absence of SCID but does not help one way or the other with the diagnosis of CVID or X-linked hyper-IgM syndrome. The presence of cervical or peripheral adenopathy, splenomegaly, or hepatomegaly may suggest HIV, CGD, or other abnormalities. Many anatomic findings are associated with immune defects in recognizable malformation syndromes (Table 2.1), and characteristic rashes may suggest Wiskott-Aldrich syndrome (9) or the hyper-IgE (Job) syndrome (12). Secondary effects, such as failure to thrive, weight loss, and short stature, may suggest significant morbidity due to chronic or recurrent infection. Scars from incision and drainage of abscesses or from drainage or surgical reduction of enlarged lymph nodes may indicate significant morbidity from neutrophil defects (24). Autoimmune phenomena (29,32) and rheumatic complaints (33,34), including infectious or chronic arthritis, are common in patients with CVID and other primary immune deficiencies and may help indicate that an evaluation for immune deficiency is warranted, even if the number or severity of acute infections has not been excessive. Careful assessment of the tympanic membranes, paranasal sinuses, and chest is extremely important in evaluating patients suspected of having antibody deficiency syndromes, and not only should the quantity and characteristics of secretions be documented but also some attempts should be made to determine whether observed abnormalities are acute or chronic. In this regard, high-resolution (thinslice) computed tomography (CT) scans of the chest may be very helpful because observation of bronchiectasis or areas of “ground-glass” density in the lung parenchyma may suggest the presence of subclinical chronic disease, which could be due to antibody deficiency (35,36). Clubbing of the digits may also provide an important indication of chronic lung disease.
GENERAL LABORATORY SCREENING TESTS Part of "2 - Evaluation and Management of Immune Deficiency in Allergy Practice" The Clinical and Laboratory Immunology Committee of the American Academy of Allergy, Asthma and Immunology has assembled a set of practice parameters for the diagnosis and management of immunodeficiency (37). These may help provide guidelines for the allergist-immunologist and the referring physician to those screening tests that might first be ordered and interpreted by the primary physician, as compared with situations in which referral to the specialist becomes appropriate. Often, the specialist is called by the primary care physician to determine whether a patient should be referred. P.32 A review of laboratory tests already obtained by the primary care physician may yield important clues to the presence of an immune deficiency disorder and may save steps in the evaluation of patients by suggesting which of the more specialized tests are most likely to be informative. The complete blood count (CBC) and differential will help to exclude neutropenia or may indicate lymphopenia, which could be seen in SCID or Bruton agammaglobulinemia. Abnormal or decreased platelets may suggest Wiskott-Aldrich syndrome, and fragmented erythrocytes may suggest sickle cell disease. General blood chemistry panels will show low total protein but normal albumin in agammaglobulinemia. A low uric acid level may be indicative of ADA deficiency or purine nucleoside phosphorylase deficiency, two causes of SCID (16,38); whereas a low serum calcium level may suggest DiGeorge syndrome. In addition to assessing the airways and lung parenchyma, the chest radiograph should be reviewed for the absence or presence of a thymus in infants and for the possibility of a thymoma, which may be associated with hypogammaglobulinemia in adults (39). Hyperinflation with patches of atelectasis, suggestive of asthma, might suggest that additional details of the past history should be carefully reviewed in patients, particularly small children, referred because of cough or recurrent pneumonia, because similar densities seen on previous films may not have actually been due to infection. The presence of old scars and active disease should be documented. Hilar adenopathy may be seen in cellular and humoral immune defects. Abnormalities of the ribs resembling those seen in rickets might suggest ADA deficiency (13), and cardiovascular abnormalities may suggest asplenia (40) or DiGeorge syndrome (41) or may steer the workup away from immune deficiency and toward Kartagener syndrome (situs inversus and ciliary dysmotility) or cystic fibrosis.
IMMUNOLOGIC SCREENING TESTS Part of "2 - Evaluation and Management of Immune Deficiency in Allergy Practice" Initial laboratory tests that may indicate that a patient has an immune deficiency can be done in most regional laboratories and community hospitals, and the results should be available in a few days. These should include measurement of the major immunoglobulins and IgG subclasses. In adults, serum protein electrophoresis should also be included because patients with monoclonal gammopathy, multiple myeloma, or chronic lymphocytic leukemia (CLL) may have antibody deficiency with a normal total level of any given class of immunoglobulin if the paraprotein is a member of that class. Interpretation of the results of measurement of the serum of concentrations of IgG and its subclasses is often less than straightforward (37,42). First of all, age-specific norms must be used, because of the marked changes in values during the first 2 years of life. Although some laboratories may report IgG concentrations as low as 200 mg/dL as normal in 3- to 6-month-old infants, concentrations of less than 400 mg/dL frequently fail to provide sufficient protective antibody levels. Second, even within a given age group, most laboratories report a normal range whose upper limit may be twofold or more higher than its lower limit. This probably reflects the fact that the total serum IgG concentration represents the sum of hundreds of separately regulated responses rather than a single variable whose physiology requires reasonably tight control, like that of an electrolyte or the blood glucose. Concentrations of IgG, and particularly its subclasses, vary not only among individuals of the same age who have different exposure histories but also in a single individual at different times. Thus, before any conclusions are reached about the diagnosis of IgG subclass deficiency, the tests should be repeated several weeks apart, and analysis of specific antibody titers should also be considered (see later). In judging the adequacy of any given IgG concentration in a given individual, the history of exposure and the frequency of documented infections must be considered. Thus, normal individuals with
frequent exposure to pathogens and those whose host defenses are compromised by conditions that do not affect lymphocyte responses, such as cystic fibrosis and chronic granulomatous disease, often have elevated total serum IgG concentrations. This may be thought of as reflecting a physiologic adaptation or as a response to increased or persistent P.33 antigen exposure by the normal immune system. IgG concentrations within the normal range, but toward its lower limit, in patients with comparably increased frequency of infection or morbidity due to infection (but without such underlying defects) may thus actually indicate relative deficiency in specific antibodies and should be evaluated further, as explained later. In addition to those conditions in which paraproteins may conceal true antibody deficiencies within normal total IgG levels, several diseases may be associated with nonspecific polyclonal B-cell activation that may cause the total IgG or IgM level to be within the normal range or even elevated, whereas specific antibodies may actually be deficient. This occurs most often in systemic lupus erythematosus, Epstein-Barr virus infection, and HIV infection (43,44). Finding low or absent serum IgA together with low-normal or borderline levels of one or more IgG subclasses, particularly subclass 2, should also raise suspicion of more severe defects in specific antibody production than would be suggested by the total IgG concentration itself, and such patients should also be investigated further (45). Elevated serum IgE and IgA concentrations may be found coexisting with deficiency of antibodies to polysaccharides in Wiskott-Aldrich syndrome, and extremely high IgE levels may suggest, but are not by themselves diagnostic of, hyper-IgE or Job syndrome. Analysis of lymphocyte surface antigens by flow cytometry is now widely available and should be included as a screening test in all patients in whom immune deficiency is suspected (46). A CBC with differential should always accompany lymphocyte surface marker analysis so that the absolute number of any given type of cell per cubic millimeter of blood can be calculated, in addition to ratios such as CD4/CD8 (47). As with immunoglobulin determinations, age-specific norms should be used (47). The physician should be careful about what specific test is ordered because, in the era of widespread treatment of HIV, many laboratories offer a standard lymphocyte surface marker panel, an analysis that includes only the total number of T cells (CD3+) and the two major subsets of T cells (CD4+ and CD8+). Because antibody deficiency due to decreased B-cell number or function is the most common type of immune deficiency overall, a complete analysis, including enumeration of natural killer (NK) and B cells, should be performed. Analysis of these lymphocyte subsets frequently provides important clues to the actual molecular defect in many cases of SCID (see later). In addition, because patients with chronic CLL may present with antibody deficiency, the ratio of lymphocytes positive for κ as opposed to λ light chains should also be determined. Flow cytometry to determine the presence of leukocyte integrins of the CD11/CD18 family can easily confirm or exclude the diagnosis of leukocyte adhesion deficiency type I (48). Similarly, flow cytometry may be used to test neutrophils for the sialyl-Lewis X antigen, whose absence establishes the diagnosis of the more rare leukocyte adhesion deficiency type II (49). More rare deficiencies involving other arms of the immune system can also be identified and characterized at this level of testing. In patients suspected of defects in T-cell–mediated immunity, the overall functional activity of T cells is best assessed by determining the patient's ability to mount cutaneous delayed hypersensitivity reactions to recall antigens such as candida, mumps, or tetanus toxoid (37,50). Obviously, delayed hypersensitivity skin tests have little meaning in children younger than 2 years of age, who may not be adequately immunized with the antigens in question. Patients who have infections suggestive of defects in T-cell–mediated immunity should also undergo HIV screening. The CBC will give an indication of the number of phagocytes, but assessing their function requires more specialized laboratory capabilities. Complement screening should include measurement of the serum C3 concentration and the total hemolytic activity (CH50) because the former may be seriously reduced without affecting the latter. Although the CH50 is the best overall screening test for complement defects and is zero in cases of late component defects, such as those that predispose to recurrent or disseminated neisserial infections (22), the serum for this test must be handled carefully or artifactually low values will be measured. In patients with a history of bacteremia, sepsis, or hematogenously spread P.34 infection, a careful review of the peripheral blood smear, looking for Howell-Jolly bodies in the erythrocytes, may suggest anatomic or functional asplenia or severely impaired reticuloendothelial system function.
