The National Medical Series for Independent Study . 3rd edition immunology Richard M. Hyde Professor and Vice Chairman Department of Microbiology and immunology University of Oklahoma Health Sciences Center Oklahoma City, Oklahoma Williams & Wilkins Philadelphia « Baltimore + Hong Kong + London + Munich + Sydney + Tokyo A Waverly Company© writers Wikies Managing Editor: Jane Velker Development Editor: Donna Rae Siegfried Project Editor: Amy G. Dinkel Production: Laurie Forsyth Illustration: Patricia MacAllen Copyright © 1995 Williams & Wilkins 200 Chester Field Parkway Malvern, PA 19355 USA Library of Congress Cataloging-in-Pul Hyde, Richard M, Immunology ! Richard M. Hyde. — 3ed ed. P. cm. — (The National medical series for independent study) Includes index: ISBN 0-683-06231-X (alk, paper) 1. Immunology—Outlines, syllabi, ec. 2. Immunology— Examinations, questions, etc. 1, Title. I. Series IDNLM: 1. Allergy and immunology—examination questions. 2. Allergy and Immunology—outlines. QW 18H995i_ 1994] QR182.55.H94 1994 616.07°9'076—de20 DNUMIDLC {or Library of Congress 9341662 cp ication Data ISBN 0-683-06231-X (©1995 by Williams & Wilkins, Malvern, PA Printed in the United States of America. All rights reserved. Except as permitted under the Copyright Act of 1976, no part of this publication may be reproduced or distributed in any form or by any means or stored in a data base or retrieval system, without the prior written permission of the publisher. 10987654321Contents Preface ix Acknowledgments xi To the Reader xiii 1 Introduction to Immunity and the Nonspecific Immune System 1 1 tmmunity 1 Ml, Mechanical, chemical, and physiologic factors 3 I. Phagocytosis 5 IV. Humoral factors 10 V. Lymphocytic cells 13 2 Antigens and Immunogenicity 23 1. The immune response 23 I. Immunogenicity 25 I. Molecular differences in epitope structure 30 3 Immunoglobulins 39 1. Immunoglobulins 39 1. General immunoglobulin structure 39 I, Structure and function of specific immunoglobulins 45 IV. Antibody diversity 51 V. Plasma cell dyscrasias 58 4 The Complement System 67 1. Complement 67 UN. Pathways of complement activation 69 IIL The membrane attack pathway (the common pathway) 75 IV. Biologic consequences of complement activation 76 V. Regulatory mechanisms 81 5 The Immune Response System 89 |. Exposure to an antigenic substance 90 I. The lymphoid system 9110 u IIL Cells involved in the immune response 95 IV, Events in the induction of the immune response 102 \V. Intracellular events occurring during cell maturation 106 VI. Phases of the humoral immune response 107 Immune Regulation 1. Introduction 117 11. Immunosuppression 119 MI. Tolerance 123 IV. Immunopotentiation 126 Immunization |. Introduction 137 IL Active immunization (immunoprophylaxis) 138 IIL. Passive immunization (immunotherapy) 142 IV. Experimental immunization procedures 143 \V. Adverse reactions to vaccines 145 Laboratory Methods In vitro antigen-antibody reactions 154 1 Procedures involving ect Jemonsation and observation of reactions 156 I, Complex serologic procedures 159 IV. Assays of immune competence 163 VL. Identification of specific allergens in immediate type hypersensitivity reactions. 167 Vi. Detection of immune complexes 168 Vil. Production and use of monoclonal antibodies 169 Immunologic Mechanisms of Tissue Damage 1. Introduction 179 IK, Immediate hypersensitivity ype W reactions 180 II. Cytotoxic (type M) reactions 185 IV. Immune complex-mediated (type Il) reactions 187 V. Cell-mediated (type IV) reactions; delayed hypersensitivity and cell-mediated cytotoxicity 190 Autoimmune Diseases |. General considerations 204 IL Representative autoimmune diseases 208 Immunodeficiency Disorders 1. General considerations 226 Il, Phagocytic cell defects 227 Il, Becell deficiency disorders 229 IV. T-cell deficiency disorders 232 17 137 153 179 203 225\. Combined B-cell and T-cell deficiency disorders VI. Secondary immunodeficiency conditions 234 Vil. Complement deficiencies 236 12 Transplantation Genetics and Immunology 1. Introduction 245 II. Histocompatibility gene complex 246 I. Clinical transplantation immunology 252 13 Tumor Immunology 1. Neoplasms 268 IL, Tumor-associated antigens 269 I, Immune response to tumor antigens 271 IV. Immunologic factors favoring tumor growth 275 V. Immunotherapy 277 Comprehensive Examination Appendix Index 233 245 267 287 305 307Preface This text was developed as a syllabus to be used in an immunology course taught to second- year medical students. This third edition has been modified considerably, both in light of new developments in this rapidly moving field and with valued feedback from students and col- leagues. In particular, the chapters on the immune response and immunologic mechanisms of regulation have been dramatically revised to update information on the cytokines and mem- brane receptors that are pivotal in these events. Case studies and vignettes have been added to each chapter to emphasize clinical and practical aspects of the material under study. The text first presents a review of native immunity, immunogenicity, the mechanisms and products of immune responses, and practical considerations of immunization and the labo- ratory procedures employed in the diagnosis of disease. The remaining chapiers are concerned with defects in native and acquired immunities, with diseases in which the immune system has been inappropriately activated or is autoreactive, and with transplantation and tumor immu- nology. This book presents a concise coverage of the fundamentals of immunology in a readily un- derstandable manner. The material is presented in outline format, with key points emphasized by boldface. Complex material is presented in figures and tables to give the student useful sum- aries. Because the vignettes and clinical cases may contain data that students have not yet ‘encountered in their education, a table of laboratory values has been added as an appendix, to assist interpretation of these data. Clinical relevance is stressed, both in the text and in the study questions and explanations. Richard M, HydeAcknowledgments | would like to express my appreciation to Jim Harris of Harwal Publishing, who developed the concept for this series, and to Debra Dreger, for her guidance and support during the prep- aration of this text, The assistance of Donna Siegfried and Martha Cushman in text and illus- tration development is also acknowledged. Appreciation is extended to Amy Dinkel for manu: script editing, Many colleagues have contributed to the preparation of this book, Dr. D. Rex Billington served as the catalyst for the preparation ofthe original manuscript; he contributed innumer- able ideas on organization and format and was invaluable in our efforts to evaluate the teaching, effectiveness of the text. Valuable advice on content was obtained from Dr. Samuel R. Oleinick and from my sons, Roderick and Scott. Dr. Robert A. Patnode, who was the co-author for the first edition, has continued to be a valued friend and colleague as subsequent editions hhave evolved. Karl Wagenhauser, MD, assisted in the development of the clinical cases and Vignettes for this edition. The contributions of my wife Ruth are also acknowledged. Her patience and support were the foundations upon which I was able to complete this text This book is dedicated to the many students, both past and present, who contributed to its preparation through their penetrating questions, thoughtful comments, and constructive critTo the Reader Since 1984, the National Medical Series for Independent Study has been helping medical students meet the challenge of education and clinical training. In today's climate of burgeoning, information and complex clinical issues, a medical career is more demanding than ever. In. creasingly, medical training must prepare physicians to seek and synthesize necessary infor- mation and to apply that information successfully. The National Medical Series s designed to provide a logical framework for organizing, learn- ing, reviewing, and applying the conceptual and factual information covered in basic and clinical sciences. Each book includes a comprehensive outline of the essential content of a discipline, with up to 500 study questions. The combination of an outlined text and tools for self-evaluation allows easy retrieval of salient information. All study questions are accompanied by the correct answer, a paragraph-length explanation, and specific reference to the text where the topic is discussed. Study questions that follow each chapter use the current National Board format o reinforce the chapter content. Study questions. appearing at the end of the text in the Comprehensive Exam vary in format depending on the book. Wherever possible, Comprehensive Exam questions are presented as clinical cases or scenarios intended to simulate real-life application of medical knowledge. The goal of this ‘exam is to challenge the student to draw from information presented throughout the book All of the books in the National Medical Series are constantly being updated and revised. The authors and editors devote considerable time and effort to ensure that the information required by all medical school curricula is included. Strict editorial attention is given to accuracy, or ganization, and consistency. Further shaping of the series occurs in response to biannual dis Cussions held with a panel of medical student advisors drawn from schools throughout the United States. At these meetings, the editorial staff considers the needs of medical students to learn how the National Medical Series can serve them better. In this regard, the Williams & ‘Wilkins staff welcomes all comments and suggestions.Chapter 1 Introduction to Immunity and the Nonspecific Immune System Vignette A fussy, 18-month-old boy presents with 1. Is there any cli significance labored breathing, a cough producing tothe size ofthe patient? copearsamliforhisage, His eg iym, 2+ Dag the histor of recutent in panic membrane is inflamed and he has Sections suggest anything of im- fales in his let lower lobe, His mother portance? repos that the child was normal unl 3: Why di the child begin to have approximately 6 months of age, at which infections at 6 months of age? time he began to experience respiratory problems and repeated sinopulmonary Infections. IMMUNITY (RESISTANCE) is the sum of all naturally occurring defense mech: that protect humans from infectious disease. There are two types of resistance mecha- nisms: nonspecific (innate) and specific (acquired), examples of which are presented in Table 1-1. Impairment of these mechanisms increases an individual's susceptibility to re- current infections often caused by “opportunistic” organisms (L.e.,. organisms not ordi narily considered pathogenic). When host defenses are diminished, these opportunists, can produce disease. A] Nonspecific immunity i innate. 1. The physiologic mechanisms of nonspecific immunity exist throughout the animal king dom as inherent, oF innate, qualities of the species. These mechanisms do not exhibit specificity —that is, they do not depend on specific recognition of a foreign material: A single defense barrier affords protection against many different potential pathogens. The emponent ofthe nonspecific immune system are discussed in sections IV ofthis chapter, TABLE 1-1, Resistance Mechanisms ‘Types of Resistance Examples Nonspecific (innate) ‘Mucous membranes Phagocytic cells Enzymes in secretions Interferon Specific (acquired) ‘Naturally acquired Placental transfer of antibody (passive) Recovery from disease (active) Anificially acquired Administration of antitoxin (passive) Vaccination (active)2 [.chaper 11 2, The complement system is a series of serum proteins that interact sequentially with one another to augment resistance. Once activated, the components of the complement system often work in conjunction with the innate immune system to rid the body of invading microbes by enhancing inflammation and phagocytosis, Activation of the ‘complement cascade can occur by either of two pathways. a. The classical pathway is activated by antibody complexed with its homologous anti- gen. b. The alternative pathway is triggered by microorganisms and other materials, usually without the participation of antibody. Specific immunity is developed as a result of exposure to a variety of agents capable of inducing an immune response (ie., immunogens), such as vaccines, microbes that colo- nize the body, and macromolecules in the diet. 1. The specific immunologic response has two components and three essential charac- ter a. Components (1) Primary immune response. Initial exposure toa particular infectious agent, or immunogen, is followed by an induetion phase, during which precommited lymphocytes proliferate and mature into antibody-secreting plasma cells (in the ‘case of humoral immune responses; see |B 3 a) or specifically active T cells that secrete various mediators (lymphokines) on subsequent contact with that agent (inthe case of cell-mediated immune responses; see | B 3 b) (2) Secondary (anamnestic) immune response. On further contact with that same immunogen, increased resistance develops through the abundant production of specific antibodies or sensitized lymphocytes. b. Character (1) The ability to distinguish self from fr (2) Specificity, which isthe selectivity shown by antibodies and lymphocytes of the specific immune system in reacting only with matching (homologous) im- munogens (3) Immunologic memory, which allows antibodies and sensitized lymphocytes to remember their homologous immunogen (sometimes referred to as an antigen) and react with i ater 2, Specific immunity may be acquired by natural or artificial processes. a. Examples of naturally acquired immunity include the immunity that develops dur: ing convalescence from an infection, and the placental passage of antibody from mother to fetus. b. Examples of artificially acquired immunity include vaccination or injection of gamma globulin for the induction of an immune state, 3. Specific immune responses are mediated by two interrelated and interdependent mechanisms. As immunologic barriers to infection, humoral and cell-mediated re- sponses usually work together to destroy pathogenic microorganisms or neutralize their toxic products . Humoral immunity primarily involves bursa- or bone marrow-derived (8) lympho- cytes, or B cells. Immunoglobulins, as proteins in the plasma fraction of the blood, Comprise the humoral (ie., soluble) component ofthe specific immune system, (1) The 8 cell expresses specific immunoglobulin on its surface. When this surface immunoglobulin (sigM) interacts with its homologous antigen, the B cell is trig- gered to proliferate and differentiate into plasma cells. (2) The plasma cells excrete vast quantities of immunoglobulin that is specific for the same antigen that originally triggered the B cell b, Cell-mediated (cellular) immunity primarily involves thyrmus-derived (T) lympho- cytes, or T cells. The T cell expresses on its surface a receptor molecule that is struc ‘urally similar to an immunoglobulin and is similarly specific for is particular ho- mologous immunogen, but is not secreted from the cel.Induction immunity andthe Nonipecie mune System 3 (1) When the T-cell receptor contacts its homologous immunogen, proliferation and differentiation of the T cell and its progeny are stimulated. The end product of this developmental process is a variety of T-cell subsets with different functions. (2) These T cells, which reside in the peripheral blood and lymphoid tissues, com- prise the cellular (.e., T-cell-mediated) component of the specific immune sys- tem. Interaction of these specifically sensitized lymphocytes with their homolo- ‘20us ligand (antigen) triggers the release of a variety of lymphokines. Acquired immunities are not always protective. The immune response may be inappro- priate, resulting in allergies (e.g., hay fever), or it may be directed against one of the body's own constituents, resulting in an autoimmune disease (e.g., thyroiditis). Thus, hu- ‘mans exist in an immunologic thunderstorm; some of the lightning bolts are beneficial, others are not (Figure 1-1). [EBD MecHanicat, CHEMICAL, AND PHYSIOLOGIC FACTORS contribute to non- specific immunity. 'A.] Mechanical barriers to infection contribute to innate immunity by inhibiting the attach- ‘ment and penetration of infectious agents (Table 1-2). 1. Intact skin is the first line of defense against infection. Consisting of the keratinized ‘outer layer of dead cells and the successive layers of epidermis, undamaged skin is vir- tually impenetrable to all but a few organisms. 2, Mucus coats the epithelial cells of the mucosa, preventing contact between many pathogens and areas not covered by skin. Microorganisms and other particles are trapped in the viscous mucus and are removed by other mechanisms. For example: a. Beating of cilia of epithelial cells in the respiratory tract removes contaminating microorganisms that become trapped in the mucus. This mechanism can be dam- aged by air pollutants, smoking, and alcoholism, all of which may predispose an individual to lower respiratory tract infections. b. Coughing and sneezing dislodge and help to expel the mucous blanket. 3. Shedding of cells that carry microbes provides a mechanical cleansing action, 4, Saliva, tears, perspiration, urine, and other body fluids assist in flushing microbes from the body. FIGURE 1-1. The acquired immune response (i. specific immunity) has both good and bad conse- ‘quences, some of which are shown here, Natura, nonspecific resistance (Le, innate immunity) isthe foun- ‘dation on which the adaptive responses have an effect. Fav fe Undesirable Arges a a ell Iiecteu ape SS ASETge— Micirevune cecnses consict_ <= ‘pro-cancerous grins EE rat rejection Enjthroblastosis fetals4 [chap 1 WA TABLE 1-2. Examples of Nonspecific Defense Mechanisms fe Protective Actions Skin Physical barrier Fatty acid secretions Oral cavity Enzymes and antibodies in saliva Flow of saliva to throat Epithelial cells and mucous layer Respiratory system Turbinations and hairs in nasal passage, which help trap microbes ‘Mucous secretions containing enzymes and antibodies, which help inactivate microbes Giliary beating, which moves mucous blanket up to esophagus, ‘where itis swallowed Gastrointestinal system Low pH of stomach Enzymes and antibodies in secretions Peristalsis Eyes Lysozyme in tears Flushing action of tears Fars ‘Antimicrobial properties of cerumen Genitourinary system pH of vagina pH of urine Flushing action of urine “Al of there bay sites ar ao protected by thei normal loa, which compte wth pathogen or adhesion sites and nutes 5. Vomiting, diarrhea, and other body functions also eliminate pathogenic organisms. ‘These functions, however, also may act as vehicles for spreading disease. Chemical barriers to infection. Numerous substances found in body secretions provide a natural defense against microorganisms that invade the body (Table 1-3). The acid pH of most physiologic secretions prevents colonization by microorganisms. 1. For example, urine and vaginal secretions, as well as the hydrochloric acid in the stom- ach, maintain acidic microenvironments. 