DETAILED IMMUNOLOGIC LABORATORY EVALUATION Part of "2 - Evaluation and Management of Immune Deficiency in Allergy Practice" Although frank hypogammaglobulinemia, neutropenia, and complete deficiency of a component of the classic complement pathway can be detected by the screening laboratory tests described previously, more detailed testing is necessary to detect more subtle immune deficiencies. This level of testing is also frequently necessary to characterize severe defects more completely. Because of the possibility that clinically significant antibody deficiency may be present even when the total serum concentrations of the major immunoglobulin classes and IgG subclasses are normal, specific antibody production should be assessed in all cases in which the clinical presentation suggests recurrent bacterial infections, particularly of the respiratory tract, unless the major immunoglobulin classes themselves are absent or severely depressed. Specific antibody titers should be measured against polysaccharide as well as protein antigens (51,52). Although measurement of isohemagglutinins may be used to screen for the ability to produce antibodies against polysaccharides (the A, B, or both blood group substances in patients of other blood groups), the availability of measurement of antibodies against specific bacterial antigens (see later) has decreased dependence on those assays. In cases in which pathogens have been isolated and identified (e.g., from effusions at the time of insertion of tympanostomy tubes, endoscopic drainage of paranasal sinuses, or expectorated or induced sputum samples), antibodies against those specific organisms should also be measured. In addition, antibodies against common immunizing agents should be measured. We usually request measurement of antibodies against tetanus and diphtheria toxins and several pneumococcal
polysaccharides as well as H. influenzae type B polysaccharide (42,51). Testing for these and additional antibody titers are available in many commercial laboratories and are sometimes referred to as a humoral immunity panel. An advantage of using these particular antigens is that they are contained in readily available, welltested vaccines, which often have already been given to or will be clinically indicated for the patients in question, so that exposure to the antigen is definite. Obtaining titers before, as well as 4 to 8 weeks after, immunization allows comparison of the response to each antigen. The absence of a threefold rise in titer after immunization or failure to achieve protective levels indicates that the patient is unable to mount specific antibody responses. This may be seen either with protein or polysaccharide antigens and may indicate a failure to process properly or recognize an entire class of antigens, such as in what has been termed specific polysaccharide antibody deficiency, or certain particular antigens in what may be considered a “lacunar” defect. In some rare cases, patients already receiving immunoglobulin infusions may require assessment of their own specific antibody production, which may be difficult because antibodies against many common antigens will have been acquired passively. In most cases, the immunoglobulin therapy can be stopped for a few months so that the patients can be immunized and their own antibody production measured while they are being reassessed clinically. If this is not possible, special test antigens, such as keyhole limpet hemocyanin and the bacteriophage φX174, can be obtained from specialized centers (53). Because most individuals and plasma donors have not been commonly exposed to these antigens, commercial immunoglobulin preparations do not contain antibodies against them, and they can be used to assess de novo specific antibody formation. Specific T-cell function is most commonly tested by measuring the incorporation of 3H-thymidine into the newly formed DNA of rapidly proliferating lymphocytes after cultures of peripheral blood mononuclear cells are stimulated in vitro (54). Lectins, proteins generally derived from plants that bind specific polysaccharides, commonly present in surface glycoproteins on P.35 human cells and are frequently used as the stimuli in such assays. Because these proteins stimulate most human lymphocytes, regardless of prior antigen sensitization, they are called mitogens, and tests using them should be referred to as lymphocyte mitogen proliferation assays. Plant lectins often used as stimuli for mitogen proliferation assays include concanavalin A, phytohemagglutinin, and pokeweed mitogen. Incorporation of 3H-thymidine, a low-molecular-weight precursor, into highmolecular-weight cellular DNA in newly proliferating lymphocytes serves as the basis for the measurements, and the results may be expressed as the amount incorporated (in counts per minute) or as the ratio of incorporation in parallel cultures of mitogen-stimulated versus unstimulated lymphocytes, also referred to as the stimulation index. Mitogen stimulation tests are useful even in newborns who have not received any immunizations and may be particularly informative about lymphocyte function and immune competence in babies with partial T-cell deficiency, such as those with DiGeorge syndrome (55). Disadvantages of these tests include the requirements for several milliliters of blood, which may be prohibitive for small newborns; time constraints that may be imposed by the laboratory to facilitate isolation of the mononuclear cells during normal working hours; and the fact that the cells must be cultured for several days (usually 48 to 72 hours) before they are “pulsed” with 3H-thymidine to assess its incorporation. To surmount these difficulties, many laboratories are now using flow cytometry assays based on the appearance on the lymphocyte plasma membrane of early activation markers such as CD69 (56). Mixed lymphocyte cultures, in which a patient's (or potential donor's) T cells are stimulated by a relative's lymphocytes that have been irradiated to prevent them from proliferating, are also used to test T-cell competence and to determine histocompatability in cases in which bone marrow transplantation is contemplated. Staphylococcal enterotoxins are also often employed as stimuli in proliferation assays because they function as “superantigens,” which stimulate broad families of T cells by binding to parts of their T-cell receptors other than the antigen-binding site. The response to these superantigens is thus also independent of prior antigen sensitization. The Cowen strain of Staphylococcus aureus may be used as a T-cell–independent stimulus for B-cell proliferation. T-cell proliferative responses to recall antigens may also be assessed using similar techniques, although because a smaller number of T cells will respond to any given antigen than to the more broadly reacting mitogens discussed previously, these tests commonly involve 4- to 5-day incubation periods before the 3H-thymidine is added and its incorporation determined. Obviously, antigen responses can only be expected if it is documented that the patient has been exposed to the antigen in question. Thus, antigen stimulation tests are usually not useful in early infancy. However, if an older child is known to have received his or her scheduled immunizations, or if candidal infection has been obvious, the response to soluble candidal preparations and vaccine antigens such as tetanus toxoid may be useful. Thus, patients with normal responses to mitogens who fail to respond to candidal preparations may be considered to have chronic mucocutaneous candidiasis rather than a more pervasive T-cell defect, as might be seen in DiGeorge syndrome or HIV infection. In patients with opportunistic infections suggestive of AIDS or positive screening tests for HIV, confirmatory tests, such as Western blot, and quantitation of p24 antigen or viral load should be performed, and absolute CD4 number as well as T-cell function should be assessed as part of the detailed evaluation. Detailed laboratory analysis in patients suspected of phagocyte disorders should include assessment of neutrophil chemotaxis and the oxidative respiratory burst that accompanies phagocytosis (37,57,58). Chemotaxis is assessed by measuring the migration of polymorphonuclear leukocytes through agar gels or across filters in specially designed Plexiglas (Boyden) chambers. The oxidative burst can be assessed by the nitroblue tetrazolium test, in which a soluble yellow dye is reduced to an easily visible insoluble blue intracellular precipitate (59). This is available in most hematology laboratories. Flow cytometric assays in which oxidized products are detected by fluorescence may also be employed (58). If P.36 the CH50 was abnormal on screening, the actual deficient component can be identified in reference laboratories that stock commercially available purified complement components and test systems. These laboratories can also screen for abnormalities of the alternative pathway, which may be indicated in patients who have recurrent bacterial infections or bacteremia and sepsis but in whom antibodies and the classic pathway have been found to be normal.
MOLECULAR TESTING
DIAGNOSIS
AND
OTHER
ADVANCED
Part of "2 - Evaluation and Management of Immune Deficiency in Allergy Practice" Advanced testing designed to pinpoint the molecular lesion in cases of confirmed immune deficiency are usually performed at a university or regional research center laboratory by an immunologist specializing in such cases. However, an additional level of definition is now possible in many hospital laboratories and may aid the practitioner in providing prognostic and genetic counseling information for patients and their families. Furthermore, the practitioner should recognize the importance of defining the molecular defects in the management of immune-deficient patients because several forms of specific therapy are already available and new modalities are being developed at a rapid rate as a result in advances in understanding of the physiology of lymphocytes and cytokines as well as the genome project. Importantly, within the B-cell disorders, the pattern of X-chromosome inactivation (60) can be used to determine whether female family members are carriers of Bruton agammaglobulinemia (61). This type of analysis is also applicable to Wiskott-Aldrich syndrome, neutrophil defects, and other, but not all, X-linked disorders (60). The lack of expression on T-cells of gp-39 (CD154), the ligand for B-cell CD40, confirms the diagnosis of X-linked hyper-IgM syndrome, which might be confused with CVID in some cases (27,28). Patients with SCID should be classified as completely as possible with flow cytometry, which may be highly suggestive of the exact molecular lesion and may have important prognostic implications (62). In particular, relative preservation of B cells in SCID patients with very low T and NK cell counts may suggest deficiency of the important signaling kinase Jak 3 or the γ c chain (62), which is an important subunit of several cytokine receptors necessary for lymphocyte development. All three cell types may be present in equal numbers in autosomal recessive SCID not due to ADA deficiency. Relatively selective deficiency of CD8 cells is characteristic of deficiency of Zap 70, a protein kinase important in signaling from the T-cell receptor. The most likely defect can then be confirmed in specialized research laboratories using assays for the specific protein (Western blot or flow cytometry) or gene that is suspect. Fluorescence in situ hybridization can be used to confirm the chromosome abnormality in patients suspected of having DiGeorge or velocardiofacial syndrome, overlapping sets of anomalies that may be associated with partial T-cell deficiencies and are due to microdeletions in chromosome 22q11.2 (41). Patients with SCID, their parents, and their siblings should undergo human leukocyte antigen typing to begin to evaluate the possibility of bone marrow transplantation, which may be accompanied by minimal morbidity and may be curative in many cases (63). If there is no potential donor who matches at all loci, transplantation of T-cell–depleted marrow from a donor with a mismatch at one or more loci might be considered but is performed only at certain research centers. There may be mild or delayed presentations of SCID due to enzyme deficiencies, such as purine nucleoside phosphorylase deficiency or ADA deficiency (64). Making the correct diagnosis as early as possible is especially important in the latter because enzyme replacement with bovine ADA conjugated with polyethylene glycol (Adagen) is readily available, often results in marked amelioration of the immune defect, and can serve as a bridge until bone marrow transplantation or as long-term replacement if the patient does not have a matched donor (65,66). Anticoagulated whole blood should be sent to a research center with expertise in these assays (66) in cases of T-cell deficiency with impaired mitogen responses. Gene therapy has been used with some success in ADA deficiency and in deficiency of the γ c chain of the T-cell cytokine receptor (67); hence, early determination of the presence of P.37 the latter by flow cytometry in cases of apparent SCID in which B cells are present (62,67) is also important.