2. An acidic environment kills most pathogenic microbes while promoting the growth of nonpathogenic bacteria (e.g., lactobacilli in the vagina and intestine). Physiologic factors that contribute to innate immunity 1. Body temperature. Many organisms do not infect humans because the organisms grow poorly at 37°C. 2. Oxygen tension, which is especially high in the lungs, inhibits the growth of anaer- obes. 3. Hormonal balance. An increase in corticosteroids decreases the inflammatory response and lowers resistance to infection. Thus, people receiving corticosteroids to control autoimmune disease or graft rejection have a heightened susceptibility to infectious agents 4. Age. People who are very young (age 3 years or younger) or very old (age 75 years or older) are much more susceptible to infection because their immune responsiveness is suboptimal,Invocation to Immunity andthe Nonspecific Inmune System | 5 TABLE 1-3, Biologic Activities of Secretory Products important in Nonspecific Immunit Product, Mechanism of Action ‘Organic acids Found at low pH in sebaceous gland secretions; many microbes sus- ceptible to low concentrations Fatty acids Interfere with functions ofthe cell membrane Saliva Contains enzymes that damage the microbial cell wall and membrane and cause leakage of cytoplasm; also contains antibodies that opsonize microbes, and, with the pasticipation of complement, may lyse cells Tears Contain lysozyme, which lyses bacteria, particularly gram-positive bac- teria, by destroying the bacterial cell wall Lactoferrin Bind iron, interfering with microbial acquisition of this essential metab- and transferrin olite Lactoperoxidase Similar to myeloperoxidase; catalyzes peroxidation in bacterial cell wall and membrane Hcl Denatures proteins Bile acids Inerfere with vital functions of the cell membrane Trypsin Hydrolyzes proteins of cell membrane and wall Mucus Eniraps foreign particles; sialic acid content blocks attachment of influ- ‘enza Virus to epithelial cells Spermine [A pht-dependent polyamine found in sperm and seminal fluid; inhibits ‘growth of gram-positive bacteria [EL PrAcocvrosis contributes to nonspe ‘A.] Definition. Phagocytosis is the process by which particulate substances, such as bacteria, are ingested by a cell and destroyed. Phagocytosis is one form of endocytosis. The other form—pinocytosis—is the internalization of fluids and solutes. Phagocytosis requires: 1. Energy generated through glucose metabolism. 2. Synthesis of a new cell membrane immunity. 3. An active cytoplasmic contractile protein system ‘Types of phagocytic cells. Although neutrophils, monocytes, and macrophages are not the only cells that phagocytize, they are the most important phagocytic cells. 1. Neutrophils (polymorphonuclear leukocytes, PMNs) are granulocytes that circulate in the blood and migrate quickly in response to local invasion by microorganisms. 2. Monocytes also circulate in the blood, but in much lower numbers than neutrophils, ‘They migrate to the tissues, where they differentiate into macrophages, which reside in all body tissues. For example: a. Kupffer cells are macrophages in the liver. b. Histiocytes are macrophages in connective tissue, Movements of phagocytic cells 1, Ameboid movement. Phagocytic cells migrate in and out of blood vessels and through- ‘out the tissues, The process of cellular emigration from capillaries is called diapedesis, 2. Chemotaxis. Phagocytes move toward other cells or organisms by cytoplasmic stream- ing in response to chemical agents called chemotaxins (Table 1-4). Two bacterial con: stituents are of particular importance.6 [cnanter 1 ic TABLE 1-4. Factors Chemotactic for Neutrophils (Chemotaxin Source Comment fMet-Leu-Phe Bacteria ‘Also chemotactic for eosinophils and ma rophages Leukotriene By Arachidonic Products ofthe lipoxygenase pathway; also acid attracts macrophages and eosinophils Endotoxin Bacteria ‘Activates the alternative complement pathway to produce Ca sa Complement Causes degranulation of neutrophils; also at- tracts eosinophils and macrophages Fibrinopeptides Fibrinogen Generated via fibrinolytic pathways Histamine Mast cells Also increases capillary permeability Plateletactivating factor Mast cells, Also aggregates platelets and causes release neutrophils of serotonin and histamine Eosinophil chemotactic Mast cells Peptide released on degranulation factor Intereukin-8 Macrophage Other lymphokines may interfere with cell movement (eg., migration inhibiting factor) {Met = ormylmetNonie; La = levine Phe = phenyllanina a. The lipopotysaccharide endotoxin found in the outer membrane of gram-negative bacteria activates the complement cascade (by the alternative pathway), which pro- duces the potent chemotaxin CSa. bb. Bacteria initiate messenger RNA (mRNA) translation with N-formylmethionine (eu- karyotic cells do not). Phagocytic cells have a receptor that reacts with peptides blocked at the N-terminus with this amino acid, causing the cell to move toward the source of the signal. Ingestion and vacuole formation (Figure 1-2) 1, When a phagocyte makes contact with a particle, it engulfs the particle, surrounding it with part of its cell membrane. 2. Once the phagocyte engulls the particle, the membrane enclosing the particle com- presses and moves into the cytoplasm of the cell, forming a phagocytic vacuole, or phagosome. 3. Lysosomes, which are membrane-bound bags of enzymes, fuse with the phagosome to form a phagolysosome. Intracellular destruction. Insice the phagolysosome, the engulfed material is digested by tenzymes contained in lysosomal granules. The contents of the lysosomal granules are im. portant in breaking down ingested material and in killing microorganisms. 1. Two types of lysosomal granules exist in neutrophils. a. Primary granules (also called azurophilic granules because they stain dark blue with Wright's stain) comprise 33% of all lysosomal granules. They contain many hydrolytic enzymes, myeloperoxidase, lysozyme, and arginine-rich basic (ie., cationic) proteins b. Secondary (specific) granules comprise 67% of all lysosomal granules. They contain alkaline phosphatase, lactoferrin, and lysozyme, . The lysosomal granules of macrophages are not subdivided in this way, although they contain a similar array of enzymes and antimicrobial products. 2, The granule contents destroy foreign particles by two mechanisms: One depends on cellular glycolysis and the accompanying respiratory burst; the other does not. a. Certain proteins destroy microorganisms by oxygen-independent mechanisms.Irvodton fo Immunity an the Nonspecific Immune Sytem | 7. IgG Fe receptor Lysosomes: 030 receptor Foesidual body Elimination FIGURE 1-2. The process of phagocytosis. The electron micrascopie views (insets) demonstrate the phago- Eylosis of a mycoplasma cell by 2 neutrophil. IgG Fe receptor = receptor forthe Fc segment of immuno: tlobulin G; C3b receptor = receptor for complement component C3b. (1) Hydrolytic enzymes include cathepsin, glycosidase, phosphatase, phospholi- pase, and aryl sulfatase (Table 1-5). These enzymes are able to digest cell wall Components of some bacteria. Some enzymes also may act on viral protein Coats or envelope membranes 2) Defensins are cationic proteins—not enzymes but basic peptides containing large amounts of arginine in polypeptide form (e.g, nuclear histones)—that kill microbes by interacting with the microbial cell membrane to form channels through which essential metabolites escape. (3) Lysozyme, a mucopeptidase, attacks bacterial cell walls and renders the cell ‘osmotically sensitive. (4) Lactoferrin acts by binding iron. (6) Nitric oxide synthase activity is stimulated in phagocytic cells by the synergistic action of interferon-y (IFN) and tumor necrosis factor (TNF). The enzyme Combines oxygen with the guanidino nitrogen of t-arginine to yield nitric oxide, which is toxic for parasite, fungi, tumor cells, and some bacteria b. Oxygen-dependent intracellular killing is a by-product of the respiratory burst that accompanies phagocytosis and produces several other microbicidal oxygen metabo- lites (Table 1-6)8 [chops 1 me TABLE 1-5. Lysosomal Enzymes Enzyme Substrate ‘Aryl sulfatase Sulfate esters (e.g, slow-reacting substance (of anaphylaxis (SRS-A)) Cathepsin Proteoglycans (e.g., collagen) Cholesteryl esterase Lipoproteins Glycosidase Carbohydrates Lysozyme Peptidoglycan Phosphatase Phosphate esters Phospholipase Lipoproteins, phospholipids (1) Events in the respiratory burst are as follows. (a) Oxygen consumption increases. {b) Hexose monophosphate shunt (HMPS) activity is stimulated, (©) Hydrogen peroxide (H,0;) production increases. HO> is a reactive oxidiz- ing agent that kills microbes. (€) Superoxide anion is produced. Superoxide anion is molecular oxygen that has picked up an extra electron, (i) Superoxide anion is extremely toxic to bacteria and tissue, but its very Unstable. Itis quickly converted to H;O; by superoxide dismutase ) HO, is tll toxic to bacteria but is not as potent. The HO, is broken down by catalase (Gi flaw in the formation of superoxide anion and eventually of HO, exist in the neutrophils of people suffering from chronic granuloma tous disease (CGD). (©) Singlet oxygen is produced. In singlet oxygen one ofthe electrons has moved to an orbit of higher energy. (Hydroxy! radicals are produced. Hydroxy! radicals are highly unstable oxi- dizing agents that react with most organic molecules they encounter (@) Myeloperoxidase, in the presence of toxic oxygen metabolites (e.g, H50,), 20H- +30 3. Enzymatic generation of halogenating compounds H,0, + halide (eg.,a chloride ion) + myeloperoxidase -> hypochlorite 1. Enzymatic generation of superoxide anion HMMS = hexose monophoaphte shunt pathway of glycols: NADP, NADPH = nicotinamide adenine din ‘eoude phosphate and ts reduce form, respectivelytation ta munity and the Nonspecific mune System | 9 (6) There are several mechanisms whereby such an activated halide could damage microorganisms, including ) Halogenation of the bacterial cell wall ) Decarboxylation of amino acids with the resultant production of toxic aldehydes 3. Exocytosis. Secondary granules release their contents into the phagosome first, usually before the vacuole has completely pinched off. The contents of the secondary granules are partially expelled into the interstitial space; this reverse endocytosis is called exocy- tosis, or regurgitation 4, Inflammation and tissue destruction. When exocytosis is accelerated and primary ‘granule contents ate also released inio the extracellular space, inflammation and tissue destruction can occur. 5. Other mechanisms of phagocyte degranulation that can lead to tissue damage include: a. Reverse endocytosis caused by immune complexes deposited on basement mem- branes b. Neutrophil cell death €. Perforation ofthe cell membrane by ingested crystalline substances, such as mono- sodium urate in patients with gout Secreted products. In addition to destroying foreign particles intracellularly, phagocytic cells secrete a wide variety of biologically active compounds (Table 1-7). The macrophage is the most active cell in this process and releases the following major secretory products: 1, Factors that influence cell differentiation (e.g., colony-stimulating factor) 2. Cytotoxic factors (e.g,, TNF-a, cachectin) 3. Hydrolytic enzymes (e.g, proteinases such as collagenase, lipase, and phosphatase) 4. Endogenous pyrogen (e,, interleukin-1 {IL-1 5 - Complement components C1 to C5, properdin, and factors B, D, 1, and H of the alter- native pathway 1 TEN 7. Various plasma proteins and coagulation factors, 8. Oxygen metabolites (e.g., H02, superoxide anion} 9, Arachidonic acid metabolites (e.g., prostaglandins, thromboxanes, leukotrienes) ‘Tests for measuring phagocytic function are described in Chapter 8 1V A. Receptor molecules on phagocytic cells (Table 1-8). Several biologically important recep- tor molecules exist on phagocytes. Some interact with lymphokines and cause activation (of the cell to enhanced phagocytic activity; others increase the adherence of the cell to surfaces of other cells in the system. 1. Integrins are surface proteins found on many different cells of the body, and play a role in inflammation and in the immune response by promoting cell-to-cell interaction 2. Opsonins are substances that bind to particles and make them more susceptible to phagocytosis. Opsonins found in serum include the following: a. Split products of the complement cascade (1) Complement component C3b is the most important complement-derived opso- nin; Components iC3b, C4b, and C3b also are active in this process. 2) Phagocytic cells possess membrane receptors for these molecules (see Table 1-8 and Figure 1-2). Thus, bacteria and other foreign particles with one of these mole ‘cules on their surface will have an enhanced interaction with the phagocyte. b. Antibodies (1) Phagocytic cells have a receptor (see Figure 1-2) for the crystallizable fragment molecules, thus enhancing the strength10 [.chaper 1 mH TABLE 1-7. Secreted Products of Macrophages Products Examples Enzymes Proteinases Plasminogen activator Collagenase Hydrolases Lysozyme Lipase Phosphatase: Cilycosidase Plasma proteins -Macroglobulin fay-Antitrypsin Fibronectin Coagulation factors Complement components Oxygen metabolites Arachidonic acid metabolites Nucleotide metabolites Cell function regulators Tissue thromboplastin Factors V, Vil IX, C1, €2,€3, C4, C5 Properdin Factors B, D, 1, H Superoxide anion Hydrogen peroxide Hydroxyl radical Singlet oxygen Prostaglandin Ey Thromboxane 6 Leukotriene C, (1e., slow-reacting substance of anaphylaxis) cAMP Thymidine Interleukin-1 Interferon ee Erythropoietin Promoters of cal proliferation for T cells cel and endothelial cls inhibitors of cel plea tin of tumor cls and een mero eg Ls ‘AMP = eye adenosine monophosphate Of interaction between the cell and the antibody-coated (i.e, antibody- opsonized) particle that is being engulfed, @) The IgG isotypes IgGi and IgG3 are the most active in this process; IgG2, IgG4, and IgA also may be opsonins for phagocytosis, but are less efficient. . Other factors that enhance phagocytosis (@) Fibronectin is a glycoprotein that opsonizes and acts like glue to cause neutro- phils and their targets to stick together. (2) leukotrienes are derivatives of arachidonic acid; some of the leukotrienes are ‘opsonins. Leukotriene B, (LTB.) is chemotactic as well (3) Tuftsin, a tetrapeptide split product of an IgG-like molecule called leukokinin, is found in the spleen. Tuftsin stimulates chemotactic and phagocytic activities. [EGA Hu morat ractors contribute to nonspecific immu A] Antibody and complement. Normal serum can kill and lyse some gram-negative bacteria, This property probably isthe result ofthe combined action of antibody ang complementInroducton to nna and the Nonspecific mune System J 11 3. Intercellular Adhesion Molecules in Phagocytic Cell Membranes | Receptor Ligand Function | Integrins* 7 —_ crt C4b,C3 Aids target cell ingestion; allows factor to cleave Cab to Cid important in clearing immune connexes crs C2b, CAM) Ads target cell ingestion; important in cll adherence tovendotelal suaces crs €3b,C3dg—Sameas CRS as IcaMel Important in cell-cell interactions Fe Receptors Fey isc High aint opsonin Fe isc Moderate afnity Fe isc Low affinty Fea Wes Opsoniation ‘integrins ae cellmembrane glcoprtsns hat ac as eceptos for exraelular atx protein sucha Sonn, complement [pote an some cel surface yeopratens. In addon, phage cls have metivane bound reset or the enalie Kragment fe potion of mmuragiabul (Ck~ complement recepor LFA ~ leukocye function antigen; ICAM = intercellular adbesion molecule; FER = receptor forthe Fporion of an immunoglobulin molecule both of which are present in normal serum. The activity is destroyed by heating at 56°C for 30 minutes, owing to the inactivation of complement. The antibody is induced by sub- Clinical infections (caused by normal flora) and by immunogenic dietary components, 1. Bacteriolysis begins with the lytic action of antibody-activated complement on the li- popolysaccharide outer layer of the cell wall 4. A gram-negative bacterial cell wall contains two layers: an outer membrane layer of lipoproteins and lipopolysaccharide and an inner layer of mucopeptide (pepti- doglycan, . The antibody and complement disrupt the lipopolysaccharide layer of the cell wall. Some complement proteins assume esterase activity, which may be involved in dis- rupting the integrity of the cell wall 2. Once the lipopolysaccharide layer is weakened, lysozyme, a mucopeptidase present in serum, can enter and destroy the mucopeptide layer. 3. The membrane attack complex (MAC) of the complement system also may produce ppores in the bacterial cell membrane that cause vital cytoplasmic constituents to be lost, resulting in the death of the microbe. Nonantibody humoral factors (Table 1-9) 1. Chemotactic factors attract phagocytes; the chemotaxin C’a is an extremely important split product of C5 that is generated during complement activation, 2. Properdin is involved in complement activation by the alternative pathway, and itis believed to work to stabilize a critical complex on this complement cascade (C3bBb, a C3 convertase) 3. IFNs are proteins that usually are produced by virally infected cells, and they protect, other cells in the area. a. Type (1) IFN-a, which is secreted by macrophages and other leukocytes, is induced by viruses or synthetic polynucleotides.12 | choptor 1 w 8 TABLE 1-9. Nonantibody Antimicrobial Factors of Plasma Factor Biologic Activity* Complement components 3a ‘Anaphylotoxin that releases histamine from mast cells and ‘causes smooth muscle contraction cab Opsonization 4a Same as C3a, but 100-fold less active sa Same as C3a, but also very active in inducing chemotaxis and degranulation of neutrophils; peroxide by neutrophils Fibronectin Glycoprotein that promotes the adhesion of cells to surfaces; it functions as an opsonin also increases production of su- Interferons Group of proteins that induce the production of antiviral pro- teins in susceptible cells Bera Iysin Platelet-derived basic protein that inhibits the multiplication of ‘gram-positive bacteria Lactoferrin lron-binding protein transferrin found in macrophages, which ‘competes with microbes for the particular ion essential for bac- terial growth Lysozyme ‘Mucopeptidase that hydrolyzes the glycosidic bond between N-acetyl muramic acid and N-acetyl glucosamine in the pept sdogiycan cell wall of bacteria; loss of this structure makes the cell susceptible to osmotic lysis C-reactive protein ‘Acts as an opsonin by binding to cell wall components of bacte- ria, especially phosphorylcholine as found in Streptococcus ‘pneumoniae; i also can activate complement when thus bound ‘Many of thse factors ae refered to as “acute phate proteins.” which increase apy in concentration (perhaps as mach a 00-010 daring nections. Ts crease she res of ate synthe id nto lease of reo Medes. The see ‘phase reaction iced by nies 1 al els une nce ac. Q) IEN-B, which is secreted by fibroblasts, also is induced by viruses or synthetic polynucleotides. . Type I—also called immune IFN or IFN-y—is secreted by T cells after stimulation with the specific antigen to which the lymphocyte has been sensitized . Protective effects of IFNs (1) IENs activate cellular genes, inducing neighboring cells to produce antiviral proteins that interfere with the translation of viral mRNA. (2) IFNs block viral translation by two enzyme-mediated processes. {a) Protein kinase transfers a phosphate group from adenosine triphosphate. (ATP) to an initiation factor required for protein synthesis. This phosphoryla- tion inactivates the initiation factor, and viral protein synthesis is inhibited, (b) Oligonucleotide polymerase synthesizes adenine trinucleotide, which acti- vates an mRNA-cleaving endonuclease that prevents viral replication, (3) Other protective actions of IFNs include: (@) Enhancing T-cell activity (b) Activating macrophages (©) Enhancing the expression of major histocompatibility complex (MHC) mole- cules on cell membranes (@) Increasing the cytotoxic action of natural killer (NK) cells 4. Beta lysin is an antibacterial protein released from blood platelets when they rupture, as in clot formation. Itis active primarily against gram-positive bacteriaroducon tala andthe Nonspecific mune System J 13: 5. Lactoferrin and transferrin are proteins found in milk and serum, respectively, which ‘compete with bacteria for iron. 6. Lactoperoxidase is found in saliva and milk. Its mechanism of action is similar to that ‘of myeloperoxidase. 