EARLY MANAGEMENT OF CELLULAR COMBINED IMMUNE DEFICIENCY
AND
SEVERE
Part of "2 - Evaluation and Management of Immune Deficiency in Allergy Practice" Infants with significant defects in T-cell number or function and those with SCID are not only at great risk for infection with opportunistic pathogens but also may suffer from severe or overwhelming infection with attenuated live viruses normally used for immunization and may be susceptible to graftversus-host disease (GVHD) from transfused leukocytes. For these reasons, special precautions must be initiated as soon as this type of immune defect is suspected, while the immunologic workup is proceeding and plans for referral and definitive treatment are being formulated. First, any blood products that are given must be irradiated to prevent transfusion of viable lymphocytes that could cause GVHD. Second, live virus vaccines must be avoided. With current recommendations in the United States abandoning the use of the live attenuated oral polio vaccine and replacing it with inactivated vaccine only, polio is less of a risk. However, immunization with Bacille-Calmette-Guérin vaccine is practiced in many other countries and may lead to fatal infection. Live measles-mumps-rubella and varicella vaccines should also be avoided, and prophylaxis with varicella-zoster immune globulin should be given if infants with SCID are exposed to children with chickenpox. Trimethoprim-sulfamethoxazole or other appropriate regimens should be should be used for prophylaxis against P. carinii pneumonia (68), and prolonged courses of nystatin, systemic antifungal agents, or both may be necessary to control candidal infections. The use of passive immunization against respiratory syncytial virus and intravenous immune globulin (IVIG) should be considered, particularly in low-birth-weight infants and in those older than 6 months of age. This may need to be continued for more than a year, even in children who have received bone marrow transplants, because functional B-cell engraftment is often delayed.
MANAGEMENT OF ANTIBODY DEFICIENCY SYNDROMES
Part of "2 - Evaluation and Management of Immune Deficiency in Allergy Practice" Because half or more of all primary immune deficiencies involve defects in antibody production, management of these patients is a common part of allergy-immunology practice. Although patients with X-linked agammaglobulinemia, X-linked hyper-IgM syndrome, and other severe immunoglobulin deficiencies generally clearly require immunoglobulin replacement (see later), others with less severe deficiencies often require complex judgment processes. In deciding which form of therapy may be most appropriate for any given patient, the practitioner must consider not only the underlying diagnosis but also the exposure history, the cumulative morbidity and future risk for end-organ damage from infection, and the risks and adverse effects of the various therapeutic options. The number of days lost from school or work and other interferences with the patient's lifestyle must also be considered. Formal pulmonary function tests and CT scans may indicate progressive yet subclinical chronic lung disease despite a relative lack of symptomatic complaints or denial on the part of the patient (69). Often, antibody-deficient patients who present with repeated acute infections also have systemic morbidity, about which they may or may not complain. This may include fatigue, lack of stamina, poor weight gain (in infants), and musculoskeletal symptoms that have been attributed to other causes or ignored. Because these symptoms often improve with appropriate management of chronic infection and immunoglobulin replacement, they must be carefully evaluated in the review of systems and weighed in considering the options for therapy. Patients with a history of inflammatory bowel disease, recurrent problems with Clostridium difficile, or drug allergies may have decreased tolerance for antibiotics, which may limit the therapeutic options in their cases. Patients diagnosed with chronic obstructive pulmonary disease and those with asthma in which infection is a trigger may actually have P.38 underlying antibody deficiencies, such as the inability to respond to polysaccharides, and if so, may experience a marked amelioration of lower airway symptoms if infection is prevented with IVIG (see later) or the astute use of antibiotics. A stepwise approach to treatment may be employed across the range of severities of antibody deficiency or sequentially in any given patient. Some patients, particularly small children, with partial antibody deficiency who have not had significant permanent end-organ damage may be managed by limiting their exposure to infectious agents (e.g., by removing them from day care or preschool) and being sure that they have received all available vaccines, including the new, conjugated heptavalent pneumococcal polysaccharide vaccine, and annual immunization against influenza. Measurement of specific antibody titers after administration of these vaccines may provide reassurance for parents and referring physicians and may suggest that additional therapy is not indicated. In some cases of partial antibody deficiency, immunization, prompt and rigorous treatment of likely bacterial infections such as sinusitis and bronchitis, and verification that these are continued until the infection has been completely resolved may provide satisfactory control of infection and freedom from chronic or progressive symptoms. In other cases, parenteral or prolonged courses of antibiotics may be initiated upon suspicion of bacterial infection, if tolerated. The next step would be the use of prophylactic antibiotics. Many patients attain satisfactory freedom from infection by a once-daily dose of trimethoprim-sulfamethoxazole* (e.g., half of the total daily dose that would be used for otitis media). Other oral antibiotics, such as ampicillin or a cephalosporin, may also be used, especially in patients who are allergic to sulfonamides, but these agents are associated with a higher risk for resistant bacteria. Patients who develop diarrhea or other excessive gastrointestinal side effects, oral thrush, or vagin*l candidiasis may be poor candidates for this approach or may not tolerate it for long. Because of the possible development of antibiotic resistance, when patients on prophylactic antibiotics develop infections likely to be of bacterial origin, sensitivity of the organism should be confirmed, if possible, and the dose should be raised to the full treatment dose if the organism is sensitive. If isolation of the organism is not possible, or if it is not sensitive to the agent used for prophylaxis, a different agent should be used for treatment, for the full prescribed course; the prophylaxis regimen may then be resumed. In patients with severe antibody deficiency, in those for which antibiotic therapy is problematic, and in those in whom prophylaxis has not been satisfactory, immunoglobulin replacement therapy is indicated (70,71 and 72). The introduction of preparations of pooled immunoglobulin for intravenous use has greatly facilitated administration of doses of IgG sufficient to prevent infection satisfactorily. This has become the modality of choice for most patients, particularly in the United States. Intramuscular injections of more concentrated preparations and plasma infusions have largely been abandoned, but subcutaneous infusion is also used, particularly in Scandinavian countries (73). Most currently available preparations of immunoglobulin for intravenous use are made from the pooled plasma of thousands of donors and contain a broad spectrum of molecularly intact specific IgG antibodies of all four subclasses, with little or no IgM or IgE. The content of albumin and IgA varies. Most preparations contain sugars such as maltose, dextrose, or others, with or without glycine as a stabilizer. Because IVIG is a blood product, the possibility of transmission of bloodborne viruses must be considered. The risk for viral transmission is minimized by careful screening and selection of donors, by the processes used to purify the IgG (usually a modification of the CohnOncley cold alcohol precipitation procedure), and by specific viral inactivation steps (74). These may include the use of solvent-detergent treatment, which inactivates enveloped viruses (75), pasteurization (76), or low pH (77). Because the average half-life of IgG in the circulation is about 21 days, infusions are usually given every 3 to 4 weeks. The dose should be individualized to control infections and other P.39 symptoms but usually falls in a range of 300 to 600 mg/kg/dose, with the higher doses often being given at the longer dosing intervals. Serum IgG concentrations determined at the trough, just before the next infusion, can be used to provide an index and to assist decisions about the adequacy of dose and treatment interval but should not by themselves be used as an end point. This is particularly important in patients with CVID, IgG subclass deficiency, or selective antibody deficiencies, such as those who are unable to respond to polysaccharide antigens. These patients often require full replacement doses to remain free from infection despite having pretreatment serum IgG levels on the border of or within the normal range. Antibody-deficient patients with active acute or chronic infection may experience severe systemic symptoms, including shaking chills and spiking fevers, and inflammatory reactions at the site of
infection (e.g., the sinuses or airways) when they first receive IVIG. It may therefore be preferable to defer initiation of treatment until a satisfactory course of antibiotics is given in such patients. Infusions are generally initiated at the rate of 0.5 to 1 mg/kg/min (0.01 to 0.02 mL/kg/min of 5% solution) and increased in a stepwise manner at 15- to 30-minute intervals, as tolerated by the individual patient, until a maximum rate of 4 to 6 mg/kg/min is achieved. Occasional patients may tolerate rates as fast as 8 to 10 mg/kg/min. Most stable patients can thus complete their infusions within 2 to 3 hours. A minority of patients may experience adverse reactions during infusions, which may consist of headache, backache, flushing, chills, and mild nausea (78). In severe cases, there may be dyspnea, a sense of anxiety and chest pain. These are not true anaphylactic reactions, are not mediated by IgE, and are frequently associated with increased rather than decreased blood pressure. Such reactions can usually be treated by decreasing the rate of infusion or by administration of diphenhydramine, acetaminophen, or aspirin. Patients who demonstrate consistent patterns of reactions can be kept at slower rates for subsequent infusions or pretreated with the previously mentioned drugs. In rare cases, pretreatment with corticosteroids (e.g., 0.5 to 1 mg/kg of prednisone or intravenous methylprednisolone) may be necessary. True anaphylaxis is extremely uncommon but has been reported in a very small number of patients with IgA deficiency who have IgE antibodies against IgA (79). Because this is so rare, IgA deficiency should not be regarded as a contraindication against IVIG therapy in patients who also have significant deficiency of IgG antibodies, but slow starting rates and caution should be used with such patients. Rarely, aseptic meningitis, thrombotic events, and acute renal failure have been caused by IVIG infusions. These have generally been cases in which high doses (>1,000 mg/kg) of IVIG have been given to achieve antiinflammatory or immunomodulatory effects in patients with underlying neurologic disease or other problems and are rare in patients receiving conventional doses as replacement therapy for immune deficiencies. Late adverse reactions include headache, which occasionally may have features of migraine; nausea, and fever, and may occur up to 48 hours after the infusion. These generally respond to acetaminophen, aspirin, or other nonsteroidal antiinflammatory drugs; however, occasionally antiemetics, serotonin receptor antagonists, or other preparations more commonly used for migraines may be required. Patients with recurrent febrile reactions should be carefully evaluated for the presence of chronic infection, which should be treated with appropriate antibiotics. In many cases, IVIG infusions are so benign that they can be safely given in the home by a home nursing service or by a parent or spouse (80). We usually establish the safety, maximally tolerated rate, and need for premedication in our clinic before allowing the patient to go to home care. IVIG is not irritating to the veins, and conventional preparations are not viscous or difficult to administer; hence, indwelling venous access devices such as MediPort should not be required. If a patient is particularly sensitive to the pain of having the IV started, advance application of a local anesthetic, such as lidocaine or prilocaine (EMLA), which is available as a cream or presaturated disk, may be helpful. An advantage of the subcutaneous infusion method is that it makes P.40 self-administration feasible, especially if a small portable pump is used (81). Although prevention of acute, severe bacterial infections is the major goal of antibody replacement therapy, freedom from the symptoms of chronic infections in bronchiectasis can often be achieved, and many patients report amelioration of other symptoms such as arthralgia or arthritis when appropriate replacement has been achieved. The pulmonary status, chest CT scan results, or both in all patients with significant antibody deficiencies should be carefully documented at the beginning of therapy and followed at regular intervals, even if they become asymptomatic, because recent studies show that some patients may have progressive subclinical lung disease even when they do not complain of chronic symptoms or acute exacerbations (36). In some infants with normal lymphoid tissues and B-cell numbers in whom IVIG is started because of problems with bacterial or viral infections, the antibody deficiency may represent a maturational delay in the full range of antibody responses rather than a fixed and permanent defect. This is most likely to involve delayed development of T-independent antibody responses such as those to bacterial capsular polysaccharides. After these patients have had a satisfactory interval with a normal or decreased incidence of infections, the IVIG infusions should be stopped, and their own antibody production should be reassessed. We find it best to try such interruptions of therapy during the summer months, when the exposure to droplet-spread respiratory infection is reduced. Serum concentrations of the major immunoglobulin classes and subclasses and specific antibody titers can be redetermined after 2 to 3 months off therapy to allow sufficient catabolism of the therapeutically administered IgG so that the infant's own production can be assessed. In our experience, children whose IgG levels or specific antibody responses are not satisfactory by the time they have reached 5 years of age are not likely to improve in subsequent years, and this exercise is rarely productive above that age. In summary, immune deficiencies include a range of disorders spanning a spectrum from SCID and Xlinked agammaglobulinemia to subtle specific antibody defects. Although the former may be relatively straightforward to detect in early infancy, common variable immunodeficiency disease and specific antibody deficiencies may present with symptoms of recurrent or chronic respiratory or gastrointestinal infections at any age. Recognition of the possibility that immune deficiency may be responsible for a patient's problems is the first step in determining whether an immunologic evaluation is appropriate. The pattern of infections and the associated historical and physical features may provide important clues to the underlying diagnosis and should be kept in mind as a progression through screening and specialized and definitive laboratory tests is pursued. Therapeutic efforts aimed at minimizing the morbidity from infection or correcting the underlying problem will be suggested by the specific diagnosis and should be individualized. Because subclinical chronic infection that can lead to long-term pulmonary damage may be present (36) and because there is an increased incidence of malignancy in patients with primary immune deficiencies (5,29), close followup is necessary. With advances in our understanding of the basic pathogenesis of these disorders at a molecular level, additional specific therapies lie just over the horizon.