7. Lysozyme hydrolyzes the mucopeptide layer of the cell wall of many different bacteria, making the cell susceptible to osmotic lysis. This enzyme is present in serum, tears, saliva, nasal secretions, and other body fluids, as well asin lysosomal granules. LYMPHOCYTIC CELLS contribute to nonspecific immunity. Certain lymphocytic cells are cytotoxic against a variety of targets in the absence of any previous exposure to the targets. “Al arate cet are rane phocytes that apes to function in mmune i valance 1. Source and location a. NK cells are naturally occurring cytotoxic lymphocytes that exist in the body at birth. They are not induced by immunologic insult, although their numbers and ac- tivity can be increased by various lymphokines (e., IL-2). . NK cells arise from bone marrow precursors but are of a lineage distinct from that of either T or B cells c. NK cells comprise 10%-15% of the lymphocytes in peripheral blood and 1%~2% of the lymphocytes in the spleen. They are absent from lymph nodes, 2. Functions a. NK cells are cytotoxic for tumor cells and virally infected autologous cells. b. They also have been reported to play a role in resistance to some bacterial, fungal, and parasitic infections and to participate in regulation of the immune response through the secretion of lymphokines such as IL-2. ‘c. Recent evidence suggests that NK cells, as well as some T cells, may be responsible for antibody-dependent cell-mediated cytotoxicity (ADCC). These cells have mem- brane receptors for the Fc portion of IgG, and therefore can interact effectively with antibody-coated targets. 3. Mode of action a. NK cells kill their targets by perforating the cell membrane, causing pores to form, ‘The molecules responsible for the pore formation are called perforins. (1) After intimate cellular contact, periorins are released from granules within the cell (2) The perforins insert into the target cell membrane and polymerize (in the pres- ence of calcium ions), forming polyperforin channels within the cytoplasmic membrane of the target cell ) Depolarization, abnormal ion flux, and essential metabolite leakage from the cytoplasm are the result. (4) The membrane of NK cells contains a protein (called protectin) that binds to perforins and prevents their insertion and polymerization in the NK membrane. Thus, the NK cells are spared. . NK cells have a broad target range, and they are not subject to MHC restriction. That is, cytotoxicity by NK cells does not require that the NK cell recognize MHC molecules on the target cells c. NK cells release numerous cytokines during their interaction with the target cells, including IFN-« and IFN~y, IL-2, TNF-a, and lymphotoxin (TNF-B). A. The cytotoxic activity of NK cells can be significantly enhanced by exposure to L-2 and the IFNs. €. NK cells do not possess antigenic specificity and do not acquire immunologic mem- ory after exposure to virus-infected cells or tumor cells.14 Loupe tv 8 ‘Antibody-dependent cytotoxic cells can kill target cells without the participation of com- plement i the target cells are coated with specific antibody. 1. Functions. ADCC is thought to play a role in antitumor and antigraft immunity, and may be involved in antiviral protection as well. 2. Types of cells a. ADC has been attributed to a unique subset of lymphocytes called killer (K) cells, It recently has been proposed, however, that ADCC is really a function of NK cells, that bear the CD16 molecule (1) The CD16 molecule is found on NK cells and on a subset of T cells. (2) CD16 is a membrane receptor for the Fe portion of IgG1 and IgG3, which ex- plains the mechanism of target recognition and interaction b. In addition to NK cells, macrophages and neutrophils also participate in ADC. Lymphokine-activated killer (LAK) cells are other naturally occurring cytotoxic cells 1. LAK cells are quiescent lymphocytes that are induced into an active cytotoxic state by 2, a lymphokine. 2. The LAK cell is similar in many ways to the NK cell, but has an even broader target cell range. ‘Tumor ‘infiltrating lymphocytes (TILs) are tumor-speci T cells that have been cultured in vitro with IL-2 in the presence of a specific tumor cell to augment cell numbers. TILs dem: ‘onstrate enhanced tumoricidal activity when infused back into the donor of the Iympho- cytes and tumor cells, Vignette Revisited Much of the information about the immu- nology of the 18-month-old boy has been presented in the chapter. However, some terms or concepts that go beyond the scope of the chapter or book are dis- cussed briefly here. 1. Patients, particularly children, are of- ten amious and “fuoy” when they ave 2, The child in this simulation is having. difficulty breathing and is spitting up a mucoid, bloodtinged (*usty") mae- 3. The child is small for his age because he has spent.a great deal of his young life fighting infections instead of put- ting on weight and body mass. 4, He has an inflamed eardrum be- ‘cause he has ofitis media, which often precedes bacterial pneumo- nia, 5. Recurrent infections are a very important clinical clue to immuno- deficiency. Cystic fibrosis might also be considered in the differen- iagnosis for this patient. inally, the antibody-mediated immunity conferred from mother {0 fetus should be waning by the sixth month of life, making the child more susceptible to infec- tious agents.Case 1 ‘Mis, Watson brings her 2-year-old son Tommie to the family medicine clinic for a 72-hour checkup following a recent bout of pneumonia. Although Tommie is sub- jectively beter, wth his pulmonary infection responding well to oral penicillin ther- apy and his lung fields clearing on chest x-ray, he has developed a severe dermatitis involving his trunk and extremities. QUESTIONS 1. What organisms are most likely to cause pneumonia in otherwise healthy children? 2. What s the possible significance ofthe timing of the appearance of Tommie’s rash and the initiation of penicillin therapy? DISCUSSION Common organisms causing pneumonia in otherwise healthy individuals inelude pneu- mococcus, streptococcus, staphylococcus, and Mycoplasma pneumoniae. All but M. pneumoniae and penicillinase-producing staphylococcus species are readily treated with penicillin. Uncommon organisms that may present as pneumonia in immunocompro- mised individuals include parasites (Pneumocystis carini, fungi (Aspergillus and Candida species), viruses (cytomegalovirus, herpes simplex), gram-negative enteric bacteria, anaer- ‘bic bacteria, and bacteria considered to be nonpathogenic in healthy individuals. The appearance of the rash several days after the start of penicillin therapy is highly suggestive of penicillin allergy. However, patients with Epstein-Barr virus (EBV) infections almost universally develop a rash if given ampicillin or penicillin, Results of labs drawn on Tommie’s initial visit 3 days ago are now available and reveal a mildly elevated white blood cell count and a differential cell count with an increase in the polymorphonucleocytes and several “bands” also present. The induced sputum Gram stain contained many white blood cells and few squamous epithelial cells, indicat- ing a good sample was obtained. The sputum culture has grown out Staphylococcus epi- dermidis, an organism considered nonpathogenic in healthy ch QUESTIONS 1. What is the significance of the increase in polymorphonucleocytes? 2. How do the culture results alter further management and therapy? 3. What questions does Mrs. Watson need to address? DISCUSSION An increase in polymorphonucleocytes on peripheral blood smear along with an inerease in the total white blood cell count is suggestive of bacterial infection. The presence of band forms indicates an acute process. An increase in lymphocytes would suggest viral infection. ‘A positive culture from an adequate sample that rows an otherwise nonpathogenic organism in healthy individuals should alert the investigator to the possiblity of an immu rnadeficiency problem. Antibiotic therapy should cover a broad spectrum of organisms, and further tests should focus on ruling out vatious immunologic dysfunctions. ‘Mrs. Watson should be asked to provide an extensive, detailed previous medical history for Tommie. A history of family allergies would be useful as well asthe general health his tory of other family members. 1516 | chager Upon further questioning, Mrs. Watson reports that there is a family history of allergies. Tommie has three older sisters who are healthy. His only brother died of bacterial pneu- imonia at 4 years of age. Tommie had been a colicky baby and had repeated episodes of diarrhea. He contracted German measles from his sister at the age of 14 months, which resolved without compli Suspecting a possible the culture results the physician draws another blood sample and orders the following tests: immunoglobulin profile, nitroblue tetrazolium reduction assay, phagocytic index, and intraphagocytic ki The -generation cephalosporin, and, as a precaution- ary measure, a swab of fluid exuding from a skin lesion is sent to the microbiology lab. QUESTIONS 1. What does the history of healthy daughters and sickly sons imply? 2. What is gained by switching from penicillin to a third-generation cephalosporin? DISCUSSION ‘An inheritance pattern affecting all males and sparing most females is characteristic of an X-linked recessive genetic disorder. Females are usually carriers unless they are homozy- '30us for the allele and would therefore express the trait A third-generation cephalosporin adds coverage against penicillinase-producing strains ‘of staphylococcus and streptococcus species as well as broad gram-negative coverage. ian notes that Serratia marcescens, istic pathogen, was isolated from the skin lesions. Tommie is slightly hyper- gammaglobulinemic, but there is no elevation of immunoglobulin E (Ig). The nitroblue {etrazolium reduction assay did not react, the phagocytic index was its, and the intraphagocytickiling curve was depressed. ‘Mrs. Watson was asked to return to discuss the test results and for further genetic QUESTIONS 1. Based on the test results, what is the most likely cause for Tommie’s frequent infections? 2. How should therapy be directed in the future? 3. What are the implications of these findings for Tommie's sisters? DISCUSSION ‘The results of the phagocytic studies indicate that Tommie most likely has chronic granu- lomatous disease (CGD). His cells were unable to reduce the nitroblue tetrazolium dye during phagocytosis. The phagocytic index was normal, indicating that the neutrophils ‘were able to engulf the test organism but were unable to kill the ingested microbes. Mrs. Watson was asked to return for appropriate counseling as to the genetic implica- tions of the disease, its management, potential complications, and prognosis. The physi- cian also stressed the need for early diagnosis and aggressive therapy in future infections. A hallmark of diseases of impaired immunity, such as CGD, is recurrent infection by agents normally considered to be nonpathogenic. The physician was suspicious of a phagocytic defect because Tommie had normal levels of antibodies and intact cell- ‘mediated immunity (ie, his gamma globulin level was normal and he had no troubleIrrobtion to Immunity an she Nonspecic Imm sysem | 17 with German measles). His white blood cells were morphologically normal, thus the defect had to reside in the biochemical events that occur during phagocytosis CGD is an inherited, most commonly X-linked disease of male children and infants, characterized by recurrent infections and death at an early age. Tommie’s mother was a carrier, and half of her daughters may also be carriers. Fortunately, the cartier state can be detected by the same test used to diagnose the disease. Carriers have lowered nitroblue tetrazolium dye reduction and intraphagocytic killing abilities,1B | chsprer 1 HBstupv questions DIRECTIONS: Each of the numbered items or incomplete statements in this section is, followed by answers or by completions of the statement. Select the ONE lettered answer or ‘completion that is BEST in each case. 1. The lysosomal enzyme that degrades slow. reacting substance of anaphylaxis (SRS-A) is (A) ary! sulfatase (B) cathepsin (C) lysozyme (D) myeloperoxidase (E) acid phosphatase 2. Which of the following plasma proteins is secreted by lymphocytes? (A) C3 component of complement (B) a-antitrypsin (C) IgM (D) Fibronectin CE) Coagulation factor IX 3. Which of the following chemotaxins is a product of microbial metabolism? (A) N-formylmethionine (B) Histamine (C) Leukotriene B, (D) Fibrinopeptide (€) C5a DIRECTIONS: Each of the numbered items or incomplete statements in this section is negatively phrased, as indicated by a capitalized word such as NOT, LEAST, or EXCEPT. Select the ONE lettered answer ar completion that is BEST in each case. 4. Which of the following substances is NOT found in the cytoplasm of natural killer (NK) cells? (A) Tumor nectosis factor (TNF-a (B) Lymphotoxin (C) Lysozyme (D) TNF. (E) Perforin 5. Human plasma can kill and lyse gram- negative bacteria. This is the result ofthe pres- ence of al ofthe following EXCEPT (A) antibody against the microbe (B) complement (C) beta lysin (D) properdin (E) lysozyme ‘6. Nonantibody factors in plasma that act to ‘opsonize microbes include all of the following EXCEPT (A) Creactive protein (B) fibronectin (C) C3b (D) lysozyme 7. Antimicrobial factors that are products of secretory glands include all of the following EXCEPT (A) spermine (B) lysozyme (C) fatty acids (D) lactoferrin (E) interferon (IFN}-y 8, Most cells that function in innate resistance contain granules, which play a major role in cytotoxicity and the ability of the cell to de- stroy foreign cells. Examples of such cells i clude all of the following EXCEPT (A) neutrophils (B) natural killer (NK) cells, (C) macrophages (D) antibody-dependent cytotoxic cells (E) plasma cellsInduction to Inmunity adhe Nonspeciic mune Sytem | 19 DIRECTIONS: Each set of matching questions in this section consists ofa list of four to twenty-six lettered options (some of which may be in figures) followed by several numbered items. For each numbered item, select the ONE lettered option that is most closely associated with it. To avoid spending too much time on matching sets with large numbers of ‘options, itis generally advisable to begin each set by reading the list of options, Then, for each item in the set, try to generate the correct answer and locate it in the option list, rather than evaluating each option individually. Each lettered option may be selected once, more than once, of not at all Questions 9-11 For each characteristic, select the factor that contributes to nonspecific resistance. (A) Complement (B) Beta lysin (C) Properdin (0) Interferon WEN) (E) Interferon (IEN)-y (F) Lactofertin (G) Lactoperoxidase (H) Lysozyme 9. Secreted by T cells after stimulation with specific antigen 10. A group of serum proteins that can lyse gram-negative bacteria that have antibody on their surface 11. A product of macrophages that induces an antiviral state in neighboring cells Questions 12-13, Match each statement to the opsonin that best describes it (A) IgG (B) Fibronectin (©) C3b (0) Tuftsin (E) Leukotriene By 12. A derivative of arachidonic acid 13. A glycoprotein that reacts nonspecifically with bacteria to enhance phagocytosis20° [chapter 1 BBRANSWERS AND EXPLANATIONS 1. The answer is A [Table 1-5]. Aryl sulfatase hydrolyzes the leukotrienes C,, Dy, and E— the arachidonic acid metabolites thought to be responsible for many of the symptoms of ana- phylaxis. Slow-reacting substance of anaphy- laxis (SRS-A) contracts smooth muscle but at a slower rate than histamine. In addition, itis ‘not inhibited by antihistamines, 2. The answer is C [I 8 1 a (1); Table 1-71. ‘Antibodies immunoglobulins) are secreted by B cells and plasma cells. Macrophages are the source of the other plasma proteins (ie... C3, ‘ay-antitrypsin, fibronectin, coagulation factor IX) listed in the question, 3. The answer is A [Ill C 2 b; Table 1-4] N-formylmethionine and lipopolysaccharide endotoxin are two bacterial products that are Cchemotaxins. N-formylmethionine binds 10 the cell membrane and induces calcium ion flux and activation of the microfilaments, thus facilitating migration. The lipopolysaccharide endotoxin of the outer membrane of gram- negative bacteria activates complement by the alternative pathway, thus generating the ‘chemotactic molecules C3a and C5a, Hista- mine is also chemotactic but is a product of mast cells; C5a is the major chemotaxin of the complement cascade. Peptide split products of fibrin also are chemotactic: 4. The answer is C [V A 3 a,c]. Lysozyme is not contained in the granules of natural killer (NK cells, but is found in many body seere- Uions and in lysosomal granules of phagocytic cells, Lysozyme is a mucopeptidase that hy- drolyzes the bacterial cell wall, rendering the cell susceptible to osmotic lysis. 5. The answer is € {IV A, 8 4, 7]. Both anti- body and complement are needed for immune lysis of gram-negative bacteria, These organ- isms appear to be more sensitive to antibody: mediated lysis owing to the relatively small amount of peptidoglycan that they have in Comparison to gram-positive organisms (5%— 10% versus 70%-80%). Complement can also be activated via the alternative (nonantibody) pathway; properdin is important in stabilizing tone of the enzymes (C3 convertase; C3bBb) in this pathway. Lysozyme appears to facilitate this lytic process. Beta Iysin is not a plasma ‘component but a platelet product that is re- leased when clotting occurs 6. The answer is D [IV B 7; Table 1-9]. C-re- active protein, fibronectin, and C3b all can act 8 opsonins, C-reactive protein reacts with phosphoryicholine in the cell wall of bacteria and can activate complement as well as op- Sonize the cell. Fibronectin acts as a glue that binds cells to surfaces throughout the body. C3b is the major opsonizing protein of the ‘complement cascade; phagocytic cells have a membrane receptor for this molecule, as they do for the crystallizable fragment (Fe) portion Of immunoglobulin chains. Lysozyme hydro- lyzes the cell wall of bacteria; it does not en- hance phagocytosis. 7, The answer is E {IV B 3 b; Table 1-31 All of these antimicrobial factors are produced by secretory glands except interferon (IFNy, ‘hich is produced by T cells. Spermine is pro- duced by the prostate gland. Lysozyme is se- Creted by the lacrimal glands ofthe eye. Fatly acids help to protec the skin and are pro duced by the sebaceous glands. Lactoterin is secreted by the lacrimal and mammary glands. 8. The answer is E (1B 3 a(1); INE 1; V A.B]. All of the cells listed have granules that contain enzymes, antimicrobial compounds, and cytotoxins important in native resistance, with the exception of plasma cells, which pro- duce antibodies, a major component of adap- tive immunity. The granules of phagocytic cells contain hydrolytic enzymes and antimi- crobial proteins that aid in intzacellular de- struction of invading organisms, The granules in cytotoxic lymphocytes contain perforins, lymphokines, and hydrolytic enzymes that are cytotoxic to target cells. 9-11, The answers are: 9-E {IV 8 3 b], 10-A WAT], 11-0 [IVB 3 a1). Interferon (IFNy, also known as immune interferon, is secreted by specifically sensitized T lymphocytes when they come into contact with their homologous antigen Complement is @ group of more than a dozen serum protein that react with antibody ina cascading manner and are able to distupt the cell membrane of gram-negative bacteria ‘Macrophages secrete interferon (INF)-a dur-Iorodction to munity and the Nonspecific mane System | 21 ing an inflammatory response. The interferon induces an antiviral state in other nearby cells by inducing the production of proteins that interfere with viral messenger RNA activity 12-13. The answers are: 12-£ [Ill H 2c (2)), 13-B {Ill H 2.c (1)]. Leukotriene B, is an opso- nin produced from arachidonic acid via the lipoxygenase pathway. It is also chemotactic. Fibronectin is an adhesive glycoprotein that is important in connective tissues where it ‘cross-links collagen. One form of fibronectin Circulates in plasma and acts as an opsonin; another is a cell-surface component that medi- ates cellular adhesive interactions.Chapter 2 Antigens and Immunogeni ‘A 25-year-old white man presents to the clinic complaining of fatigue and a sore throat of 2 weeks’ duration. The patient denies having nausea, vomiting, diarrhea, cough, chest pain, and weight loss, but believes he has had a fever. His medical history is unremarkable, and he knows of no contact with a sick person. Of note on physical examination are an oral temper- ature of 101°F, marked bilateral, tender, mobile, anterior and posterior cervical adenopathy, a gray-white exudative ton- sills, and a palpable spleen, 4 ‘A monospot testis requested, and the results are positive. A diagnosis of infectious mononucleosis is made. ‘The patient is sent home for bed rest and is counseled to eat properly and not exert himself 3. The diagnosis relies on a serologic procedure called the heterophile test, What does this mean? re there other infectious diseases 1. Formulate a differential diagnosis at this point. ‘A complete blood count with differential is ordered and reveals a white blood ‘count of 14,000 with 63% atypical lym- phocytes. - What is the significance of atypical Iymphocytes (also known as Downey cells), given the patient's signs and symptoms? hich similar antigenic rela- tionships are invalved in serologic diagnosist Another heterophil antigen is thought to be involved in the pro duction of blood group A isoaggluti- 5. What is this antigen, and how ‘might it induce the production of these antibodies in individuals ‘with blood groups O and B? ‘THE IMMUNE RESPONSE is characterized by the production of proteins (called immu- rnoglobulins or antibodies) and specifically reactive lymphocytes (called thymus-derived lymphocytes, or T cells) when an animal encounters a foreign macromolecule or cell The inducing substances are called antigens (ie., antibody generators), or immunogens. A] tangent ad antigenic re wo testa are wed et inerchangebly in discussion of the immune response, 1. Immunogenicity is the inherent ability of a substance (ie., an immunogen) to induce a specific immune response. 2. The term antigen ity implies both the ability to induce a response and the ability to react with the products of that response. Antigens are the ligands that react with the products of an immune response. 2324 Lorapme 218 Epitopes (also called determinant groups, or antigenic determinants) are the sites either ‘on or within the antigen with which antibodies and T-cell receptors react. 1. Structure a. Size, Epitopes are very small (i, just four or five amino acid or monosaccharide residues) b. Conformation. The epitopes on an antigen can be linear (i.¢., continuous within the amino acid sequence of the molecule) or conformational (i.e., containing amino acids that end up in the same area on the surface of the protein but are not adjacent in the peptide chain) (Figure 2-1) . Site. Some antibody-binding sites (ie. epitopes) are on the antigen’s surface (e.g. ‘topographic epitopes), whereas others are internal. Internal epitopes are expressed, only after the antigen has been partially degraded in vivo, 2. Function. Epitopes determine the specificity of the antigen molecule, and therefore they are also known as determinant groups. Epitopes are immunoreactive (ie. able to react with their homologous antibody) only if they are spatially accessible as a result of tertiary protein structure, ‘a Paratope. Antibodies are specific for epitopes. The area of the antibody molecule that interacts with the epitope is called the paratope. The structure of the paratope is totally complementary to the epitope, as shown in Figure 2-2. b. Valence of an antigen. Antigens are multivalent. That is, an antigen molecule carries a number of different epitopes—sometimes hundreds of them—some of which spec ify antibody “A,” others that specify antibody "B,” and so forth. The valence of an antigen is equal to the total number of epitopes the antigen possesses ¢. Altering antigenicity. Antigen molecules can be artificially manipulated by altering, adding, or taking away epitopes. With each change, antigenicity is altered (1) New antigens are produced by altering epitopes. This can be done by con- jugating molecules to the antigen or by enzymatically removing portions of the antigen. Changes of this nature are thought to be important in the develop- ment of autoimmune diseases such as drug-induced systemic lupus erythema- tosus. (2) Denaturation oF hydrolysis of the protein almost always destroys conforma- tional epitopes. FIGURE 2-1. Model of epitopes on lysozyme. The Epitope t shaded areas are the specific epitopes. They are ‘composed of chain segments that are either linear (epitopes 1 and 2) oF conformational (epitopes 3-5). (Adapted from Klein |: Immunology: The Seience of Seli-Nonself Discrimination. New York, Wiley 1982, p 356) Epitope 5 Epitope 2 EpitopeAmgen and inmunogenicy | 25 FIGURE 2-2. The interaction of an epitope and its homologous paratope. the pattern of electrical charges influences the interaction between antigen and antibody. As the pattern becomes more com plementary, the force of attraction becomes propor- Uionately stronger, and the affinity ofthe two reac- tants increases, EE incnocenicrry The degree of immunogenicity of a molecule is influenced by several factors, The rela- tionship between these factors can be illustrated by the following “equation:” Immunogenicity = (foreignness) (chemical complexity) (molecular size) Foreignness. An antigen must be foreign or alien to the host with which it makes contact, 1. The greater the phylogenetic difference, the more foreign something becomes, and the greater is its immunogenicity. The use of transplant terminology helps to clarify this concept. a. Autologous antigens are found within the same individual. That is, they are not for- eign to that individual. For example, a skin graft from a man’s thigh to his chest is ‘an autograft. tis not foreign and will not be attacked by the immune system. b, Syngeneic antigens are found in genetically identical individuals (e.., individuals from an inbred strain of mice, identical twins). A graft between members of an in- bred strain is a syngeneic graft or an isograft, and is not foreign. ¢. Allogeneic antigens (alloantigens) are found in genetically dissimilar members of the same species. For example, a kidney transplant from mother to daughter is called an allograft, or a homograft. Its foreign, and therefore can induce an immune re- sponse in the recipient. @) Some alloantigens found in some members of a species and not in others are called isoantigens. @) The A and B blood group antigens are examples of isoantigens. d. Xenogeneic antigens are found in different species. For example, a transplant of a monkey's kidneys to a human is called a heterograft, or xenograft, and it is highly immunogenic. The term heterologous sometimes is used as a synonym for xenoge- 2. Heterogenetic antigens are so named because they are found in widely separated places in the phylogenetic tree. The ubiquity of these antigens is exemplified by the Forssman antigen, the breadth of occurrence of which is presented in Table 2-1 a. Heterophile antigens have in common one or more epitopes; this is the basis of their antigenic relationship. As a result, they are cross-reacting [i.e they combine (ceact} with the same specific antibody because of the presence of this shared epitope}26 | chapter 28 TABLE 2-1. Occurrence of Forssman Antigen in Various Animals and Plants Group Present Absent Plants Corn Rice Microorganisms Bacillus anthracis. Salmonella typhosa Fish Garp Cod Birds Chicken Goose Farm animals sheep Pig Rodents Guinea pig Rat Humans Blood group A al application, since it provides the basi for several clinical tests. screening test. The spirochete that causes syphilis (Treponema pall- dum) shares an epitope with a lipid called cardiolipin, which is found in heart muscle. This heterophilic antigen relationship forms the basis of a diagnostic {and inexpensive) screening test for syphilis. Individuals who are infected with T. pallidum make an antibody that reacts with cardiolipin, (2) Infectious mononucleosis screening test. The heterophile antibody response also can be used in the diagnosis of infectious mononucleosis, which is caused by the Epstein-Barr virus. Most affected patients have a serum antibody that re- acts with sheep red blood cells (RBCS). {@) Screening tests for Rocky Mountain spotted fever and typhus fever. Serum from patients with rickettsial infections agglutinate certain strains of Proteus vul- Baris, This heterophilic immune response is termed the Weil-Felix reaction, and itis useful in the serologic workup of patients with Rocky Mountain spotted fe: ver and typhus fever. 3. Tissue-specific (organ-specific) antigens. Various organs have in their makeup certain antigens that are unique to those organs. a. Thyroid has an organ-specific antigen, thyroglobulin. Any thyroid from any species contains this unique thyroid protein. An immune response to this antigen is seen in patients with Hashimoto's thyroid bb. Myelin basic protein exists in the brain tissue of all species, and it does not exist in any other organ. Myelin basic protein has been implicated in experimental allergic encephalomyelits. (1) Anallergic encephalitis may be provoked by rabies vaccines that contain cen- tral nervous system tissue, which was the case in the original Pasteur vaccine. A similar condition can be induced in experimental animals by the injection of brain tissue or purified myelin basic protein. (2) The process by which cross-reacting epitopes cause immunologic damage is, referred to as antigenic mimicry. Tissue damage occurs as a result of antigenic mimicry (.e., shared epitopes) between Streptococcus pyogenes and human heart tissues in rheumatic fever. The patient makes an immune response to the shared epitope, and the products cause damage to the heart (see II B 2 a). . Cell-specific epitopes. Certain epitopes are found on selected cells. For example, mature T cells bear the CD3 marker in their membranes, d. Sequestered antigens are organ-specific antigens that aré not normally exposed to the immune system. Immune responses do not normally occur to autologous anti- gens in the eye, for example, because these substances are inaccessible to the cells involved in the immune response. If an event occurs that releases these autoanti ens, an autoimmune disease may occur. For example, a puncture wound injury to the left eye may precipitate severe inflammation in the right eye. This is called sym- pathetic uveitis and may result in blindness. <) ‘The chemical complexity of a molecule contributes significantly to its immunogenicity Epitope diversity contributes to the degree of immunogenicity: The more varied theAmgen ad immunogenicity | 27 epitope composition of an antigen, the more likely different (individual) immune re- sponses will be induced. 1. With the exception of pure lipids, most macromolecules can be immunogens. a. Proteins are the strongest immunogens. (1) Proteins have the largest array of potential building blocks (over 20 amino ac- id). This diversity imparts epitopes of differing specificities to a molecule. The total immune response is the sum of all the individual antibodies that are pro- duced, (2) Immunogenicity can be enhanced by adding additional epitopes to the mole- cule. Lipoproteins, complex protein immunogen that exist as part of many cell membranes, induce immune responses to epitopes of both lipid and protein, . Most polysaccharides are nonimmunogenic. They can impart a unique specificity to an antigen if appropriately coupled to that carrier molecule. (1) Polysaccharides do not possess sufficient chemical diversity for full immunoge- ricity 2) In addition, many polysaccharides are rapidly degraded when they enter a host Thus, polysaccharides are not in contact with the immune apparatus long, enough to induce a response. (3) However, certain polysaccharides can be immunogens, including: (a) The capsular polysaccharides that are responsible for the protective im- ‘mune response to the pneumococcus (b) Lipopolysaccharides such as the endotoxins that occur within the cell memn- branes of gram-nogative bacteria «c. The immunogenicity of glycoproteins is best illustrated by the A and B blood group antigens and the Rh antigens. These substances are strong immunogens, and they induce an immune response to the carbohydrate epitope of the molecule. The monosaccharide differences between blood groups A and B are shown in Figure 2-3. Small polypeptides, such as insulin and growth hormone, usually are weakly immu- rogenic. Immune responses usually are induced only after prolonged exposure 10 the antigen, or by the use of adjuvants, which are substances that greatly enhance the immunogenicity of molecules without altering their chemical composition (see Chapter 6 IV'8). €. Nucleic acids are considered to be nonimmunogenic; however, when combined ‘with basic proteins they can act as immunogens. In patients with systemic lupus erythematosus, antibodies are produced to autologous nucleoproteins as well as to ible-stranded DNA ids are also nonimmunogenic, although a few (e.g., cardiolipin) can impart spect- ficity to antigens when appropriately presented to the immune system, 4, 2, Antigenic mosaic. Bacterial and mammalian cells are strong immunogens, and they present a vast array of different antigens to the host. This diversity is sometimes called the antigenic mosaic ofthe cell. Each antigen has multipe epitopes; therefore, many differing antibodies will be produced against cellular immunogens. D,] The size of the molecule is important to it ability to induce an immune response. Usu- ally, the larger the molecule, the better the immunogen (Table 2-2), although there are exceptions. For example, insulin is small but is immunogenic; carbon particles are very large but are nonimmunogenic. 1, As a general rule, molecules smaller than 5—10 kDa are nonimmunogenic. Reasonable immune responses are induced by molecules such as serum albumin (69 kDa). 2. Size is important for several reasons. a. The number and variety of epitopes increase proportionately with the size of the protein, With other macromolecules (eg, carbohydrate antigens), however, the rhumber of epitopes may increase with size but the diversity does not. These are sometimes referred 10 as "monotonously repeating” immunogen.28 [Ouper 2 wo FIGURE 2-3. Blood group antigens A and 8 differ only in the terminal sugar ofthe oligosaccharide stem." Group ‘Ahhas an N-acetyigalactosamine (NAc), and group B has a salactosamine. Genes encoding for groups A and B do fot encade the sugars but rather encode the proper gluco- SY transferase forthe placement ofthe paticular agent. Fuc = fucose: Gal = galactose; Glu = glucose b. Larger molecules are phagocytized. (1) "Antigen processing. Antibodies to most antigens are formed much more ef ciently i the antigen is first “processed” by a macrophage. This involves phago- cytosis of the antigen (see Chapter 5 1 A 1) (2) Molecules must be susceptible to intracellular catabolism within the phago- cytic cell. Antigens that are difficult or impossible to phagocytize are nonimmu- nogenic at times (see IIE 1), For example, polystyrene particles, although readily engulfed by macrophages, are nonimmunogenic because the phagocytic cell lacks the enzymes to degrade the polystyrene, 3. Haptens 2. Structure. Haptens are unique molecules sometimes called “incomplete” or “par- tial” antigens. They usually ate to small to be immunogenic b. Function, Haptens can react wit immune lymphocytes or antibodies once they are formed, but they are not immunogenic by themselves. Haptens cannot induce an immune response, but they can react with the producis of that response. TABLE 2-2. Relationship of Molecular Size to Immunogenicity Molecule ive Immunogenicity Hemocyanin +eee Gamma globulin Diphtheria toxoid Insulin Vasopressin Aspitin|Amgen ad immunogenic | 29 ‘c. Examples of haptens include antibiotics, analgesics, and other low-molecular- weight compounds, Table 2-3 lists some haptens of medical importance. Some mac romolecules are also haptens and are unable to induce an immune response when, injected into an animal in purified form. (1) Many complex carbohydrates are haptenic. For example, the capsule of the type b strain of Haemophilus influenzae does not induce an immune response in very young children (.e., younger than 2 years), who are the people at high- est risk for development of meningitis when infected with this microbe. Immu- nogenicity is gained when the capsular carbohydrate is conjugated to an outer membrane protein of the meningococcus. (2) The immune response of patients with lupus to various nucleic acids is enhanced because the nucleic acids are coupled to basic proteins in their natural state 4. A phenomenon known as the carrier effect explains how haptens acquire immunoge- nicity. fa hapten is coupled to an immunogenic carrier, the hapten becomes endowed with immunogenicity, and thus is able to induce an immune response. The biologi basis ofthe carrier effect is depicted in Table 2-4 a. The carrier molecules may be albumins, globulins, or synthetic polypeptides. }. Drugs often couple with carriers in the body and thereby acquire immunogenicity (1) Penicillin allergy. A classic example in clinical medicine isthe allergic response of some people to penicillin. The penicilloyl moiety of penicillin, which is pro- duced in the body during degradation of the antibiotic, is highly reactive and readily forms a complex with serum albumin. The penicilloyl moiety acquires immunogenicity and elicits an immune response that can be harmful—even life-threatening. Therefore, penicillin is contraindicated in certain individuals. (2) A similar sensitization occurs when an individual comes in contact with the leaves of the poison ivy plant. In this case, however, a cell-mediated immune response results. Resins rich in complex catechols known as urushiols spontane- ‘ously bind to proteins in the skin. The modified proteins ate regarded as foreign by the immune system, and an allergic contact dermatitis resus ‘Other factors that influence the strength of the immune response 1. Degradability. Molecules that are not biodegradable, such as polystyrene particles or asbestos, are nonimmunogenic because they cannot be processed in phagocytic cells of the body. Substances that are rapidly broken down in the body by plasma enzymes, however, can be nonimmunogenic or very weakly immunogenic TABLE 2-3. Selected Low-Molecular-Weight Antigens and Haptens| Haptens Molecular Weight Penicillin 320 Aspirin 180 Methyldopa 20 Urushiols (poison ivy) 211-290 Gentamicin 700 Antigens Molecular Weight Oxytocin 1000 Vasopressin 1000 Gastrin 1800 Calcitonin 3600 Insulin 000 Haptoglobin 9000 Note Most ofthe hapens sted ae involved in contact dermatitis temic allies The antigens sted are weak and they ince abo or Ima at ow levels lyn Some sil30 | chapter TABLE 2-4, The Carrier Effect Carrier Prote’ Hapten Antibody No Yes No Yes No Anticartiet only Yes (not chemically linked) ‘Anticartier only Yes (chemically linked) ‘Anticarsier and antihapten Protein molecules can impart immunogenicity to small molecules atached to them, Animals nected wit this hapten-potein tnture wil make arog: tothe hapten ony covalent Unk to he carer protocol WV ves prtoca i). Anansi ‘will occur ony fhe sme conugate employed (an uneated arer wll ntsc) Because memory cells xth the T apd Testes are necessary. 2, Structural stability. Highly flexible molecules that have no fixed shape are poor immu- rnogens. For example, gelatin is structurally unstable and does not induce much of an immune response unless the molecule is stabilized by cross-linking peptide chains. 3. Route of immunization. As a general rule, the subcutaneous or intramuscular routes of antigen injection are best for soluble substances, whereas an intravenous injection may bbe more effective for cellular immunogens, such as erythrocytes or bacterial vaccines. In practice, the intravenous route is not uséd because there exists danger of embolism and other adverse effects. 