3
Immunology of IgE-mediated and Other Hypersensitivity States C . R a ym o n d Z e i s s Jacob J. Pruzansky Division of Allergy-Immunology, Department of Medicine, Northwestern University Medical School; VA Chicago Health Care System, Lakeside Division, Chicago, Illinois
Contents • • • •
HISTORICAL REVIEW OF IgE-MEDIATED HYPERSENSITIVITY PHYSIOLOGY OF IgE ROLE OF IgE IN HEALTH AND DISEASE OTHER HYPERSENSITIVITY STATES
HISTORICAL REVIEW HYPERSENSITIVITY
OF
IgE-MEDIATED
Part of "3 - Immunology of IgE-mediated and Other Hypersensitivity States" In 1902, Richet and Portier described the development of anaphylaxis in dogs given sea anemone toxin; subsequently, anaphylaxis was described in humans after the injection of horse serum to achieve passive immunization against tetanus and diphtheria. In 1906, Clemons von Pirquet correctly predicted that immunity and hypersensitivity reactions would depend on the interaction between a foreign substance and the immune system, and that immunity and hypersensitivity would have similar underlying immunologic mechanisms (1). The search for the factor responsible for immediate hypersensitivity reactions became a subject of intense investigation over several years. In 1921, Prausnitz and Küstner (2) described the transfer of immediate hypersensitivity (to fish protein) by serum to the skin of a normal individual. This test for the serum factor responsible for immediate hypersensitivity reactions was termed the PrausnitzKüstner test. Variations of this test remained the standard for measuring skin sensitizing antibody over the next 50 years. In 1925, Coca and Grove (3) extensively studied the skin-sensitizing factor from sera of patients with ragweed hay fever. They called skin-sensitizing antibody atopic reagin because of its association with hereditary conditions and because of their uncertainty as to the nature of the antibody involved. Thereafter, this factor was called atopic reagin, reaginic antibody, or skin-sensitizing antibody. This antibody clearly had unusual properties and could not be measured readily by standard immunologic methods. Major research efforts from the 1920s through the 1960s defined its physical and chemical properties and measured its presence in allergic individuals (4,5). In 1967, the Ishizakas (6) discovered that skin-sensitizing antibody belonged to a unique class of immunoglobulin, which they called immunoglobulin E (IgE). In elegant studies using immunologic techniques, they clearly demonstrated that reagin-rich serum fractions from a patient with ragweed hay fever belonged to a unique class of immunoglobulin (6). Shortly thereafter, the Swedish researchers Johansson and Bennich discovered a new myeloma protein, termed IgND, which had no antigenic relation to the other immunoglobulin classes. In 1969, cooperative studies between these workers and Ishizakas confirmed that the proteins were identical and that a new class of immunoglobulin, IgE, had been discovered (7).
PHYSIOLOGY OF IgE Part of "3 - Immunology of IgE-mediated and Other Hypersensitivity States"
IgE Structure and Receptors The immunochemical properties of IgE are shown in Table 3.1 in contrast to those of the other immunoglobulin classes. IgE is a glycoprotein that has a molecular weight of 190,000 P.44 P.45 with a sedimentation coefficient of 8S. Like all immunoglobulins, IgE has a four-chain structure with two light chains and two heavy chains. The heavy chains contain five domains (one variable and four constant regions) that carry unique, antigenic specificities termed the epsilon (ε) determinants (Fig. 3.1A). These unique antigenic structures determine the class specificity of this protein. Digestion with papain yields the Fc fragment, which contains the epsilon antigenic determinants, and two Fab fragments. The Fab fragments contain the antigen-combining sites. The tertiary structure of the Fc fragment is responsible for the protein's ability to fix to the FcεRI receptors on mast cells and basophils (8).
T AB L E 3 . 1. Immunoglobulin isotypes
F I G . 3 . 1 . A: T h e h e a v y c h a i n d o m a i n s t r u c t u r e o f I g E . T h e b i n d i n g s i t e f o r t h e h i g h - a f f i n i t y m a s t c e l l r e c e p t o r FcεRI and the low-affinity IgE receptor FcεRII is shown. B: The structure and characteristics of the surface receptors for immunoglobulin Fc regions. i, inducible; bars, disulfide bridge; NK, natural killer; v.hi, very high; Ig, immunoglobulin. (Adapted from Roitt I. Essential immunology. 8th ed. Oxford, UK: Blackwell Science, 1994:57, 61, with permission.) The FcεR1 receptor is the high-affinity receptor for IgE found on mast cells, basophils, eosinophils, and human skin Langerhans cells (9). Cross-linking of high-affinity receptor-bound IgE by allergen results in the release of mediators from mast cells and basophils. Molecular biologic techniques have been used to clone the gene encoding the e chain of human IgE (ND) and to determine the site on IgE that binds to its receptor (10). Recent studies have localized this site to the Cε3 heavy chain domains (11). The high-affinity receptor for IgE is composed of an α chain, a β chain, and two γ chains, and it is the α chain that binds IgE (Fig. 3.1B). The crystal structure of the α chain has been determined giving insights into the interaction of IgE with its receptor at the molecular level (12). The β and γ chains are involved in signal transduction when the receptors are aggregated by the crosslinking of IgE, resulting in mediator release (13). Recent studies have delineated the central role that IgE molecules in the circulation play in determining the number of FcεRI receptors on mast cells and basophils (14,15) and consequently the release of mediators from these cells. With infusion of anti-IgE monoclonal antibody in allergic subjects, there was a significant reduction in serum IgE levels, with a dramatic fall in basophil FcεRI number and mediator release (14). A low-affinity FcεRII receptor (CD23) has been localized to B lymphocytes, monocytes and macrophages, and platelets and eosinophils. The receptor has an A form found only on B lymphocytes and a B form found on all cells expressing CD23. The expression of this receptor is markedly upregulated on all cell types by interleukin-4 (IL-4) and IL-13. Binding of IgE to this receptor places IgE at the center of activation of many important effector cells (16). The role of CD23 in regulation of the IgE response is complex, having both positive and inhibitory effects (13).
Sites of IgE Production, Turnover, and Tissue Localization With the advent of a highly specific reagent for detecting IgE, antibody against the Fc portion of IgE (anti-IgE), the sites of production of this immunoglobulin could be examined by fluorescent-labeled anti-IgE. It was found that lymphoid tissue of the tonsils, adenoids, and the bronchial and peritoneal areas contained IgE-forming plasma cells. IgE-forming plasma cells also were found in the respiratory and intestinal mucosa (17). This distribution is similar to that of IgA. However, unlike IgA, IgE is not associated with a secretory piece, although IgE is found in respiratory and intestinal secretions. The traffic of IgE molecules from areas of production to the tissues and the circulation has not been established. Areas of production in the respiratory and intestinal mucosa are associated with the presence of tissue mast cells (18). With the development of techniques to measure total IgE in the blood and the availability of purified IgE protein, investigators were able to study the metabolic properties of this immunoglobulin in normal individuals (19). The mean total circulating IgE pool was found to be 3.3 µg/kg of body weight, in contrast to the total circulating IgG pool of about 500,000 µg/kg of body weight. IgE has an intravascular half-life of only 2.3 days. The rate of IgE production was found to be 2.3 µg/kg/day. It had been known for several years that the half-life of reaginic antibody in human skin as determined by passive transfer studies was about 14 days. This was reconfirmed with studies that investigated the disappearance of radiolabeled IgE in human skin. The half-life in the skin was found to be between 8 and 14 days (6). The basophil and mast cell-bound IgE pool needs to be investigated thoroughly, but it has been estimated P.46 P.47 that only 1% of the total IgE is cell bound. Direct quantification of specific IgE in the blood, in contrast to specific IgE on the basophil surface, indicates that for every IgE molecule on the basophil, there are 100 to 4,000 molecules in circulation (20).