4. Adjuvants. Nonspecific stimulation of the immune response can occur with the use of adjuvants. The mechanisms by which adjuvants exert their biologic effect are multiple (see Chapter 4 1V 8). a. Adjuvants can act as depots and prolong the period of exposure to the immunogen. b. The adjuvant may be an irritant that increases the inflammatory response at the site of immunogen administration, Some adjuvants may amplify the proliferation of immunologically committed cells. MOLECULAR DIFFERENCES IN EPITOPE STRUCTURE form the basis of the ex- uisite specificity of the immune response. [TA] itereces in portion of charged proups within a lecle datethe pelo the immune response products. 1. This aspect ofthe specificity ofthe immune response has been studied in relation to haptens, as illustrated in Figure 2-4 a. Haptens can be, for example, side chains of benzene rings, or substituted benzene rings. Attaching haptens to protein molecules renders them immunogenic. The im mune response can then be mounted against both the carrier protein and the hapten, b. All of the substituted groups shown are in the para position; however, the para substituted group is different in each case. (1) The immune response generated by any one of the conjugated hapten groups is cross-reactive with the others. That is, an antibody produced against one reacts with either of the other haptens. (2) The host can recognize the para position but not the substituent groups (Le., as long as these remain chemically similar). «. On the other hand, the host can distinguish between ortho and para positions (see Figure 2-4), and the antibodies formed are not cross-reactive. 2, The extreme specificity of the immune response is used in the identification of several biologically important molecules in human serum (Table 2-5). Differences in glycosidic linkages between monosaccharides also can be important in determining the immunologic specificity of antibodies.Amigos and immunogenicity | 31 CH Br a | Nip NMp NH e coon FIGURE 2-4. (4) Examples of cross-reactive substituted benzene haptens. Antibody rise against any one ofthe haptens shown wl crosseact withthe otter two haptens-A hapten with a Ca boxyl group in he para poston, however, would not react with COOH antibody agains! the iusrated haptns, 8) Examples of non Cross reacive subaltuled benzene haptens, The change from NH; Nhe parato ortho substtion produces a new spect. 1. A clinically relevant example is provided by the capsular antigens of pneumococcus, ‘which is one of the major causes of labar pneumor 2, Pneumococcus (a Streptococcus pneumoniae organism) has more than 80 different immunologic types of capsular polysaccharide. An antibody to one type does not react with an antigen of another type. The capsular polysaccharides are structurally different, which accounts for their antigenic differences, a. The type Il pneumococcal capsule has glucose in 1,4;1,6 linkages as its disaccha- ride building block. The antibody directed against pneumococcus type Il is directed against this glucose polymer. b. This specificity is known because the antibody reacts with glucose 1,4;1,6 linkages, regardless of where they are found (ie., it reacts with those found in glycogen). ‘c. The reaction is so specific that it can be used to determine if the 1,4:1,6 linkage ex ists in unknown polysaccharides. ifferences in amino acid residues in the epitopic region of proteins are critical to th immunologic specificity. J 1. Another clinically relevant example is the ability to distinguish very small changes in protein compounds, such as the various insulin molecules. 2. For example, patients with diabetes who use bovine insulin for maintenance sometimes become insulin tolerant, and the insulin is no longer effective. a. Insulin, a weak immunogen, has a molecular weight of approximately 6 kDa and is composed of two chains, an A chain and a B chain. Bovine and human insulin vary by three amino acid residues sitting side by side on the A chain . In insulin tolerance, antibodies are formed to this linear tripeptide epitope, and they inactivate the molecule. ‘c. The treatment for this antibody inactivation of the insulin is to switch from beef to pork insulin, because the three amino acids are different.TABLE 2.5. Detection of Hormones in Human Serum Based on Antibody Recognition of Specific Epitopes Molecular Pair Structure Digitoxin/Digoxin Digitoxin Thyroxine/Triodothyronine om, J smigncoon Thyroxine ‘Other examples: Comicosterone/Deoxycorticosterone Cyclic adenosine monophosphate! ‘oyclic guanosine monophosphate ge Digoxin y z Triiodothyronine Digoxin contains a hydroxyl group at catbon 12 Thyroxine has iodine atthe 5 position Of the second benzene ring, Corticosterone has a hydroxyl group at carbon 11 Cyclic GMP has an amino group on ‘carbon 2 and a change af amino ‘group for oxygen ue or)1. The differential diagnosis based on the patients signs and symptoms includes Ebstein-Barr virus (EBV), cytomegalo- virus (CMV), secondary syphilis, HIV infection, Iymploma, and Hodgkin's jisease. ‘A high percentage of patients with infectious mononucleosis who are given ampicillin or amoxicillin will develop an extensive morillform rash. W this occurs and EBV infection hhas not been suspected and, therefore, not tested for, the incorrect conclusion may be drawn that the patient is aller- ic to penicillin, 2. Downey cells are abnormal Iympho- ‘tes that have a foamy cytoplasm. ‘They are characteristic of infectious mononucleosis and probably reflect the tropism ofthis group of viuses for cells ofthe lymphocytic series. EBV is ‘one cause of infectious mononucleosis. Jn response to the vius, the patient produces several diferent antibodies, Some of which will neutralize the vius and hasten recovery. ‘One of the antibodies produced dur- ing the infection agelutinates sheep erythrocytes because of the presence of a shared (heterophil) epitope between the red blood cell and the virus. A re- fated virus, CMV, causes a similar dis- ‘ease but does not induce the produc- tion of these same antibodies. 3. Heterophilic antibodies are those that are induced by one immunogenic stimulus and react with the inducing agent as well as with unrelated ant gens because of the presence of com- ‘mon (shared) epitopes, Antigens ad immunogenic 4. Heterophilic relationships are use- ful in the serologic diagnosis of various infectious diseases and reptesent a more convenient, and Usually less expensive, test than ‘would be possible using the infec- tious agent itself. Two commonly used antigens are cardiolipin, used inthe diagnosis of syphilis (which is caused by the spirochete Treponema pallidum, which can- not be cultivated in the laboratory) and the antigens that react with antibodies in rickettsial diseases, such as Rocky Mountain spotted fever, Ricketsal pathogens are also dificult to grow. 5. The Forssman antigen could be responsible for the production of anti-A isoagglutinins in individuals ‘with blood groups O and B. For ‘example, this heterophil antigen is present in corn and could be im- ‘muunogenic once absorbed from the intestinal tract. Another possi- ble stimulus could come from bacteria that have the Forssman antigen in their cell walls. If these ‘microbes colonize the mucous ‘membranes of the body, they ‘could induce an anti-A fesponse ‘The A antigen is not found in peo- ple with blood group O or B; therefore, these individuals would respond to the antigen by produc- ing homologous isoagalut 33Case 2 The director of a hospital laboratory must prepare an antiserum specific for gentam- icin to use in radioimmunoassay (RIA). A staff physician would use the antiserum to measure the level of the antibiotic in patient blood samples. The most cost-effective way to provide this service would be for the laboratory to produce its own antise- rum reagents, ‘A protocol for producing the anti-gentamicin antibody was found and considered for use by this laboratory: 1. Inject an adult rabbit intramuscularly with 5 mg of gentamicin in sterile physiologic saline. 2. Repeat the injection 10 days later. 3. Test bleed the rabbit 10 days after the last injection and determine the anti- gentamicin titer of the serum by RIA. 4. If the antibody titer is low for efficient use in RIA, repeat steps 13 until a satisfactory antiserum is reached, QUESTIONS 1. Why would you not be surprised ifthe rabbit did not respond to the gentamicin? 2. What can be done to increase the titer of the antibody in the serum? 3. Why might it not be a good procedure to use only one rabbit in the protocol you have followed? DISCUSSION a4 Gentamicin is a hapten and, therefore, unable to elicit a response in the rabbit. Although the gentamicin could combine with carrier proteins in the rabbit, the probability is slim. IF it did, you would not expect to see a high serum titer but rather a cell-mediated response. The hapten should be chemically conjugated to a high-molecular-weight cartier protein, such as bovine serum albumin (BSA) or, even better, keyhole-limpet hemocyanin (KLH). The gentamicin-BSA/KLH would still not produce the desired response because soluble antigens are, as a rule, not highly immunogenic. An adjuvant such as Freund's complete adjuvant (FCA) should be given intramuscularly for the first injection, followed by at least {wo more injections in saline given subcutaneously before the frst test bleed. Sometimes not enough animals are used in the immunization protocol because of the desire to save money, and time is wasted. At least two rabbits should have been used, and preferably three, since the out-bred rabbits do not respond in a uniform manner and the tone rabbit used might not have been capable of responding strongly to this particular antigen.Amigens and nmunogenicty | 35 stupy questions DIRECTIONS: Each of the numbered items or incomplete statements in this section is followed by answers or by completions of the statement. Select the ONE lettered answer ‘or completion that is BEST in each case. 1. An antigen that occurs in phylogenetically 4. The capacity of a molecule to react speciti- Unrelated species i elerred to as cally with a product of induced lympho cell {A) allogeneic differentiation is known as oo (A) specificity (©) isogenele (8) antigen ester (C) affinity eee (D) avidity 2. The sites in or on antigens with which anti-._(E) immunogenicity bodies react are called 5. Substances that are immunogenic only (A) haplotypes when coupled to a protein cartier are called (B) isotopes (C) epitopes (A) opsonins (D) idiotypes. (B) haptens (C) adjuvants 3. To be immunoreactive, an epitope must be (D) toxoids (A) part of a globular protein (E) epitopes (B) linear (C) electronegative (D) spatially accessible (E) part of a glycoprotein ECTIONS: The incomplete statement in this sect by the capitalized word. nis negatively phrased, as indicated ect the ONE lettered answer or completion that is BEST. 6. The degree of immunogenicity is influenced by all of the following EXCEPT (A) molecular size (B) degree of foreignness (C) electronegativity (D) chemical composition (E) presence of adjuvants36 [orp 2 DIRECTIONS: Each set of matching questions in this section consists of a list of four to ‘twenty-six letlered options (some of which may be in figures) followed by several ‘numbered items. For each numbered item, select the ONE lettered option that is most closely associated with it. To avoid spending too much time on matching sets with large numbers of options, itis generally advisable to begin each set by reading the list of options. Then, for each item in the set, try to generate the correct answer and locate it in the option list, rather than evaluating each option individually. Each lettered option may be selected fonce, more than once, of not at all Questions 7-9 Questions 10-12 For each disease, select the most appropriate Match each characteristic to the most appro- antigen, priate substance. (A) Cardiolipin (A) Penicilloyl moiety of penicillin (B) Sheep red blood cells (SRBCs) (B) Adjuvant (C) Thyroglobulin (C) Carrier protein (D) Proteus vulgaris (D) Lipopolysaccharide (E) Myelin basic protein (E) Lipoprotein (F)_ Streptococcus pyogenes (F) Forssman antigen (G) Double-stranded DNA (G)_ Type Il pneumococcal carbohydrate cap- (H) Urushiol sule (H) Hemocyanin 7. Systemic lupus erythematosus 10. Outer membrane protein of the meningo- 8. Infectious mononucleosis coccus 9. Poison ivy 11. A hapten that is too small to be immuno- genic 12. A heterophilic antigen found in corn and human type A Rh-positive red blood cells,Amgen and immavogenicty | 37 BBANSWERS AND EXPLANATIONS 1. The answer is C [Il B 2]. Heterogenetic, or heterophilic, antigens occur in phylogeneti- cally unrelated species—that is, in widely sep- arated species in the plant or animal kingdom, (0f across kingdoms, The Forssman antigen, for ‘example, is found on sheep red blood cells (SRBCs), guinea pig tissues, corn, and certain bacteria. Allogeneic antigens are present in some, but not all, members af a species. In- bred animals are said to be antigenically syn- geneic. Antigens that can generate an immune response in genetically different members of the same species but not in the member carry- ing it are called isogeneic. 2. The answer is C (18). The sites on antigens with which antibodies react are called epi- topes, or antigenic determinant groups. An epitope comprises approximately four to six amino acids or monosaccharide unis. Epitopes are distributed throughout the surface of an antigen and appear in a repeating manner, so that any particular epitope appears on the anti- gen surace several times. In addition, a given antigen is likely to have several immunologi- cally diferent epitopes; for example, bovine serum albumin has seven different immunoiog- ically specific epitopes. This array of epitopes is refered to as the epitope mosaic or determi- nant group mosaic of an antigen. The valence Cf an antigen is the total number of epitopes on that antigen tht is, the number of antibody molecules with which that particular antigen potentially could react. 3, The answer is D [1 B 2]. An epitope must be on the outside of a molecule if the anti- body is to react with it, Otherwise, spatial in- terference prevents the molecular interaction. Initiat surface accessibility is not necessary for immunogenicity, however, because the cell or molecule may be broken down somewhat ‘during phagocytic cell “processing.” This ex- poses internal antigens or epitopes to the im= ‘munologic apparatus of the host. 4. The answer is B [1A 21. Antigenicity is de- fined as the capacity of a molecule to react with the immunologic product that it induced {ie either a humoral antibody ora sensitized iymphocyte. The term antigenic” also often is used to describe a molecule that can induce an immune response. itis more accurate, however, to use the term “immunogenic” to describe molecules that are able to induce immune responses because some molecules (eg, haptens) can react but are not able to induce an immune response—that is, they are genic but not immunogenic. Affinity and avidity are terms that describe the strength of interaction between antigens and their homol- ‘ogous antibodies. 5. The answer is B [Il D 3]. Haptens cannot induce the production of antibodies unless they are coupled to a carrier. They are able to react with the antibodies once formed but are rot immunogenic by themselves. Complex hhaptens react with their homologous antibody ina visible manner, usually precipitation. The reaction of antibodies with simple haptens can be detected only indirectly (eg., in agglutina tion inhibition assays}. Opsonins are complex molecules, usually proteins (e.g., antibodies), that sensitize foreign materials to phagocyto- sis, Adjuvants enhance the immunogenicity of substances. Toxoids are chemically modified toxins that are still immunogenic but have lost their toxicity 6. The answer is C [ll A, E 4]. The charge of the molecule does not aifect its immunogen ity. The relative ability of a molecule to in duce an immune response (i... its degree of immunogenicity) is influenced by its molecu lar size, chemical nature, and degree of for- eeignness. These attributes are not simply addi- tive, nor directly factorial, since a molecule of limited size may be highly immunogenic if it has sufficient complexity. Nonspecific stimula- tion of the immune response can occur with adjuvants, which can enhance immunogenic: ity without altering chemical composition. 7-9, The answers are: 7-G [II C 1 e}, 8-8 (IIB 2b (2), 9-H [ILD 4 b (2)). The detection of antibodies against double-stranded DNA is serologic evidence of autoimmunity. There is 2 high correlation with the presence of these antibodies and the disease systemic lupus erythematosus, Patients with infectious mononucleosis caused by the Epstein-Barr virus commonly produce an antibody that agglutinates sheep red blood cells (SRBCs). Cytomegalovirus ‘causes a similar disease but does not induce this same heterophilic antibody. ‘The poison ivy plant has in is leaves a cate-3B [Chane 2 cchol called urushiol, which is a potent aller- ‘gen that induces a cell-mediated immune re- sponse in most individuals on skin contact 10-12. The answers are: 10-C {Il D 3 ¢(1)], 11-A {ILD 4b (1)}, 12-F IIB 2; Table 2-1) Haemophilus influenzae type b is nonimmu- rnogenic in children younger than 2 years. If the molecule is attached to a carrier protein, such as outer membrane proteins of the men- ingococcus or tetanus toxoid, it gains immu- nogenicity.. The antibiotic penicillin is a common aller- gen. Itis estimated that more than 20% of the United States population is allergic to this an- tibiotic, The penicilloyl moiety is often the epitope that is recognized by the immune sys- tem. The Forssman antigen is a heterophile anti- gen found throughout the phylogenic tree. It is likely to be the antigen that induces blood group O and 8 humans to produce anti-A iso- hemagglutinins,Chapter 3 Immunoglobulins Vignette Some antibodies have a unique ability 3: Is there any survival benefit to to pass through physiological barriers this selective secretion? that are impervious to other immuno- slobulins, which greatly increases their 4, How does secretory IgA get Concentration in certain body fluids. into secretions like colostrum? 5. Does this have any survival 1. Why does this happen? benefit? 2. Why is IgG the only matemal anti- ‘body that crosses the human. placenta’ Is the placental barrier ‘operative in other species? IMMUNOGLOBULINS (Ig), or ANTIBODIES (Ab), are glycoproteins present in the ‘gamma-globulin fraction of serum. The concentration of immunoglobulins in the body is divided equally between the intra- vascular and extravascular (primarily lymphatic) compartments. Every day, 25% of the antibody concentration exchanges as the tissues are bathed in plasma proteins. Immunoglobulins are produced by B lymphocytes (B cells) or plasma cells in response to exposure to an immunogen. They react specifically with that antigen in vivo or in vito; therefore, immunoglobulins are a part of the adaptive immune response (specifically, hu- ‘moral immunity) GENERAL IMMUNOGLOBULIN STRUCTURE. The basic structural unit of an im- _munoglobulin molecule consists of four polypeptide chains linked covalently by disul- fide bonds (Figure 3-1). The four-chain, monomeric immunoglobulin structure is com- posed of two identical heavy (H) polypeptide chains and two identical light (L) polypeptide chains. Ad} Heavy (th and light (1) chains 1. H chains have a molecular weight of 50 to 75 kilodaltons (kDa), which is approxi- mately twice that of L chains. H chains contain approximately 400 amino acids, which is twice the number in L chains. ‘a. Amino acid differences in the carboxy-terminal portion of the H chains identify five antigenically distinct H-chain isotypes, which form the basis for the five classes of immunoglobulin molecules (Table 3-1), b. H-chain classes + and «are subdivided into subclasses of molecules (see Table 3-1) ‘The subdivision is based on the greater similarity of amino acid sequence shown by 3940 | chapter 5A ‘Variable domain ‘otheavy chain arable domain ight chain Constant domain light chain Joining region votod ae versity region Joining region “ Hinge region Constant domains __ of avy enain| wowod ag Carboxy termini ght chain ‘of amigen FIGURE 3-1. Basic unit (monomer ofthe IgG molecule, consisting of four polypeptide chains linked co- valenily by disulfide bonds (5-5), with intrachain disulfide linkages as well, The loops correspond to do- ‘mains within each chain. V = variable domain; C = constant domain; L = light chain; H = heavy chain; CHO = carbohydrate side chain. The inset shows the hypervatiable complementarty-determining regions {as shaded. The framework regions of each variable domain in the paratopic region are not shaded. Similar hypervariable regions are found inthe a and B chains ofthe T-cell receptor for antigen,tren | 41 TABLE 3-1. Properties of Human Immunoglobulins eG eA tM gD tg | Hehain ¥ @ * 8 € H-chain subclasses Wye Yee ray = > H-chain allotypes* Gm (25) Am (2) ‘Molecular weight (kDa) 150 160-400" 900,180,190 J.chain = + + a Carbohydrate (%) 3 7 12 ae ‘Serum concentration (meal) 1200 200 130 3 005 Serum half-life (days) at 6 10 3 2 Functions ‘Complement activation ++ - Che = (classic pathway) Opsonization thet ' - So 6 Antiviral activity = tee + = 2 Mast cel sensitization - - - + chin = bea chain chain ~ joining chain "he number in prentheres = he amber of now allahypes, Al merunoglobulins have Ke chain alltypes, {igo3 hae hale ot? ys "Virani polymere forms found in serum, subclasses of the same class than is shown by different classes. The H-chain sub- classes determine immunoglobulin subclasses; 1 corresponds to 1gG1; a1 to IgA1, and so forth, 2. L chains are composed of approximately 200 amino acids, They are of two types—x or A—based on their structural (antigenic) differences. a. All immunoglobulin classes have both x and 4 chains. However, a given immuno- slobulin molecule may contain either two identical « chains or two identical chains, but never a « chain and a chain combined. The proportion of « to X chains in human immunoglobulin molecules is approximately 3:2 b. A chain subtype. There is no isotypic variation in x chains. However, there are four distinct A chains, giving rise to four distinct subtypes (so called to avoid contusion with the H-chain subclasses that distinguish immunoglobulin isotypes). All four subtypes are present in each of the immunoglobulin classes. fide (-S-$-) bonds hold together the four polypeptide chains in immunoglobulin mol- ecules, There are two types of disulfide bonds. 1. Interchain bonds occur between H chains (H-H), between H and L chains (H-L), and between L chains (L-L) isulfide bonds between heavy chains occur primarily in the hinge region of the immunoglobulin molecule (see I! G), but they also occur in the carboxy-terminal portion of the heavy chain. There can be from 1 to 15 interchain disulfide bonds, depending on the class and subclass of the immunoglobulin molecule b. Only one disulfide bond connects H and L chains. Bonding between heavy and Tight chains occurs in all immunoglobulin molecules except IgA2m(1), which lacks an interchain disulfide bond. . Single light-chain bonds occur in IgA2m(1). Such bonds can also occur under pathologic conditions (e.., in the Bence Jones protein seen in the urine of some patients with multiple myeloma). 2, Inteachain bonds occur within an individual chain, and they are stronger than inter- chain bonds, The number of intrachain disulfide bonds varies depending on the num- ber of domains in the molecule. a. Light chains have two intrachain bonds. b. Human 7, a, and 8 heavy chains have four intrachain bonds. ‘¢. Human p and € heavy chains have five intrachain bonds. 342 | chaper 3c Each H chain and each L chain has a variable (V) and a constant (C) region. The V re- gion, which lies in the amino-terminal portion of the molecule, shows a wide variation in amino acid composition. Except for minor inherited differences, the C region, which lies in the carboxy-terminal portion of the molecule, demonstrates a much more uniform (con- stant) amino acid sequence. 1. The V regions associate with appropriate constant regions, so that a variable H-chain region (V4) does not occur in an L chain, and vice versa. However, a particular Vi- chain sequence may occur in more than ‘one H-chain class (i.e., y, @, 1, 8, €). Thus, during class switching in an immune response, such as when B ceils change their pro- duction from to y heavy chains (ie., from producing IgM to IgGl, only the constant portion of the H chain changes and the antibody specificity remains the same. 2. Hypervariable regions are particular areas within the variable regions that are highly variable in amino acid sequence. These hypervariable regions, often called comple- ‘mentarity-determining regions (CDRS), occur at similar amino acid positions in an oth= erwise relatively invariant molecule. They are short polypeptide segments lying near amino acid positions 30, 50, and 95 in the variable regions of both L and H chains a. The hypervariable regions are important in the structure of the antigen-binding site (ie, the paratope). L chains have three hypervariable regions and H chains have {our (although only three of the four have been associated with epitope recognition) b. There is extreme variability in the amino acids found in the hypervariable regions (Figure 3-2). However, the peptide sequences that intervene between the hypervari- able regions, called framework regions (FRs), are relatively constant in amino acid sequence. consists of a series of globular regions, or domains, enclosed by disulfide bonds. These domains comprise the major part of the V and C regions. cont [cone| ‘cons| FRI FR2 FAS FRA Variability ‘Amino acid number FIGURE 3-2, Amino acid variability in the variable region of the light chain of an immunoglobulin mole- cule. The variability index used is an arbitrary scale ofthe number of different amino acids found in each position if 100 diferent light chains were analyzed, FR = framework region; CDR = complementarity Setermining regionImmurogabulns | 43 1. Domains consist of approximately 110 amino acid residues. Peptide loops of 60 to 70 ‘amino acid residues enclosed by intrachain disulfide bonds represent the central portion of each domain. The amino acid sequences of the loops show a high degree of homology (i.., the sequences are very similar) 2. Each H chain has four or five domains: one in the variable region (V,,) and three or four in the constant region (C1, C2, C43, and C4). The +, a, and 8 H chains have four domains (one variable and three constant). The 1 and € H chains have five do- ‘mains (one variable and four constant) 3. Each L chain has two domait region (Cy) ne in the variable region (V,) and one in the constant ‘The paratope is the area of the immunoglobulin molecule that interacts specifically with the epitope of the antigen. The paratope is formed by a very small portion of the entire immunoglobulin molecule. Folding of the polypeptide chains brings the hypervariable regions of the Vj, and V domains into close proximity. This folding cteates a three= dimensional structure that is complementary to the epitope (see Figure 3-1). ‘The hinge region is the portion of the H chain between the C,,1 and C,,2 domains (see Figure 3-1), There is no homology between the hinge and other immunoglobulin heavy chain domains, and its sequence is unique for each immunoglobulin type and subclass. IgM and IgE do not possess a hinge but have one more C,, domain, 1. In this region, interchain disullide bonds form between the arms of the Fab fragments, preventing them from folding and, therefore, rendering this portion of the molecule ly susceptible to fragmentation by enzymatic attack. 2. The hinge region is highly flexible and allows for movement of the Fab arms in relation to each other. This mobility explains why native antibody molecules do not activate complement, whereas those in an immune complex do. The native molecule is not in the appropriate configuration. Fab and Fe fragments. Early studies of immunoglobulin structure used proteolytic en. zymes, particularly papain and pepsin, to degrade immunoglobulin molecules into defin- able fragments. 1. Papain splits the monomeric basic unit into three fragments of approximately equal size atthe hinge region. It breaks the heavy chains above (i.e., on the amino-tert side) the interchain disulfide bonds (see Figure 3-1). a. Two Fab fragments (fragment antigen-binding) each contain an entire L chain and the amino-terminal half of the H chain. The Fab portion of the H chain, which contains the Vj, and C41 domains, i called the Fd fragment. A Fab fragment is monovalent; thats, it possesses only one antigen-binding site. Therefore, unlike bivalent molecules, a Fab fragment Cannot facilitate antigen precipitation or aggluti- nation b. One Fe fragment (fragment erystallizable) comprises the carboxy-terminal portion of the H chain. The two heavy chains are held together by disulfide bonds. The Fe por- tion of the molecule has several properties: (Q) Ie binds the Ciq component of complement and activates the complement cas- ‘cade (see Chapter 41 1). 2) It contains carbohydrate G) It reacts with Fc receptors on various cells ofthe body; therefore, it dictates logic activities of the molecule {e.g., whether a given immunoglobulin can cross the placenta, sensitize mast cells to degranulate when they contact allergen, or ‘opsonize bacteria and other cellular elements for phagocytosis (Table 3-2). 2. Pepsin digests away most ofthe Fc fragment below (on the carboxy-terminal side) the interchain disulfide bonds in the hinge region (see Figure 3-1), leaving one large piece termed the Flab')2 fragment, which consists of two Fab fragments joined by disulfide bonds. This fragment has two antigen-binding sites. Thus, itis bivalent, possessing the ability to bind and precipitate or agelutinate an antigen.44 (cuter 3H TABLE 3-2. The Distribution of Receptors on Various Cells of the Body and the Biologic Significance of Their Presence Immunoglobulin Cellular i | Receptor presence Biologic significance Fea Neutrophils ‘Opsonization: protection of body surfaces by inhibition of adherence Cf microbes; neutralization of toxins and enzymes Poly-lgk Secretory Mucosal Serves as a receptor for dimeric IgA ‘component epithelial cells and facilitates passage through the of IgA cell and secretion of IgA dimers in sigA form | Fee Ise Basophils Involved in atopic allergies; cross- Mast cells linking in cell membrane by allergen triggers degranulation and release of vasoactive compounds Eosinophils kills metazoan parasites Fey IgG Neutrophils (+) Opsonization; different receptors ‘Monocytes (++) react with varying avidity with indi vidual antibodies, thus Fe, Ril reacts | with IgG, 3, & 4, but not 2 Natural killer (NK}_ ADCC cells Trophoblasts Responsible for IgG transport across the placenta, | Few IgM Monocytes Opsonization (2) Fe = fragment ceyalizable; g = lnmunoglbulin; R = receptor. There are diferent eceptos for may of hve Gente by Roman mums ©, Fel co on 30th Immunoglobulins are glycoproteins. From 3% to 13% of the immunoglobulin molecule is composed of oligosaccharides, which usually are attached to the immunoglobulin at one ‘or more locations in the C,, regions (i.e, the C}2 or C3 regions) 1. N-Glycosidic bonds usually link an N-acetylglucosamine in the carbohydrate moiety to an asparagine residue in the polypeptide chain at the Cj region. The linkage occurs in the Golgi complex via N-acetylglucosarnine-asparagine transglycosylase, a transferase enzyme, 2, The half-life of antibodies in the circulation (see Table 3-1) depends on the status of the oligosaccharide side chains. 1. The oligosaccharide side chains terminate in galactose, to which sialic acid is fre- quently attached. When antibodies in the circulation have the sialic acid removed by enzymes such as neuraminidase, they become susceptible to degradation in the liver. b. The terminal galactose binds to a receptor on hepatocytes and the entire molecule is internalized by the cell. Degradation then occurs because of the proteolytic en zymes in the lysosomes ofthe cell J chains are small proteins that connect the two or more basic units in polymer noglobulins (.e., in IgM, IgA). They are named J chains because of their je 1. Structure. The J chain is a glycopeptide chain that is covalently linked to the carboxy- terminal portions of the H chains. itis synthesized by B lymphocytes during maturation and is incorporated into polymeric immunoglobulins before they are secreted from the plasma cell.Immunoglobulins J 45, 2. Function, The J chain is thought to be involved in the synthesis, polymerization, and secretion of IgA and IgM polymers. Membrane immunoglobulins, Presumably all immunoglobulins exist in both membrane and secreted forms. In the membrane they function as antigen receptors. IgM and IgD are found in the membrane of mature B lymphocytes. After the cell interacts with antigen, it proliferates and matures into a cell that is actively secreting immunoglobulin, The switch from membrane-bound monomeric IgM to secreted IgM is accompanied by an increase in J-chain synthesis. Membrane forms of IgG and IgA are thought to function as antigen re- ceptors on memory B cells. The membrane forms of the immunoglobulins are larger than their serum counterparts because of the presence of transmembrane and cytoplasmic ‘components attached to the carboxy-terminal end of the heavy chain. The added struc- tures are encoded by two exons located at the 3’ end of the heavy chain gene. Immunoglobulin superfamily. The immunoglobulin molecule is a member of a very large family of proteins that share a similarity of structure and function and have arisen from a common ancestral gene. 1. Structure. Structurally, all members of the family have significant amino acid sequence homology and contain at least one domain. A domain is a segment of a polypeptide chain that is folded together and stabilized by disulfide bonds. 2. Function, The members of this superfamily are involved in cellular interactions. Most ‘occur in cell membranes and serve as recognition molecules. Some of the members of the family are listed in Table 3-3. ADDR structure ano FUNCTION OF SPECIFIC IMMUNOGLOBULINS ‘A.] Isotypic variation, Immunoglobulins fall into five classes (isotypes), based on certain structural differences (see Table 3-1). Each class also has certain unique biologic and chemical properties, ‘TABLE 3-3. Members of the Immunoglobulin Superiamily [Mote Immunoglobulin T-cell receptor MHC Class tee and 6 Class a 82 microglobulin © 1,2, 3,4, 8, and others Other cell LAR receptors Poly-tgR FeyRI (CD = chat of ciferemiaion antigens fund on lymphocytes ad aber (ely the bay Fe = rapmentenstalizble, fg = rane: >] [o]0) Jo)-)-)5)-]-]o]0) 12bases 2a bases ‘Spacer Heptamer lo|]o]-Jop>Jo _, Site o Vid recombinase action NS ve [ins] us | ns FIGURE 3-7. A schematic representation of the joining of immunoglobulin x chain variable (V) and joining ()) gene segments. The alignment of the heptamer and nonamer components of the DNA strand is essential for gene recombination, The 2-base strand makes one ful turn around the 12-base strand, which brings the recombination signal sequences together. Then, the enzymatic cleavage and ligation of the DNA strand. ‘occurs and the two genes are joined. A= adenine: C= cytosine; G = guanine; INS = intervening non- ceoding sequences (introns); T'= thymidine ins | vs | ws | vy \ OPP PP PEELC b. During T-cell development in the thymus, the genes for the T-cell receptor undergo [DNA rearrangements much like those for the immunoglobulin molecule. (1) The variable region of the T-cell receptor’s e chain involves rearrangements of V and J gene segments. (2) The variable region of the receptor’s B chain involves rearrangements of V, J, and D gene segments, Mechanisms contributing to antibody diversity 1. Chance recombination creates a large amount of antibody diversity 2. Ifall events occurred randomly, somatic recombination in the DNA followed by RNA splicing (see Figures 3-5 and 3-6) could produce: (1) More than 1000 varieties of x L chains Q) A similar number of X L chains (G) Perhaps as many as 20,000 varieties H chains56 | chaper 3 v8 bb, The random combining of H and L chains greatly expands the immunologic reper- toire, 2. Imprecise joining of the V, D, and J genes can also be a source of immunoglobulin diversity. Whenever genes join, imprecision in joining can occur. 3. N-region additions can produce changes in the specificity and reactivity ofthe immu- noglobulin molecule, a. N regions are very short peptides of variable sequence, often found near the third hypervariable region of the H chain (1) This eegion, the most variable region of the immunoglobulin molecule, contains amino acids encoded by the Dy, gene region. (2) Furthermore, because the Dy, segment is very small, the N region contains two joining boundaries, where exons V and | join D at opposing ends. b. Immature lymphoid cells contain an enzyme, terminal deoxynucleotidy|trans- ferase, which catalyzes the generation of N regions by the addition of nucleotides at the 3” end of the DNA strands. 4, Extensive mutation involving variable-region genes produces even further diversity. a. Mutations can occur by several mechanisms. (1) Point mutations. These single-nucleotide substitutions can occur in the variable region of a functional immunoglobulin gene. (a) Most mechanisms that generate diversity are active before any exposure to antigen, (b) The somatic mutation theory of antibody diversity suggests that a small number of genes diversify during lymphocyte differentiation—that is, in the pre-B-cell or B-cell stage—either by point mutation or by recombination events, @_ These mutations occur after the cells have encountered antigen, espe- Cially during intensive immunization. ‘The nucleotide substitutions are found only in variable domains of either H or L chains, predominantly in the hypervariable regions, (2) Gene conversion. in some immunoglobulin gene families (e-g., chicken d-chain Benes), it appears that stretches of nucleotides are translocated from one V-gene segment to another in the same V locus. This process may also contribute to the diversity of mammalian variable domains. . The antibodies produced by mutations in variable-domain genes may confer a selec tive advantage to the lymphocyte if they possess a higher affinity (i.e., stronger at- traction) for the antigen. {0) Cells coated with highafinity antibody are better able to capture the immuno- gen and perpetuate the immune response, (2) A characteristic of prolonged immunization is the increase in antibody affinity for the antigen as the duration of exposure to the antigen increases, because the longer time of exposure increases the chance that a “good” mutation will Immunoglobulin class switching (isotype switching) 1. During the immune response, plasma cells switch from producing IgM to IgG or to another immunoglobulin clas 2. There is alteration inthe L chain or in the variable portion of the H chain; thus, there is no change in antigen-binding specificity. 3. The switch involves a change in the H-chain constant domains (C,,). a. Chromosome 14 contains C,, gene sequences for the nine H-chain isotypes; these ate designated Cy, Cy, Cyr Ce and so forth b. When the Hechain gene is assembled, all but one of the Cy, gene sequences is se- lected, thereby producing the desired isotype. When constant-region genes loop outIrvmanoglobatns | 57 during the switch, the cell loses its ability to produce that immunoglobulin class. ‘Thus, a cell that has switched to IgG will not be able to revert to IgM synthesis, but could switch again to IgA or IgE, for example, because these gene loci are down- stream from the IgG loci . In the process, DNA rearrangement and RNA splicing place the remaining Cy gene sequence adjacent to the V/D/} exon (see Figure 3-7) 4, Repetitive sequences in H.