IgE Synthesis Major advances in the understanding of IgE synthesis have resulted from human and animal studies (21,22,23,24,25,26,27 and 28). Tada (21) studied the production of IgE antibody in rats and found that IgE antibody production is regulated by cooperation between T lymphocytes (T cells) and B lymphocytes (B cells). The T cells provide the helper function, and the B cells are the producers of IgE antibody. In human systems, it became clear that IgE production from B cells required T-cell signals that were unique to the IgE system (22). In 1986, Coffman and Carty (23) defined the essential role of IL-4 in the production of IgE. The pathway to IgE production is complex, requiring not only IL-4 and IL-13 but also T- and B-cell contact, major histocompatibility complex (MHC) restriction, adhesion molecules, expression of FcεRII (CD23) receptors, CD40 and CD40 ligand interaction, and the terminal action of IL-5 and IL-6 (24). IL-4 acts on precursor B lymphocytes and is involved in the class switch to ε heavy chain production (22). IL-4 and IL-13 are not sufficient to complete the switch to functional ε messenger RNA, and several second signals have been described that result in productive messenger RNA transcripts (25,26). In the absence of those signals, sterile transcripts result. A key physiologic second signal is provided by CD4+ helper T-cell contact. This contact signal is provided by CD40 ligand on activated T cells, which interacts with the CD40 receptor on IL-4–primed B cells and completes isotype switching to IgE (24). Recent studies indicate that IgE synthesis is critically dependent on the IL-4 receptor α chain and nuclear factors such as NF-κB and Stat 6 (27). Another cytokine, interferon-γ (IFN-γ) suppresses IgE production, acting at the same point as IL-4 (24). This complex set of interactions is shown in Fig. 3.2.
FIG. 3.2. The molecular control of the IgE response. Interleukin-4 (IL-4) and IL-13 are the most important cytokine inducers of IgE production acting at the IL-4 receptor α chain and through nuclear factor, Stat 6. Interferon-γ (IFN-γ) is the most important inhibitor of IgE synthesis. CD154, the T-cell ligand for CD40 on the B cell, promotes IgE transcription through nuclear factor, NF-κB. Antigen presented to the T-cell receptor (TCR) by class II major histocompatibility complex molecules on the B cell initiates this complex process. (Adapted from, Cory DB, Kheradmand F. Induction and regulation of the IgE response. Nature 1999;402s:18– 23, with permission.) P.48 During terminal differentiation of IgE B cells to plasma cells producing IgE, IgE-binding factors have been described that either enhance or suppress IgE synthesis (28). During the secondary IgE response to allergen, allergen-specific B lymphocytes capture allergen by surface IgE, internalize and degrade it, and present it to T cells as peptides complexed to class II MHC molecules. This leads to T-cell–B-cell interaction, mutual exchange of cytokine and cell contact signals, and enhanced allergen-specific IgE production.
ROLE OF IgE IN HEALTH AND DISEASE Part of "3 - Immunology of IgE-mediated and Other Hypersensitivity States"
IgE in Health The fetus is capable of producing IgE by 11 weeks' gestation. Johansson and Foucard (29) measured total IgE in sera from children and adults. They found that cord serum contained 13 to 202 ng/mL and that the concentration of IgE in the cord serum did not correlate with the serum IgE concentration of the mother, which confirmed that IgE does not cross the placenta. In children, IgE levels increase steadily and peak between 10 and 15 years of age. Johansson and Foucard illustrate well the selection of population groups for determining the normal level of serum IgE. Studies of healthy Swedish and Ethiopian children showed a marked difference in mean IgE levels: Swedish children had a mean of 160 ng/mL, and Ethiopian children had a mean of 860 ng/mL (30). Barbee and coworkers (31) studied the IgE levels in atopic and nonatopic people 6 to 75 years of age in Tucson. IgE levels peaked in those aged 6 to 14 years and gradually declined with advancing age; male subjects had higher levels of IgE than female subjects (Fig. 3.3).
FIG. 3.3. Serum IgE as function of age and sex among whites in the United States. Geometric means and upper 95% confidence intervals are plotted against age for males and females with positive and negative results from skin tests. Double cross-hatched area represents overlap of total IgE levels between the two groups of subjects. Age-related declines in serum IgE are significant in all groups. (From Knauer KA, Adkinson NF. Clinical significance of IgE. In: Middleton E, Reed CE, Ellis EJ, eds. Allergy principles and practice. St. Louis: CV Mosby, 1983, with permission.) Several roles for the possible beneficial effect of IgE antibody have been postulated. The presence of IgE antibody on mast cells in the tissues that contain heparin and histamine points to a role for IgE in controlling the microcirculation, and a role for the mast cell as a “sentinel” or first line of defense against microorganisms has been advanced. The hypothesis is that IgE antibody specific for bacterial or viral antigens could have a part in localizing high concentrations of protective antibody at the site of tissue invasion (32,33). The role of IgE antibody has been studied extensively in an experimental infection of rats with the parasite Nippostrongylus brasiliensis. IgE antibody on the surface of mast cells in the gut may be responsible for triggering histamine release and helping the animal to reduce the worm burden (34). In experimental Schistosoma mansoni infection in the rat, IgE is produced at high levels to schistosome antigens. IgE complexed to these antigens has a role in antibody-dependent cellmediated cytotoxicity, whereas eosinophils, macrophages, and platelets are effector cells that damage the parasite (35). IgE and IgE immune complexes are bound to these effector cells by the IgE FcεRII receptor, which has a high affinity for IgE immune complexes. Effector cells triggered by FcεRII receptor aggregation result in release of oxygen metabolites, lysosomal enzymes, leukotrienes, and platelet-activating factor. These observations in animals have relevance to human populations, where the IgE inflammatory cascade may protect against helminth infections (35).
IgE in Disease The Atopic State and the T H 2 Paradigm
Extensive evidence has accumulated that may define the underlying immunologic basis for the atopic phenotype, that is, individuals with allergic asthma, allergic rhinitis, and atopic eczema (24). The atopic condition can be viewed as a TH2 lymphocyte-driven response to allergens of complex genetic and environmental origins (36). The reciprocal action of IL-4 and IFN-γ on IgE production led to several studies on the T-cell origin of these cytokines. Mosmann and Coffman (37) described two distinct types of helper T cells in murine systems and defined them as TH1 or TH2 cells by the pattern of cytokine secretion. TH1 cells produced IL-2, IFN-γ, and lymphotoxin. TH2 cells produced IL-4, IL-5, IL-6, and IL-10. A significant body of evidence has further defined the role of TH2 cells in the human atopic state related to IL-4 production, IgE synthesis, and the maturation and recruitment of P.49 P.50 eosinophils by IL-5 and the maturation of IgE B cells by IL-5 and IL-6 (24,36). T cells having the TH2 cytokine profile have been cloned from individuals with a variety of atopic diseases, (24) have been identified in the airway of atopic asthmatic patients, and have been implicated as fundamental to persistent airway inflammation in asthma (38,39). Once a TH2 response is established, there is downregulation of TH1 cells by the cytokines IL-4 and IL-10. TH1 cells are capable of downregulating TH2 cytokine secretion through the reciprocal action of IFN-γ on TH2 cells, a physiologic control that is abrogated by the predominant TH2 cell response in the atopic individual (40) (Fig. 3.4).
FIG. 3.4. The TH2 cell paradigm in allergic disease. The interaction of allergen, dendritic cell, and cytokine environment causes naïve CD4+ T cells to differentiate to the TH2 phenotype with the capacity for enhanced secretion of cytokines that drive and maintain the allergic inflammatory response. The established TH2 response downregulates the influence of TH1 cells and the inhibitory effect of interferon-γ (IFN-γ), by the action of cytokines IL-10 and IL-4. These cytokine pathways are under complex genetic control that defines the atopic phenotype. (Adapted from Holgate ST. The epidemic of allergy and asthma. Nature 1999;402s:2–4, with permission.) The expression of the atopic state is dependent on genes that control the TH2 response, total IgE production, and specific IgE responsiveness to environmental allergens. High serum IgE levels have been shown to be under the control of a recessive gene, and specific allergen responses are associated with human leukocyte antigens (41). The chromosomal location and identification of these genes are under intense investigation (42). The recent observation that mast cells and basophils produce IL-4, leading to IgE synthesis (24), adds an amplification loop that maintains the atopic state with continued exposure to allergen, leading to mast cell and basophil activation and mediator and cytokine release with enhanced and sustained IgE production. Studies of the interaction of histamine releasing factor (HRF) and human basophil histamine release revealed that there may be two kinds of IgE: IgE that reacts with HRF (IgE+) and IgE that does not (IgE–) (43). The amino acid sequence of this HRF has been determined (44). These observations may add to the role of IgE in several diseases in which no definable allergen is present.
Measurement of Total IgE Several early studies evaluated the role of IgE in patients with a variety of allergic diseases (29,30 and 31). Adults and children with allergic rhinitis and extrinsic asthma tend to have higher total serum IgE concentrations. About half of such patients have total IgE concentrations that are two standard deviations above the mean of a normal control group. Significant overlap of total serum IgE concentrations in normal subjects and in patients with allergic asthma and hay fever has been demonstrated (Fig. 3.3). Therefore, the total P.51 serum IgE concentration is neither a specific nor sensitive diagnostic test for the presence of these disorders. Total serum IgE has been found to be markedly elevated in atopic dermatitis, with the serum IgE concentration correlating with the severity of the eczema and with the presence of allergic rhinitis, asthma, or both. Patients with atopic dermatitis without severe skin disease or accompanying asthma or hay fever may have normal IgE concentrations (45). Total IgE concentrations have been found to be markedly elevated in allergic bronchopulmonary aspergillosis.