-chain genes permit immunoglobulin class switching 2. The switch is accomplished by recombination between special repetitive switching sequences inthe intron upstream from the constant-region exons. b. The transcriptional enhancer in the intron between the J, cluster and C, exon is upstream of the switching sequence. Therefore, every switch carries the enhancer with it, and active transcription is ensured regardless ofthe H-chain class expressed, Nonproductive rearrangements 1. The H or L chains are formed during the pre-B-cell phase of tion, 2. In the formation of a functional gene, DNA segments must be joined properly to ‘ensure that the correct reading frame is maintained. (1) Ifthe joining is erroneous (off by one or two nucleotides), the downstream se- quences are out of frame, preventing translation into a functional polypeptide. (2) Such erroneous rearrangements occur frequently, and in this case the cell con- tinues to rearrange and join its immunoglobulin gene segments until a func tional immunoglobulin is made; rearrangement then ceases, 'b. The appearance of an intact H chain in the cytoplasm seems to be the signal for the fend of H-chain gene shuffling. . Its also the signal for commencement of L-chain gene rearrangement. d. The trial-and-error process is repeated until a functional L chain, either x or d, is produced. smunologic matura- 2. The pre-B cell then enters the B-lymphocyte stage of development, in which the H and L chains are joined by disulfide bonds and are expressed in the B-cell mem- brane. 3, Itis likely that many lymphocytes have nonproductive rearrangements and are of no value to the immune system. This appears to be the price that must be paid to preserve the generation of immunologic diversity by such an elaborate system Allelic exclusion and clonal rest producing cells. tion. These phenomena are peculiar to antibody- 1. Allelic exclusion is the expression, in a single cel, of only one allele ata particular locus. The process of allelic exclusion results in the synthesis of molecules with identi- cal variable regions because the expressed messenger RNA (mRNA) is derived from a single chromosome. Therefore, the antibodies produced by each B lymphocyte are identical in thei specificity a. Two or more alleles frequently exist at the genetic loc forthe various components of H and U chains b, Because of alielic exclusion, only one H-chain gene and one L-chain gene can be expressed in any given B lymphocyte (1) In individuals who are heterozygous for allotypic forms of H or L chains, indi- vidual 8 cells express one or the other allele, but not both (2) When a single H-chain gene or L-chain gene is successfully assembled and ex- pressed, all other genes of that type are prevented from undergoing rearrange- ‘ment in the same cell5B [chapter 3 WE 2. Clonal restriction ‘a. On its surface each B cell expresses multiple identical copies of an antibody that is, specific for a single epitope. b. When a B cell divides, the chromosomes in its progeny cells bear the selected al- lelic genes, and these genes do not undergo any further V/J or V/D/} rearrangements This is why all of the immunoglobulin molecules produced by a given clone (a B lymphocyte and its progeny) are identical in epitope specificity and in « or A chain isotype. PLASMA CELL DYSCRASIAS represent a group of diseases characterized by the over production of immunoglobulins, or their fragments, by a single clone of plasma cells. The abnormal monoclonal product is called M component (paraprotein) Fy oven 1. Clinical effects. The most important of the plasma cell dyscrasias is multiple myeloma. Also notable are Waldenstrém’s macroglobulinemia, benign monoclonal gammopathy, primary amyloidosis, and heavy chain disease. These diseases cause malfunction of the immune system with resultant increases in susceptibility to infec- tious disease. 2. Therapy for a plasma cell dyscrasia includes the following a. Administration of prednisone, with or without cytotoxic drugs (e.., cyclophospha- mide) b, Removal of tne excess protein eM in Waldensrbm's macroglobulineia) via plasmapheresis, le myeloma. This plasma cell tumor in bone marrow overproduces a single class of immunoglobulin; most cases involve IgG 1. Characteristics a. The most characteristic feature of multiple myeloma is the demonstration of M com= ponent in the blood, urine, or both. The M component consists of intact monoclonal antibodies (usually IgG or lgA), H chains, or L chains, alone or in any combination, b, In some cases, a substance called Bence Jones protein, which is a dimer of L chains, is found in the urine. This abnormal immunoglobulin fragment is a pyro- ‘globulin and can be irreversibly precipitated by heating acidified urine to 56°C. 2. The clinical features and complications of multiple myeloma vary widely. Some of the more common manifestations include: a. Fatigue and weakness ». Bone pain and pathologic fractures ‘¢. Bone marrow iniiltation by an abundance of abnormal plasma cells with resultant normochromic, normacytic anemia 4d. Renal abnormalities, which possibly result in chronic renal failure “C] Waldenstromys macroglobulinemia isa lymphocytic lymphoma characterized by overpro- luction of lymphocytes and plasma cells. 1. Characteristics, The most distinguishing feature ofthis disorder isa high level of mono Clonal IgM in the serum. Bence Jones proteins also may be seen in the urine. 2, Clinical features and complications. Because of the high serum IgM, patients have a hyperviscosity of the blood, which may be severe. Anemia, Iymphadenopathy, and chronic lymphocytic leukemia also are common clinical features,Immunoglobulins | 59 ‘Amyloidosis is a disease characterized by deposition of an abnormal protein (i. amy- loid) in the vascular endothelium of various organs of the body. The clinical features de- pend on the site and extent of amyloid deposition. 1. Two types of amyloidosis have been defined a. Primary amyloidosis is a plasma cell dyscrasia characterized by the accumulation of amyloid fibrils, which are composed primarily of immunoglobulin L-chain fragments, and are therefore called amyloid light chain (AL) protein. This type of amyloidosis Often is associated with multiple myeloma, b. Secondary amyloidosis refers to amyloidosis that accompanies or follows a chronic infection or inflammation. The amyloid fibrils in secondary amyloidosis, called amy- loid A (AA) protein, bear no structural resemblance to the AL protein seen in pri mary amyloidosis, 2. Clinical features. Amyloid deposits, whether they are AL or AA, are very resistant to proteolytic degradation, and therefore are not cleared from the site of deposition. The accumulation of amyloid in tissues thus is ireversible, leading to cell death and tissue necrosis, The effect depends on the site of deposition 2 In primary amyloidosis, amyloid is deposited where the tumor cells are located b. In secondary amyloidosis (.e., reactive amyloidosis), the deposits are found in the Ther splot and Kelneys This for of amylidoa associ with sutommnse diseases and with chronic infectious processes such as tuberculosis. Heavy chain diseases 1. Characteristics. These rare malignancies are characterized by a serum paraprotein con- sisting of incomplete H chains without L chains; ¥, a, and x heavy chain diseases have been described. 2. Clinical features. The abnormal Hi chain shows deletion of the hinge region, or partial deletion of the Fd portion, or a combination of these abnormalities. Errors in immunoglobulin gene rearrangement are thought to contribute to the genes of several B-cell malignancies. 1. Follicular lymphoma a. In many patients with follicular Iymphoma—the most common B-cell cancer—a putative proto-oncogene (called bci-2) on chromosome 18 is translocated into the H-chain gene region on chromosome 14. b. Its thought that the close proximity of bcl-2 to the active H-chain gene region en- hances the expression of the proto-oncogene and, thus, contributes to malignant transformation, 2. Burkit’s lymphoma a. In the malignant cells ofthis B-cell cancer, a portion of chromosome 8 containing the cellular proto-oncogene c-myc is translocated into chromosome 14 so that ‘c-myc lies right next to the H-chain gene region. b. The c-myc proto-oncogene is thought to be activated as a result of this proximity to the genetically active H-gene region.60 Chapter 3 “Antibodies cross normally impervious barriers because the epithelial cells have specific transport mechanisms. ‘Thus, syneytiotrophoblastic cells have a receptor for the y heavy chain; they selectively absorb this immunogiobu- lin, internalize it, and secrete it into the fetal circulation. They recognize ‘n0 other immunoglobulin class. This ‘prevents infants from having atopic diseases, such as asthma, at birth be- ‘cause the mother's IgE does not get ito the fetal blood. IgA is similarly internalized by epithelial cells that have a poly-Ig receptor. The IgA is transported inter- ] ‘The alternative pathway is considered to be a primitive defense system—a bypass mecha- nism that does not require C1, C4, and C2 (Figure 4-2). 1. The alternative pathway can be triggered immunologically e.g, by aggregated IgA or 'gG4) or nonimmunologically c.g, by certain microbial cell products).he Complement Sytem | 73 D ‘Amplitcation loop oe ‘Biochemical os —_ we/ So Enzymatic on ~~ i ween {is inctvated by factors H and | GY bound to activator surtace, opsonization (5 convertase) Ce 00+ co—> oytolais Membrane attack pathway) FIGURE 4-2, The altemative pathway of complement activation. a. Activation by immunoglobulins occurs when antibodies that are unable to interact with Clq in their native state are aggregated, either chemically or by mild heating, The biologic significance of this pathway of activation is unclear. b. Activation by bacterial products has immense biologic significance: It represents a primitive defense mechanism by which the body can activate inflammatory pro- cesses and opsonize pathogens for phagocytic destruction . Substances capable of activating complement by the alternative pathway include: (1) Polysaccharides of microbial ori {@) Lipopolysaccharides of gram-negative bacteria (e.g., endotoxins) (6) Teichoic acids of gram-positive bacteria (e.g., adhesion molecules of some pathogens) (©) Zymosan from yeast cell walls @) Surface components of some animal parasites (e,, Schistosoma mansoni lar vae) d. These cell wall components (polysaccharides) appear to protect C3b from inactiva- tion by the regulatory proteins H and I (ie, factors H and I) [see VA 1; 8 2] 2. The initial recognition event for alternative pathway activation is the presence of C3, specifically C3b, which is continuously present in very small amounts in normal serum, 2. Inhibition. Initiation by C3b is prevented by interaction with factors H and | (1) 1 C3b binds to a nonactivator (.e., nonprotected) surface, it reacts with factor H and becomes inactivated through the combined actions of factors H and L (2) 1 C3b binds to an activator (ve, protected) surface, its ability to bind to factor H is diminished, and it can activate the alternative pathway. C3 has two molecular forms in the body: () Native C3, which has an intact thioester bond @) C3(H,0), in which the thioester bond has been hydrolyzed ¢. Factor B (the C3 proactivator) (1) The surface-bound, protected C3b interacts with factor 8 to form C3bB, a imagnesium-dependent complex.74 [.chopter 4D (2) In the presence of magnesium ions, C3(H:0), ike C3b, can also bind to factor B (3) Factor B is analogous to C2 ofthe classical pathway. {a) The genes for factor B and C2 are adjacent on human chromosome 6 (6) C2 probably arises from duplication ofthe factor B gene. 4, Factor D is a serine protease resembling CTs. (1) Factor B, when bound to C3b [or C3(H,O)], is susceptible to enzymatic cleav- age by factor D. (2) Two fragments are formed, Ba and Bb. (a) The Ba fragment is released; itis chemotactic for neutrophil (6) An active site is exposed in the Bh fragment, which remains bound to C3b, forming the enzymatically active C3bBb complex. CHDBD ste alternative pathway C3 convertase (i) The C3b molecule serves to bind substrate, which may be either more C3 molecules or C5 molecules, and hold it for enzymatic action of the activated serine protease, Bb in (P) isa protein that stabilizes the C3bBb complex. the presence of properdin, the dissociation of Bb is slowed, stabilizing the C3BBD complex (2) When stabilized by properdin, the C3BBH complex becomes a C3 convertase that cleaves C3 and generates more C3b. (3) This creates a positive feedback loop that amplifies the complement cascade. (4) As more C3b is generated, the complex expands, with numerous C3b mole- cules attached to a single Bb (C3B,BB, where n > 1), and becomes a C5 Conwertase capable of cleaving C3 and thereby initiating the membrane attack pathway 3. Cobra venom factor (CVF) has the interesting property of activating the alternative complement pathway. 2. Like C3b, CVF complexes with Bb Bb. The CVF-Bb complex that is formed is resistant to factors H and | and is capable of continuously activating the conversion of C3 to C3b, leading to complement deple- tion Many of the components of the complement system are enzymes; therefore, the poten- tial for self-amplification is tremendous. 1. In the classical pathway, as a consequence of amplification, approximately 1200 C3b molecules can be deposited on a membrane near the site ofa single IgM molecule a, One IgM molecule is sufficient to bind and activate one C1q molecule; the single Cis molecule is then capable of cleaving approximately 100 C4 molecules. b. Approximately 20 C4b fragments bind to the cell membrane and cleave about 100 2 molecules «. Although only four to six of the C2b fragments are captured by C4 to form CABID, this enzyme complex (ie., C4b2B) can activate thousands of C3 molecules 2. The alternative pathway represents other options for amplification. 1. As stated earlier, approximately 1200 C3b fragments bind the cell membrane in the Classical pathway. Each of these can interact with altemative pathway components (factors B and D) to form another molecule of the C3-cleaving enzyme, C3bBD. b. The C3 convertase of the altemative pathway (ie., C3Bb), once itis stabilized by properdin, can cleave thousands of C3 molecules and form many C3b molecules that are capable of initiating the altemative pathway. ‘c. When these C3b molecules become fixed to an activator surface, more factor B binding sites are exposed d. These C3b molecules also can bind C5 and hold it in a position favorable for Bb hydrolysis, thus initiating the membrane attack pathway.The Complement Syne | 75 THE MEMBRANE ATTACK PATHWAY (THE COMMON PATHWAY). The conver- gence of the classical and alternative pathways occurs at the point of C5 activation, Activation of the membrane attack pathway is initiated by C5 convertase. 1. C5 convertase is: a. CaBIDIB in the classical pathway b. C3B,Bb in the alternative pathway 2. These C5 convertases are the only components in the attack pathway that have enzy- matic activity. a. Thus, substrate cleavage of C5 into CSa and C5b occurs only once in the attack se- quence, , Subsequent steps involve spontaneous binding and polymerization of intact pro- teins ‘There are five components in this portion of the pathway. 1. C5 convertase cleaves C5 into a smaller C5a fragment and a larger C3b fragment. ‘a. C5a, an anaphylatoxin and chemotactic factor, is released into the surrounding fluid ‘medium. b. C5b is the first component of the membrane attack complex. As such, itis the re- ceptor for C6 and C7, and it initiates assembly of the terminal components, C8 and C9. 2. Unstable C5b binds to C6 to form a stable C5b6 complex. C5b6 then binds to C7 to form the metastable trimolecular C5B67 complex, which attaches to the target cell membrane without inflicting any damage. 3. Formation of the membrane attack C8 and C9 complex (MAC) begins when C8 at- taches to the membrane-bound C567 complex to form C5b678. The addition of C9 to the complex forms C5b6789, the MAC. ‘a. The MAC is an amphiphilic (ic., loving both lipid and water) molecular complex composed of 1 molecule each of C5b, C6, C7, and C8, plus 12 to 18 molecules of polymerized C9 (see Figure 4-1) (1) In plasma, C9 is in soluble, monomeric form. 2) When C9 associates with cell-bound C5b678 complexes, it undergoes a change in shape and polymerizes. This process requires a divalent cation, such as cal- cium, magnesium, of zinc. b. CB and C9, the final components of the complement cascade, are responsible for the membrane damage that causes cell lysis ‘c. C9 shares a great deal of amino acid homology with the perforin molecule, as evi- denced by antibodies produced against C9 cross-reacting with perforin, Perforin causes membrane damage when itis released from the cytoplasmic granules of T lymphocytes and NK cells, Because C9 is homologous to perforin, C9 can also cause the same type of membrane damage. 4. Cell Isis can occur when the C5b678 complex forms. In the absence of C9, tiny (3 nm) pores form in the membrane, and the cells become somewhat leaky, ‘a. The polymerization of C9 greatly accelerates cell lysis. b. The C9 polymer forms a cylindrical transmembrane channel 15 to 16 nm long, with an internal diameter of 8 to 12 nm and a wall thickness of 2 nm (see Figure 4-3 in- Set). Atlached to the channel is the C5b678 complex. «c. The hollow cylindrical channels of the MAC are in the lipid bilayer of the cell mem= brane. d. The channels coalesce into leaky patches, and the surface of the membrane is cov- ered with craters and looks like the surface of the moon (see Figute 4-3) . The channels allow the passage of electrolytes and water across the membrane, leading ultimately to osmotic lysis of the target cells. Thus, the process of cell lysis is nonenzymatic.76 | chapter 4 ui 8 Effect of membrane attack complex (MAC; C5b6789) on cell membrane. inset shows a sche ‘matic diagram of the transmembrane channel formed by polymerized C9 molecules, which allow water and fons to cross the cell membrane, leading to cell ists. The top of the C9 polymer Cylinder is hydrophilic and s capped by ang that remains ouside the cel The other end of he cinder pop and ors the transmembrane channel (1) Target erythrocytes lose potassium, imbibe sodium and water that flow into the cell through the pores, swell, and lyse. 2) I the target cell is nucleated, the cell membrane becomes porous; low- molecular-weight and then high-molecular-weight substances pass unimpeded. ‘These cells usually do not lyse {. The lysis caused by the MAC is restricted to those cells on which the initiating events of the complement cascade have occurred. Cells located nearby escape damage because of the following, (1) In solution, the MAC is extremely unstable and quickly loses its cytotoxicity. (2) There are MAC inhibitors in plasma (e.., S protein) and in cell membranes [exg., homologous restriction factor (HRFII BEM srotocic consequences oF COMPLEMENT ACTIVATION (Table 4-4) AJ Overview 1. During complement activation, a number of materials with important biologic activities are generated ‘a The cleavage products (split products) of C3, C4, and C5 appear to be the most important complement components in terms of biologic functions associated with the inflammatory response. . CB and C9 play a major role in membrane attack and the lytic process. 2, Cell receptors that have complement fragments for their homologous ligands have sig- nificant biologic roles (Table 4-5). a. Some receptors (e.g., CRI, CR2, CR3, CR4) react with larger fragments, such as C3, and promote opsonization and, therefore, phagocytosis.