Measurement of Specific IgE Since the discovery of IgE in 1967, it is possible not only to measure total IgE in the serum but also to measure IgE antibody against complex as well as purified allergens. One of the first methods described by Wide and coworkers (46) was the radioallergosorbent test (RAST). Allergen is covalently linked to solid-phase particles, and these solid-phase particles are incubated with the patient's serum, which may contain IgE antibody specific for that allergen. After a period of incubation, the specific IgE present binds firmly to the solid phase. The solid phase is then washed extensively, and the last reagent added is radiolabeled anti-IgE antibody. The bound anti-IgE reflects amounts of specific IgE bound to the allergen. The results are usually given in RAST units or in units in which a standard serum containing significant amounts of IgE specific for a particular allergen is used as a reference. Specific IgE antibody detected by RAST in the serum of patients whose skin test results are positive to an allergen has been shown to cover a wide range. Between 100-fold and 1,000-fold differences in RAST levels against a specific allergen are found in skin-reactive individuals. In studies of large groups of patients, there is a significant correlation between the RAST result, specific IgE level, and skin test reactivity. However, individuals with the same level of specific IgE antibody to ragweed allergen may vary 100-fold in their skin reactivity to that allergen (47). The RAST concept has been extended to the use of fluorescent- and enzyme-labeled anti-IgE, which obviates the need for radiolabeled materials. Although RAST and other specific IgE measurement technologies have clarified the relationships between specific IgE in the serum and patients' clinical sensitivity, these tests do not replace skin testing with the allergens in clinical practice because skin testing is more sensitive.
It is possible to estimate the absolute quantity of specific IgE antibody per milliliter of serum against complex and purified allergens (48,49). Using one of these methods to measure IgE antibody against ragweed allergens, Gleich and coworkers (48) defined the natural rise and fall of ragweed-specific IgE over a 1-year period. In this population of ragweed-sensitive individuals, the IgE antibody specific for ragweed allergens varied from 10 to 1,000 ng/mL. A marked rise of specific IgE level occurred after the pollen season, with a peak in October followed by a gradual decrease. Specific IgE level reached a low point just before the next ragweed season in August (Fig. 3.5). It is also possible to measure basophil-bound, total, and specific IgE against ragweed antigen E. There are between 100,000 and 500,000 molecules of total IgE per basophil (50) and between 2500 and 50,000 molecules of specific IgE per basophil (20).
FIG. 3.5. ragweed Unginger J Allergy
Levels and changes of IgE antibodies to ragweed allergens in 40 untreated allergy patients. The pollination season is indicated by the black bar on the abscissa. (From Gleich GJ, Jacob GL, JW, et al. Measurement of the absolute levels of IgE antibodies in patients with ragweed hay fever. Clin Immunol 1977;60:188, with permission.)
OTHER HYPERSENSITIVITY STATES Part of "3 - Immunology of IgE-mediated and Other Hypersensitivity States"
All immunologically mediated hypersensitivity states had been classified into four types by Gell and Coombs in 1964. This classification has been a foundation for an understanding of the immunopathogenesis of clinical hypersensitivity syndromes (51). This schema depends on the location and class of antibody that interacts with antigen resulting in effector cell activation and tissue injury. In type I, or immediate, hypersensitivity, allergen interacts with IgE antibody on the surface of mast cells and basophils, resulting in the cross-linking of IgE, FcεRI receptor apposition, and mediator release from these cells. Only a few allergen molecules, interacting with cell-bound IgE, lead to the release of many mediator molecules, resulting in a major biologic amplification of the allergen–IgE antibody reaction. Clinical examples include anaphylaxis, allergic rhinitis, and allergic asthma. P.52 In type II, or cytotoxic, injury, IgG or IgM antibody is directed against antigens on the individual's own tissue. Binding of antibody to the cell surface results in complement activation, which signals white blood cell influx and tissue injury. In addition, cytotoxic killer lymphocytes, with Fc receptors for IgG, can bind to the tissue-bound IgG, resulting in antibody-dependent cellular cytotoxicity. Clinical examples include lung and kidney damage in Goodpasture syndrome, acute graft rejection, hemolytic disease of the newborn, and certain bullous skin diseases. In type III, or immune complex, disease, IgG and IgM antigen–antibody complexes of a critical size are not cleared from the circulation and fix in small capillaries throughout the body. These complexes activate the complement system, which leads to the influx of inflammatory white blood cells, resulting in tissue damage. Clinical examples include serum sickness (after foreign proteins or drugs), lupus erythematosus, and glomerulonephritis after common infections. In type IV, or delayed-type, hypersensitivity, the T-cell antigen receptor on TH1 lymphocytes binds to tissue antigens, resulting in clonal expansion of the lymphocyte population and T-cell activation with the release of inflammatory lymphokines. Clinical examples include contact dermatitis (e.g., poison ivy) and tuberculin hypersensitivity in tuberculosis and leprosy. The classic Gell and Coombs classification has been adapted by Janeway and colleagues (52). Subsequently, Kay further expanded the adaptation (53). Type II reactions have been divided into two different subtypes. Type IIa are characterized by cytolytic reactions, such as are produced by antibodies causing immune mediated hemolytic anemia, whereas type IIb reactions are characterized by cell-stimulating reactions such as are produced by thyroid-stimulating antibody in patients with Graves disease or antibodies to the high-affinity mast cell receptor in chronic idiopathic urticaria. The latter antibodies cause mast cell activation. Type IV reactions are divided into four subtypes. Type IVa1 reactions are mediated by CD4+ TH1 cells causing classic delayed-type hypersensitivity reactions, such as allergic contact dermatitis or tuberculin reactions. Type IVa2 reactions are mediated by CD4+ TH2 cells resulting in cell-mediated eosinophilic hypersensitivity as occurs in asthma. Type IVb1 reactions are mediated by cytotoxic CD8+ cells that mediate P.53 graft rejection and Stevens-Johnson syndrome. Type IVb2 reactions are mediated by CD8+ lymphocytes that can produce IL-5, resulting in cell-mediated eosinophilic hypersensitivity, usually in association with viral mucosal infection.
4 Biochemical Mediators of Allergic Reactions Stephen I. Wasserman Department of Medicine, University of California, San Diego, La Jolla, California, Department of Medicine, University of California, San Diego Medical Center, San Diego, California
Contents • • • • • •
MEDIATOR-GENERATING CELLS ACTIVATION OF MAST CELLS AND BASOPHILS MEDIATORS MEDIATOR INTERACTIONS THE ROLE OF THE MAST CELL AND ITS MEDIATORS IN TISSUE HOMEOSTATIC ROLE OF MAST CELLS
Recent research has expanded the understanding of the cells and mediators relevant to diseases of immediate-type hypersensitivity. The biologically active molecules responsible have been identified, and a thorough biochemical and structural elucidation of diverse lipid mediators has been accomplished. The activity of mediator-generating cells and their diverse products has been assigned a central role in both immunoglobulin E (IgE)-mediated acute and prolonged inflammatory events. This chapter places in perspective the mediator-generating cells, the mediators themselves, and these newer concepts of their roles in pathobiologic and homeostatic events.
MEDIATOR-GENERATING CELLS Part of "4 - Biochemical Mediators of Allergic Reactions" Mast cells and basophilic polymorphonuclear leukocytes (basophils) constitute the two IgE-activated mediator-generating cells (1,2). Mast cells are heterogeneous, and both connective tissue and mucosal types have been recognized (3) (Table 4.1). The latter predominate in the lamina propria of
the gastrointestinal tract and in the peripheral airways and alveolar septa. Both occur in the upper airway and nose, and the connective tissue subtype dominates in the skin (4).
T AB L E 4 . 1. Human mas t cell heterogeneity Mast cells are most closely related to mononuclear leukocytes (5) and are richly distributed in the deeper region of the central nervous system, the upper and lower respiratory epithelium, the bronchial lumen, the gastrointestinal mucosa and submucosa, bone marrow, and skin (6,7). They are especially prominent in bone, dense connective tissue adjacent to blood vessels (particularly small arterioles and venules), and peripheral nerves. In the skin, lungs, and gastrointestinal tract, mast cell concentrations approximate 10,000 to 20,000 cells/mm3 (8). They develop from CD34+ bone marrow precursors through the action of stem cell factor (kit-ligand SCF), which binds to a specific receptor (c-kit, CD117) (9). Precursor cells exit the marrow and terminally differentiate in tissues under a variety of local influences, such as interleukin-3 (IL-3), IL-4, IL-6, IL-9, IL-10, and factors from fibroblasts (5,10,11), but are inhibited by transforming growth factor B (12). Mast cells are large (10 to 15 mm in diameter) and possess a ruffled membrane, numerous membrane-bound granules (0.5 to 0.7 mm in diameter), mitochondria, a mononuclear nucleus, and scant rough endoplasmic reticulum. Ultrastructurally, human mast cell granules display whorl and scroll patterns (13). Basophils, most closely related to eosinophils, are circulating leukocytes whose presence in tissue is unusual except in disease states (14). They originate in bone marrow and constitute 0.1% to 2.0% of the peripheral blood leukocytes. Basophils possess a polylobed nucleus and differ from mast cells in their tinctorial properties, their relatively smooth cell surface, and their granule morphologic makeup, which is larger and less structured than that of the mast cell. Their growth is responsive, not to SCF, but rather to IL-3 P.56 and granulocyte-macrophage colony-stimulating factor (GM-CSF).
ACTIVATION OF MAST CELLS AND BASOPHILS Part of "4 - Biochemical Mediators of Allergic Reactions" Mast cells and basophils possess numerous high-affinity intramembranous receptors (FcεRI) for the Fc portion of IgE. The number of such receptors is upregulated by exposure of the mast cell or basophil to increased amounts of IgE (15). The bridging of two or more such Fc receptors by antigen cross-linking of receptor-bound surface IgE molecules leads to cell activation and rapid release of preformed granular constituents and to the generation of unstored mediators. Mast cell responsiveness may be heightened by exposure to SCF or other cytokines (16,17), whereas basophils are primed to respond by GM-CSF, IL-1, IL-3, and IL-5 (18). Other important secretagogues include a family of histamine-releasing factors (19) and complement fragments C3a and C5a. The secretagogue-induced activation of mediator release is noncytolytic, a process termed stimulussecretion coupling. In vitro, extremely complex intertwined and potentially interacting systems have been identified, some of which may play roles in cell activation (20). An additional complexity is added as stored granule-associated mediators are regulated independently from unstored newly generated mediators. In IgE-mediated activation, receptor bridging is accompanied by protein tyrosine phosphorylation, an increase in intracellular calcium, protein kinase C translocation, G protein activation, and cyclic adenosine monophosphate generation. At the same time, membrane phospholipids are metabolized to generate monoacylglycerols, diacylglycerols, and phosphorylated inositol species, which facilitate protein kinase C function and liberate Ca2+ from intracellular sites. While these biochemical events are underway, adenosine triphosphate (ATP) is catabolized, and adenosine is liberated, which, in turn, activates a mast cell adenosine receptor to enhance granule
release. Finally, the cell gains control over mediator release, the process stops, and the cell regranulates (21). Although initiated at the time of IgE and antigen activation, the generation of cytokines is expressed over a time frame of hours to days. Both mast cells and basophils are important sources of a variety of inflammatory cytokines, as described later. After the initiating event of allergen binding, cytokine synthesis proceeds through activation of such signaling pathways as the STAT and NF-κB–regulated processes, with P.57 gene transcription evident within hours and protein secretion occurring subsequently (22). Recent work has added further complexity to mast cell and basophil activation. Mast cells possess a receptor for IgG, FcεRII, which can modulate mediator release (23), and these cells also respond to endotoxin through engagement of a toll-like receptor complex. The presence of these additional modulatory pathways suggests that mast cell and basophil mediators participate in inflammatory conditions in which IgE may not be present.
MEDIATORS Part of "4 - Biochemical Mediators of Allergic Reactions" Whatever their final metabolic interrelationships, the early biochemical processes lead to the generation of a heterogenous group of molecules termed mediators. Some mediators are preformed and are stored in the granules of the cell; others are generated only after cell activation and originate in the cytosol or membrane. Mediators are classified in this chapter by their proposed actions (Table 4.2 and Table 4.3), although some mediators subserve several functions.
T AB L E 4 . 2. Mast cell vas oac tive and spasmo genic mediato rs
T AB L E 4 . 3. Mast cell mediators affec ting c ell migration
Spasmogenic Mediators Histamine, generated by decarboxylation of histidine, was the first mast cell mediator to be identified, and it is the sole preformed mediator in this functional class. It is bound to the proteoglycans of mast cell and basophil granules (5 and 1 mg/106 cells, respectively) (24,25). Histamine circulates at concentrations of about 300 pg/mL with a circadian maximum in the early morning hours (26). Histamine excretion exceeds 10 mg/24 hours; a small fraction is excreted as the native molecule, and the remainder as imidazole acetic acid or methyl histamine. Histamine interacts with specific H1, H2, and H3 receptors (27,28). H1 receptors predominate in the skin and smooth muscle; H2 receptors are most prevalent in the skin, lungs, and stomach and on a variety of leukocytes; and H3 receptors predominate in the brain. The biologic response to histamine reflects the ratio of these receptors in a given tissue. H1 histamine effects include contraction of bronchial and gut musculature, vascular permeability, pulmonary vasoconstriction, and nasal mucus production (29,30). By its H2 pathway, histamine dilates respiratory musculature, enhances airway mucus production, inhibits basophil and skin (but not lung) mast cell degranulation, and activates suppressor T lymphocytes. Both H1 and H2 actions are required for the full expression of pruritus, cutaneous vasodilation, and cardiac irritability (27). The H3 actions of histamine suppress P.58 central nervous system histamine synthesis. Increased levels of histamine have been reported in the blood or urine of patients with physical urticaria, anaphylaxis, systemic mastocytosis, and antigeninduced rhinitis and asthma (31).
Platelet-activating Factor Platelet-activating factor (PAF) is a lipid identified structurally as 1-alkyl-2-acetyl-sn-glyceryl-3phosphorylcholine (32). This mediator is generated by mast cells, eosinophils, and monocytes. Degradation of PAF occurs by the action of acetyl hydrolase to remove acetate from the sn-2 position. PAF causes aggregation of human platelets, wheal-and-flare permeability responses, and eosinophil chemotaxis (33); PAF also contracts pulmonary and gut musculature, induces vasoconstriction, and is a potent hypotensive agent. Effects mediated by PAF also include pulmonary artery hypertension, pulmonary edema, an increase in total pulmonary resistance, and a decrease in dynamic compliance. In addition, PAF is capable of inducing a prolonged increase in nonspecific bronchial hyperreactivity in vivo (34).
Oxidative Products of Arachidonic Acid Arachidonic acid is a C20:4 fatty acid component of mast cell membrane phospholipids, from which it may be liberated by the action of phospholipase A2 or by the concerted action of phospholipase C and diacylglycerol lipase. At least 20 potential end products may be generated from arachidonic acid by the two major enzymes, 5-lipoxygenase and cyclooxygenase, which regulate its fate.
Cyclooxygenase Products Prostaglandin (PG) D2 is the predominant cyclooxygenase product generated by human mast cells, whereas human basophils do not generate this molecule. The production of PGD2 from PGH2 is glutathione dependent and is blocked by nonsteroidal antiinflammatory drugs and dapsone. It is a potent vasoactive and smooth muscle reactive compound that causes vasodilation when injected into human skin, induces gut and pulmonary muscle contraction, and, in vitro, inhibits platelet aggregation (35). PGD2 is thought to be responsible for flushing and hypotension in some patients with mastocytosis and to be an important mediator of allergic asthma (36). PGD2 is further
metabolized to PGJ2, a natural ligand for peroxisome proliferator-activated receptor-γ (37), a nuclear receptor important in diabetes and atherosclerosis. Immediate IgE antigen–activated PGD2 production is dependent on the constitutive expression of cyclooxygenase 1. Later and more prolonged PGD2 synthesis occurs after antigen challenge of sensitized cells that are stimulated with SCF and IL-10 (38).
Lipoxygenase Products Human mast cells generate 5-lipoxygenase products of arachidonic acid, starting with an unstable intermediate, 5-HPETE (which may be reduced to the monohydroxy fatty acid), 5-HETE, or (through leukotriene synthetase) LTC4 by addition of glutathione through the action of LTC4 synthase. The initial product of this pathway is LTC4, from which LTD4 may be generated by the removal of the terminal glutamine, and LTE4 by the further removal of glycine. A polymorphism in the LTC4 synthase gene is thought to alter the amount of this mediator generated during biologic reactions (39). The biologic activity of the sulfidopeptide leukotrienes occurs by its binding to two specific receptors termed Cys LTR I and II (40,41). Degradation is rapid and is accomplished by various oxygen metabolites. Clinically useful inhibitors of 5-lipoxygenase or the Cys LTR I receptors are available and demonstrate efficacy in clinical asthma (42). No clinically available inhibitor of Cys LTR II has been assessed in vivo, and the contribution of this receptor to the physiologic manifestations of LTC4, LTD4, or LTE4 remains speculative. Leukotrienes are potent and possess a broad spectrum of biologic activity (43). They induce whealand-flare responses that are long lived and are accompanied histologically by endothelial activation and dermal edema. In P.59 the airway, they enhance mucus production and cause bronchoconstriction, especially by affecting peripheral units. In humans, LTD4 is most active, LTC4 is intermediate, and LTE4 is the least potent. LTE4 has been implicated as an inducer of nonspecific bronchial hyperreactivity. It has been suggested that LTD4 augments airway remodeling (44), possibly by stimulating matrix metalloproteinase release or activity. All depress cardiac muscle performance and diminish coronary flow rates. LTC4 and LTD4 have been recovered from nasal washings and bronchial lavage fluids of patients with allergic rhinitis or asthma, whereas LTE4 has been recovered from the urine.
Adenosine
The nucleoside adenosine generated from the breakdown of ATP is released from mast cells on IgEmediated activation (45). In humans, circulating blood levels of adenosine are 0.3 µg/mL and are increased after hypoxia or antigen-induced bronchospasm. Adenosine is a potent vasodilator, inhibits platelet aggregation, and causes bronchospasm on inhalation by asthmatics. Adenosine, acting through a cell surface receptor, probably the A2b and A3 subtypes (46,47) enhances mast cell mediator release in vitro and potentiates antigen-induced local wheal-and-flare responses in vivo. Adenosine binding to its receptor is inhibited by methylxanthines.
Chemotactic Mediators Several chemotactic molecules have been characterized by activities generated during IgE-dependent allergic responses. Most remain incompletely characterized. A new family of cytokines has been described; these cytokines, called chemokines, have chemoattractant activity for leukocytes and fibroblasts (Table 4.4). In the C-X-C or α chemokines, the cysteines are separated by one amino acid, whereas the cysteines are adjacent in the C-C or β chemokines. Most α chemokines attract neutrophils, whereas β chemokines attract T cells and monocytes (some also attract basophils and eosinophils). The C-X-C chemokines that attract neutrophils include GRO-α, GRO-β, IL-8, NAP-2 and PF-4. The C-C chemokines that attract eosinophils include eotaxin, MIP-1α, MCP-2, MCP-3, and RANTES. IL-8, MIP-1α, and RANTES are also cell chemoattractants for both mast cells and basophils.
T AB L E 4 . 4. Chemokines causing chemoattraction
Neutrophil Chemotactic Factors
High-molecular-weight (HMW) factors are the most prominent neutrophil-directed activities noted. HMW-NCF (neutrophil chemotactic factor) is released into the circulation soon after mast cell activation (48). Its release in asthmatic patients is antigen dose dependent, inhibited by cromolyn, and accompanied by transient leukocytosis. LTB4 and PAF are potent chemotactic agents capable of inducing neutrophil exudation into human skin, and they induce production of oxygen radicals and lipid mediators. Histamine also alters neutrophil chemotactic responses.
Eosinophil Chemotactic Factors The most potent and selective eosinophil-directed agent is PAF, (33) which induces skin or bronchial eosinophilia. Other, less active eosinophil-directed mast cell products include the tetrapeptides Val or ala-gly-ser-glu (eosinophil chemotactic factor of anaphylaxis P.60 [ECF-A]) (49) and others having a molecular weight of 1,000 to 3,000. The latter ones have been found in the blood of humans after induction of physical urticaria or allergic asthma. ECF-A is capable of inducing PAF production by eosinophils (50).
Mediators with Enzymatic Properties Two important proteases are found in human mast cells and not basophils. Tryptase (51), a tryptic protease of 140,000 daltons, is present in all human mast cells. It constitutes nearly 25% of mast cell granular protein and is released during IgE-dependent reactions. It is capable of cleaving kininogen to yield bradykinin, diminish clotting activity, and generate and degrade complement components such as C3a and a variety of other peptides. Tryptase is not inhibited by plasma antiproteases, and thus its activity may be persistent. It is present in plasma in patients experiencing anaphylaxis and in those with systemic mastocytosis. The amount and ratio of α and β subtypes have proved useful markers in these disorders (52). Its true biologic role is unclear, but it enhances smooth muscle reactivity and is a mitogen for fibroblasts, increasing their production of collagen (53,54). A chymotryptic protease termed chymase is present in a subclass of human mast cells, particularly those in the skin and on serosal surfaces, and has thus been used as a marker to identify connective tissue mast cells. It cleaves angiotensinogen to yield angiotensin, activates IL-1, and is a mucus secretagogue. Other enzymes found in mast cells include carboxypeptidase and acid hydrolases.
Structural Proteoglycans The structural proteoglycans include heparin and various chondroitin sulfates.
Heparin
Heparin is a highly sulfated proteoglycan that is contained in amounts of 5 pg/106 cells in human mast cell granules (55) and is released on immunologic activation. Human heparin is an anticoagulant proteoglycan and a complement inhibitor, and it modulates tryptase activity. Human heparin also may be important in angiogenesis by binding angiogenic growth factors and preventing their degradation, and it is essential for the proper packaging of proteases and histamine within the mast cell granule.
Chondroitin Sulfates Human basophils contain about 3 to 4 pg of chondroitin 4 and 6 sulfates, which lack anticoagulant activity and bind less histamine than heparin. Human lung mast cells contain highly sulfated proteoglycans, chondroitin sulfates D and E, which accounts for the different staining characteristics of these mast cells.
Cytokines Although cytokines traditionally have been viewed as products of monocyte-macrophages or lymphocytes, it has become clear that mast cells (56) generate many, including tumor necrosis factor-α (TNF-α), IL-1, IL-1ra, IL-3, IL-4, IL-5, IL-6, IL-9, IL-13, IL-16, and GM-CSF (57,58 and 59) in an NF-κB–dependent process (22). These molecules may be central to local regulation of mast cell growth and differentiation and may also provide new functions for mast cells in health and disease. Basophils are also a prominent source of IL-4 and IL-13 (56). The preponderance of these mast cell and basophil cytokines can be categorized as proinflammatory (IL-1, IL-6, TNF-α); possess properties important in IgE synthesis (IL-4, IL-13); stimulate eosinophil growth, longevity, localization, and activation (IL-3, IL-5, and GM-CSF); and participate in airway remodeling (IL-9).
MEDIATOR INTERACTIONS Part of "4 - Biochemical Mediators of Allergic Reactions" The mediators generated and released after mast cell activation have been isolated, identified, and characterized as individual factors, whereas physiologic and pathologic events reflect their combined interactions. Given the number of mediators, the knowledge that many have yet to be purified (or even identified), and the lack of P.61 understanding of appropriate ratios of mediators generated or released in vivo, it is not surprising that there are no reliable data regarding these interactions in health or disease. The number and type of mast cell mediator interactions are potentially enormous, and their pathobiologic consequences are relevant to a variety of homeostatic and disease processes. The best clues to the interaction of mediators are the known physiologic and pathologic manifestations of allergic diseases. It is hoped that the valuable tool of gene knockouts in mice will elucidate critical individual and interactive roles of these molecules.
THE ROLE OF THE MAST CELL AND ITS MEDIATORS IN TISSUE Part of "4 - Biochemical Mediators of Allergic Reactions" The most compelling evidence for the role of mast cells and mediators in human tissue is derived from experiments in which IgE-dependent mast cell activation in skin is caused by specific antigen (or antibody to IgE). The participation of other immunoglobulin classes and immunologically activated
cells, and thus of other inflammatory pathways, is excluded in such studies by using purified IgE to sensitize nonimmune individuals passively. Activation of cutaneous mast cells by antigen results initially in a pruritic wheal-and-flare reaction that begins in minutes and persists for 1 to 2 hours, followed in 6 to 12 hours by a large, poorly demarcated, erythematous, tender, and indurated lesion (60). Histologic analysis of the initial response shows mast cell degranulation, dermal edema, and endothelial cell activation. The late reaction is characterized by edema; by infiltration of the dermis by neutrophils, eosinophils, basophils, lymphocytes, and mononuclear leukocytes; and in some instances by hemorrhage, blood vessel wall damage, and fibrin deposition of sufficient severity to warrant the diagnosis of vasculitis. Similar studies of lung tissue responses, employing passive sensitization or mast cell–deficient subjects, have only been possible in mice. In humans, a similar dual-phase reaction is experienced by allergic patients who inhale antigen, but the participation of immunoglobulins other than IgE and of activating cells other than mast cells cannot be excluded, therefore complicating assessment and preventing unambiguous assignment of any response to a particular immunologic pathway. Such challenges result in an immediate bronchospastic response followed by recovery, and, 6 to 24 hours later, by a recrudescence of asthmatic signs and symptoms (61). The mediators responsible for these pathophysiologic manifestations have not been delineated fully, but clues to their identity can be derived from knowledge of the effects of pharmacologic manipulation, by the identification of mediators in blood or tissue fluid obtained when the inflammatory response occurs, and by the known effects of isolated mediators. Pharmacologic intervention suggests that the initial phase is mast cell dependent in both skin and lung tissues. The initial response in skin may be inhibited by antihistamines, and in the lungs by cromolyn. In both tissues, corticosteroids effectively inhibit only the late response, reflecting its inflammatory nature. Histamine, TNF-α, tryptase, LTD4, PGD2, IL-5, and both neutrophil and eosinophil chemotactic activity are found soon after challenge. The late response is associated with leukocyte infiltration and cytokine release, but not with a unique profile of released mediators. The exact genesis of the early and late reactions is speculative. The concerted action of the spasmogenic mediators histamine, adenosine, PGD2, leukotrienes, and PAF seems sufficient to account for all of the immediate pathophysiologic (anaphylactic) responses to antigen. This concept is supported by the knowledge that the early response occurs before a significant influx of circulating leukocytes. However, mast cell mediators or mediators from antigen-reactive T lymphocytes, epithelial cells, or macrophages may induce such changes, either directly or indirectly. In response to mediators, vascular endothelium, fibroblasts, and a variety of connective tissue and epithelial cells then could generate other inflammatory and vasoactive mediators. The late phases in lung and skin tissue are likely to represent the residue of the early response as well as the contribution of active enzymes, newly arrived plasma inflammatory cascades, various cytokines (particularly those inducing endothelial expression of adhesion molecules) (57), and the influx of activated circulating leukocytes. Of direct relevance to P.62 leukocyte recruitment are GM-CSF, IL-3, and especially IL-5, which promote eosinophil growth, differentiation, migration, adherence, and activation (62). The late inflammatory response is relevant to the progression of asthma in that patients experiencing the late responses have exacerbation of their nonspecific bronchial hyperreactivity, whereas this phenomenon does not occur after isolated early responses.
HOMEOSTATIC ROLE OF MAST CELLS Part of "4 - Biochemical Mediators of Allergic Reactions" Mast cell mediators likely are important in maintaining normal tissue function and participate in the expression of innate immunity. Because mast cells are positioned near small blood vessels and at the host–environment interface, and are thus at crucial sites for regulating local nutrient delivery and for the entry of noxious materials, the potential regulatory role of mediators is obvious. They are likely to be especially important in the regulation of flow through small blood vessels, impulse generation in unmyelinated nerves, and smooth muscle and bone structural integrity and function. The ability to recruit and activate plasma proteins and cells may also provide preimmune defense against host invasion by infectious agents. Such a role is most apparent in parasitic infestation but is also likely in the case of other insults. Moreover, the recognition of mast cell heterogeneity implies that differences in mast cells relate to locally important biologic requirements. Although the homeostatic and pathophysiologic role of mast cell mediators is understood imprecisely, the broadening understanding of their chemical nature and function provides a useful framework for addressing their role in health and disease.
5 Antihistamines J o n a t h a n A. B e r n s t e i n Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio
Contents • • • • • • •
HISTORICAL PERSPECTIVE H1 RECEPTOR HISTAMINE ANTAGONISTS DUAL-ACTION ANTIHISTAMINES OTHER AGENTS WITH ANTIHISTAMINE PROPERTIES CLINICAL USE OF ANTIHISTAMINES ADVERSE EFFECTS OF H1 RECEPTOR ANTAGONISTS TOLERANCE
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SYMPATHOMIMETICS H2 RECEPTOR ANTAGONISTS H3 RECEPTOR ANTAGONISTS CONCLUSIONS
Histamine receptor antagonists (antihistamines) can be categorized in terms of their structure, pharmaco*kinetics, pharmacodynamics, and clinical utility. Second-generation, nonsedating H1 receptor antagonists, many of which have been derived from first-generation agents, have added a new dimension to the treatment of allergic disorders. During the past several years, a new field of pharmacoepidemiology has also emerged, largely as a result of postmarketing surveillance of these newer H1 antagonists. In fact, investigations into the adverse drug reactions associated with the second-generation agent terfenadine have served as prototypes for the design of current long-term surveillance studies monitoring the safety of drugs in a variety of clinical situations.
HISTORICAL PERSPECTIVE Part of "5 - Antihistamines" Histamine, or b-imidazolylethylamine, was first synthesized by Windaus and Vogt in 1907 (1). The term histamine was adopted because of its prevalence in animal and human tissues (hist, relating to tissue) and its amine structure (2,3) (Fig. 5.1) Dale and Laidlaw (4), in 1910, were the first to recognize histamine's role in anaphylaxis when they observed a dramatic bronchospastic and vasodilatory effect in animals injected intravenously with this compound. Subsequently, histamine was found to be synthesized fro