‘he Complement Sytem | 77 TABLE 4-4, Principal Inflammatory Activites of Activated Complement Proteins and Their Fragments Component Activity 3a, C4a, C5a___Anaphylatoxin—releases histamine, serotonin, and other vasoactive ‘compounds from mast cells, increasing capillary permeability 3b, IC3b, 4b Immune adherence and opsonization—binds antigen-antibody com- plexes to membranes of macrophages and neutrophils, enhancing phago: {eytosis; also binds complexes to erythrocytes, facilitating removal by the liver and spleen Csa ‘Chemotaxis and chemokinesis—attracts phagocytic cells to sites of inflammation and increases their overall activity ca,c9 ‘Membrane damage—transmembrane channels form, permitting flux of cytoplasmic constituents, Mammalian cells swell and burst; bacterial cells become leaky and lose vital intracellular metabolites, but usually do not burst Ba Neutrophil chemotaxis Bb Macrophage activation—causes macrophages to adhere to surfaces and spread on them b. Other receptors (e.g., C3a, C4a, C5a) ate specific for the smaller a” fragments that play a role in inflammatory processes. . Complement receptors on human erythrocytes provide an important mechanism for clearing immune complexes from the circulation, They bind the complexes and transport them to the liver and spleen, where they are engulfed by phagocytic cells Cleavage products of C3 and C5 1. C3a and C5a are anaphylatoxins. 2. Shared characteristics (@) C3a and C5a react with specific receptors (e,, C3aR and C5aR) on basophil and mast cell membranes (a) This reaction causes the release of pharmacologic mediators of inflamma- tion (eg, histamine) ina manner that is similar to mediator release by ie (b) The mediators cause smooth muscle contraction and increased vascular permeability, shich are effects that can be counteracted by antihistamines and anaphylatoxin inactivator. (2) C3a and C5 also appear to have the ability to contract smooth muscle and lary permeability directly (i.e., without the mediation of mast cells and basophil) b. C3a appears to function as an immunoregulatory molecule, demonstrating suppres- sive activity in immunoglobulin synthesis as well as in T-cell functions. ‘¢. C3a is much more active than C3a on a molar basis and, in addition to anaphyla- toxin activity, has a wider range of biologic activity, including the following: (1) CSais the major complement-derived chemotactic factor in serum, (a) Chemotaxis. C5a causes neutrophils and macrophages to migrate toward the site where C5a is generated (i.e., chemotaxis) (b) Chemokinesis. C5a also increases the overall activity of phagocytic cells (e., chemokinesis). (2) C5a causes neutrophils to adhere to the endothelium of vessels and to one an- ‘other, which leads to neutropenia (3) C5a activates neutrophils, which triggers a bactericidal oxidative burst and de- granulation.78 | chop 4 WB TABLE 4-5. Complement Receptors of Human Cells Receptor Ligands Cell Types Functions cri Cab, Erythrocytes, phagocytes, Opsonin: cofactor in factor | cleavage 3b, Bees, eosinophils (of C3b to C3dg; clearance of antigen— ica antibody compiexes* cra iCab, —_B cells, some T cells, Immunoregulatory; atachment site for Ga Nk ceils Epstein-Barr virus Gag cra iCab ——_-Phagocytes. ‘Opsonin; cofactor in iC3b degradation cra 3b, Phagocytes Not established; presumably an opso- iC3b, niin Cds C3aR ca Mast cells, basophils, Release of histamine and other media- eosinophils, smooth tors muscle, phagocytes caar Cha Mast cells, basophils May be same as C3a receptor; same effects as C3al Mast cells, basophils Same effects as C3a Neutrophils Causes PMN release from marrow stores kell = natural ler el: PMN = polmorphonucesr nearophi mune complexes ind rapt Yo enttroyses ty mune adherence) andar anor to the ve and pen, where mac rophages sep the compen fom the eel sure and eur he entry toe ereulton (4) C5a stimulates the production of leukotrienes (e.g, slow-reacting substance of anaphylaxis (SRS-A)] by mast cells. d. CSa-des-arg is C5a without the carboxy-terminal arginine, which is removed by anaphylatoxin inactivator. C5a-des-arg lacks the anaphylatoxin activity of C5a, but it retains chemotactic and neutrophil-activating ability 2. The generation of C3b and the coating of target cells by C3b are perhaps the major biologic functions of complement. ‘a. The role of C3b in the activation of the alternative complement pathway has been described previously (see I! D) b. C3b is a major factor in opsonization. (1) Several cell types have surface receptors for C3b and iC3b, including neutro- phils, eosinophils, monocytes, macrophages, 6 cells, basophils, and primate erythrocytes. {@) In the case of phagocytic cells, a coating of C3b on particles such as bacte- ria or antigen-antibody complexes promotes the attachment and ultimate ingestion of the particles. (b) Clinical importance. Individuals who lack C3 or whose phagocytic cells do not bear a C3bR molecule suffer from recurrent pyogenic infections. (2) The biologic significance of the presence of C3b receptors on B cells is not en- tirely clear, but they may play a role in the induction of certain humoral im- mune responses. €. C3b-coated cells also tend to aggregate (1) This aggregation of C3b-coated cells (.e., immune adherence) may promote phagocytosis. (2) Immune adherence of platelets to C3b can also activate the clotting system (see IVG 2d),the Complement system J} 79 3. Other C3 split products a. C3e is derived by proteolytic cleavage from C3c, which, in tur, is derived from C3b by the action of factors H and I. C3e provokes a release of neutrophils from bone marrow, which causes prompt leukocytosis. b. C3d, another cleavage product of C3b, can interact with CR2 receptors on B Iym= phocytes (see Table 4-5) and acts as a growth factor for these cells. c. C3 nephritic factor (C3NeF) is a pathologic component of the altemative pathway. (1) CANE is found in the circulation of patients with mesangiocapilary glomerulo- nephritis. (2) Itacts as an antibody against the C3bBb complex and leads to a marked hypo- complementemia, 4, The C5B67 complex has been reported to have chemotactic activity. However, C567 is rapidly inactivated in serum, and therefore itis not clear whether the complex plays an important chemotactic role, (C4 and its cleavage products 1. Activated C4 molecules attach to membranes in proximity to the C1 site. The binding (of C1-and C4 by a virus-antibody complex can neutralize virus activity. The C4 mole- cules may prevent viral attachment to target cells, 2. C4a has anaphylatoxi activity 3. C4b receptor sites exist on several cell types (e.g., phagocytic cells, lymphocytes, and primate erythrocytes), which suggests that C4b is involved in opsonization. €2. C2 cleavage has been reported to be linked to the production of a kinin-tike molecule that increases vascular permeability and contracts smooth muscle. C2 is thought to be in- volved in the symptoms seen in HANE (see V B 1 b). Ba and Bb. These two factors, which are generated exclusively by the alternative pathway, hhave important biologic functions. 1. Ba is chemotactic for neutrophils. 2. Bb activates macrophages and causes them to adhere to and spread on surfaces. C8 and C9 1. Function. C8 and C9 are responsible for the membrane damage that causes the lysis of bacteria and other cells. 2. Clinical importance. Absence of C8 is accompanied by an increased incidence and/or severity of Neisseria meningitidis and Neisseria gonorrhoeae infections. Interaction of the complement system with other systems is a common characteristic of the complement cascade. 1. Complement activation, kinin generation, blood coagulation, and fibrinolysis all are physiologic processes that occur through the sequential activation of enzymes in cas- cading fashion, These cascades interact with each other, sharing some activators, in- hibitors, and cell membrane receptors. 2, Tissue injury can initiate each of the cascades. For example, neutrophils are attracted to the site of injury by the chemotactically active C5a that is cleaved from C5 by pro- teases released from lysosomes (Figure 4-4) a. Lysosomal enzymes from damaged cells cleave precursor molecules to generate in flammatory compounds. b, Cascade-initiating factors can be activated by contact with negatively charged sur- faces, such as heparin or lipid A of the lipopolysaccharide from gram-negative bacte- ria. Hageman factor (HF; coagulation factor Xl) thereby activated, producing Ha. (1) Ha has serine protease ac80 | chapter « w G. [Peng mtn onan) | RRR TIT a aR Be Ame ae Gina AY} é wt eee p es Ser eats alee Cas Oss 5 Gis Bes Os FIGURE 4-4. Biologic activites of component split products that contribute to an acute inflammatory re- sponse. This response is usually protective, but if exaggerated it may lead to tissue destruction, The inflam ‘matory response, spurred on by microbial invasion o° tissue damage, occuts in three stages. In pavement. ing, the endothelial cells become sticky, and the polymorphonuclear cells (PMNS) adhere, C3a is active in this stage. In tansudation, proteins leave the lumen of the capillaries, CSa and C3a act on mast cells during this stage to cause mediator (eg, histamine) release. In exudation, cells leg, PMNs and red blood cells (RBCs}} leave the capillaries via iapedesis. Once the cells leave the capillaries, opsonization and phagocy: tosis can occur. @) Inthe kinin cascade, HFa activates prekallikrein, to form kallikrein (@) Kallikrein in turn generates bradykinin from high-molecular-weight kinino- gen, (b) Bradykinin is a potent vasoactive peptide that causes pain, increases vascu- lar permeability, and causes vasodilation. 3) Kallikrein is able to activate HF, and HFa has autoactivating capabilities; thus this system, like the complement system, has an efficient amplification loop. To prevent too much vasodilation, C1 inhibitor blocks HFa to stop the loop. The three proteins—HFa, prekallikrein, and high-molecular-weight kininogen—are also the initiating factors required for the intrinsic blood coagulation and fibrin- olytic pathways (1) Ha, by activating coagulation factor XI to factor Xla, ig cascade. (2) Kallikrein stimulates the plasminogen activator to generate plasmin, which is a protease that hydrolyzes peptides and esters of arginine and valine. Plasmin has a variety of biologic effects (a) Plasmin can activate HF and can initiate the complement cascade. Plasmin activates certain complement components (e.8., C1 and C3). ) Plasmin enzymatically activates C's. (Gil) Plasmin generates the anaphylatoxin C3a from C3. (b) Plasmin also releases chemotactic and vasoactive fibrinopeptides from brin. 4d. immune complexes (e.¢,, antigen—antibody complexes) added to blood or plasma activate the clotting system. This is caused by’ (1) Clumping of C3b-coated platelets (ie., immune adherence) (2) Release of clot-promoting platelet factors tes the blood clot- 3. Inhibiting factors a. Cl esterase 1 (CT INH) of the complement system is an important contro! mechanism in various cascading processes (see V B 1), b. In addition to inhibiting the serine protease activity of CTs, CT INH inhibits HFa, factor Xla, plasmin, and kallikrein.the Complement Sytem | BT REGULATORY MECHANISMS. Complement activation is associated with potent bio- logic functions that, if let unchecked, exhaust the complement system and can cause significant damage to the host. Uncontrolled activation of the complement system is pre- vented by several types of protein-complement interactions, A] some sem potine esymatily tack complement component, herby 1g them. 1. Factor I, the C3b inactivator, isa serine protease. 1a. Factor | cleaves C3b free in solution or on the surface of cells (1) The degradation products are unable to function in the C4bIB3B complex. (2) One degradation product, C3c, is converted to C3e b. Factor | also degrades C4b, but only in the presence of the C4b binding protein (C4bBP; see VB 3), 2. Anaphylatoxin inactivator (e., serum carboxypeptidase N) enzymatically destroys the biologic activity of C3a, C4a, and C5a by removing the carboxy-terminal arginine. Some serum proteins bind to, and thus inhibit, complement components. 1. CT INH isa plasma ay-globulin. 2. Inhibitory effects. C1 INH inhibits the enzymatic activity of CT esterase by dissoci ating the subunits Clq, Clr, and C1. I also inhibits plasmin, kallikrein, activated HF, and coagulation factor Xia, b. Clinical importance. Deficiency of CT INH results in hereditary angioneurotic edema (HANE), which is an inherited defect characterized by transient but recurrent episodes of local edema due to uncontrolled CTs activity. 2, Factor H acts with factor | to bind C3b and to allow factor 10 cleave the @ chain of C3b. This leads to formation of cleavage product iC3b, which does not interact with the alternative pathway components. 3. CAbBP controls the activity of cell-bound C4b. As with the interaction of factor H and C3b, C4bBP binds to C4b and allows factor | to cleave the @ chain of C3b. 4. S protein (vitronectin) protects the target cell from lysis by binding to the developing fluid-phase C5b67 complex. ‘a. The S protein-C5b67 complex cannot penetrate the target cell membrane. b. 5 protein does not stop the MAC from forming, but prevents it from penetrating the lipid layer of the cell membrane. Regulatory proteins in cell membranes 1. Decay-accelerating factor (DAP) and membrane cofactor protein (MCP). DAF and MCP have the same functions as factor H and C4bBP. However, DAF and MCP inacti- vate membrane-bound C3b and Cb, whereas factor H and C4bBP act on C3b and Cab in solution. a. DAF is a membrane glycoprotein found on many cells ofthe body. (1) Function. DAF prevents assembly of the two C3 convertases by promoting dis- sociation of C2b from Cab and Bb from C3b. (2) Clinical importance. DAF is deficient or absent in cells from people with parox- ysmal nocturnal hemoglobinuria (PNH). The red blood cells ofthese patients have increased sensitivity to complement-mediated lysis b. MCP is an integral membrane protein that serves as a cofactor forthe proteolytic inactivation of Cb and C3b by factor | 2. Homologous restriction factor |HRF; also called C8 binding protein (C8BP)]. HRF, which is found on many cells of the body, binds to CB and prevents the interaction of C8 with cell-bound C567,82 | choper 4 VC TABLE 4-6. Summary of the Biologic Functions of the Complement System Inflammation ‘Mast cell degranulation Chemotaxis 7 Vascular permeability PMN margination and diapedesis ‘Smooth muscle contraction PMN, NK, and macrophage activation Clearance of immune complexes Cell Iysis ‘Gram-negative bacteria Protozoa Enveloped viruses Viral neutralization Opsonization These functions ate atated by ethe the temave antibody dependent) o lis pathways (antbodymeditd and hence ar part the ante as wel the acquit Systems nos instances the prteipation of anbody wl accentuate te eect.Vignette Revisited 4, The classical pathway is activated via antigen-antibody complexes. ‘The alternative pathway may be triggered either immunologically, ‘when antibodies that are unable to. interact with Ciq in their native state are aggregated, or nonimmu- nological, by certain cell prod- ‘ucts such as lipopolysaccharides of ‘gram-negative bacteria, teichoic acids of gram-positive bacteria, or “zymosan from yeast cell walls. ‘The biologic functions of comple- ‘ment include the following: membrane attack complex (C5-C9) formation pro- ‘motes lysis of cells and kills mictoor- ganismns; C3b or iC3b on the surface of ‘microorganisms binds to CRI (and CR3, CRA) on neutrophils and mac- rophages, promoting phagocytosis; Ca, C3a, and C4a stimulate mast cell histamine release and smooth muscle contraction; C5a is a chemoattractant for neutrophils and activates neutrophil oxidative metabolism; complement activation on immunoglobulin mole- cules inhibits immune complex forma- tion; C3b on immune complexes binds to CRI on erythrocytes, and the im: mune complexes are cleared from the circulation as the erythrocytes traverse the liver and spleen (Table 4-6). 2. Even when properly regulated and activated, the complement system ‘can cause significant Hssue damage, ‘especialy in immune complex dis- ‘eases. |mmune complex glomerulo- nephritis and systemic vasculitis may result from the deposition of antigen—antibody complexes in glomeruli and blood vessel walls. ement activated by the immu- ‘noglobulin in these deposited im- mune complexes initiates the acute et responses that destroy. the glomeruh or vesel walls, leading to thrombosi ischemic damage to ‘issues, and scarring. The Complement Stem | A variety of deficiencies in many of the complement proteins have been described and are mostly attributable to genetic mutations. Defects in classical pathway activation have been associ- ated with systemic lupus erythematosus, slomerulonepbaritis, pyogenic infections, and vasculitis, Alternative pathway acti- vation defects most commonly result in pyogenic infections. Abnormalities in membrane attack complex formation are associated with disseminated Neisseria infections, systemic lupus erythematosus, and glomerulonephrits, Deficiencies in complement regula- tory proteins are associated with abnor mal complement activation and a vari- ‘ty of related clinical abnormalities. Hereditary angioedema is an autosomal dominant deficiency in C1 inhibitor manifested by acute, intermittent attacks of skin and mucosal edema. Paroxysmal nocturnal hemoglobinuria is a ccomplement-mediated intravascular hemolysis caused by deficiencies in the integral membrane protein regulators decay-accelerating factor (OAF), homo! ‘ogous restriction factor (HRP), and (CD59, Abnormalities in factor lor factor H result in deregulated classical path- ‘way activation, with consumption of (C3, and are associated with pyogenic infections, vasculitis, and glomerulone- phrtis.Case 4 ‘The patient is a 14-year-old white female prostitute who, 4 days ago, experienced an episode of shaking chills followed by perspiration and fever, These symptoms abated later that day but have recurred daily since. She has recently developed ten- ddemess in her right wrist and a small sore on her right index finger. The patient says she has not experienced vaginal discharge, diarrhea, dysuria, swollen joints, sore throat, jaundice, or abdominal pain Physical examination reveals a well-developed young woman with fever (her temperature is 40.3°C), a shaking chill, and regular respiration of 20. Her blood pressure is 110/70, and her heart rate is 135. The lesion on the index finger is brown at the center, is slightly raised, and is surrounded with a small erythematous fing. Extension ofthe thumb causes pain and palpable erepitus. Thickening and redness are noted around the wrist. The remainder ofthe physical examination is uncemarkable Laboratory data reveal the following values: normal urinalysis; complete blood count (CBC) with Hct 35%, Hgb 11.4 gid, and white blood cells (WBCs) 19,500 with polymorphonuclear leukocytes (PMN) 90%, bands 5%, lymphocytes 4%, and monocytes 1%; normal radiologic and chemical studies QUESTIONS 1. Which of the above data are helpful? 2. In what way? DISCUSSION At this point, the diagnosis is unclear. The most important finding is the elevated white cell count, with 90% PMNs and 5% bands, which suggests an acute infection. ‘The patient is placed on antibiotics and is admitted to the hospital. Specimens are sent to the laboratory for culture and sensitivity assays. ria gonorrhoeae is isolated from the cervix and the blood. QUESTIONS 1. What treatment is recommended? 2. Is there any underlying problem that contributed to the severity of this infection? DISCUSSION The patient has gonorrhea, and, because she is high risk, may well have more than one infection Her infection is caused by a penicillinase-producing N. gonorrhoeae (PPNG). PPNG strains are not very common in the United States, but their incidence is increasing (20+ %) in metropolitan areas. The organism is susceptible to ceftriaxone, and this isthe drug of choice. Tetracycline or doxycycline may be added to the treatment to combat (Chlamydia trachomatis, an organism that commonly coinfects with gonococeus, Gonococcal infections are usually asymptomatic in women and are restricted to urethri- tis oF cervctis in the few women who are sympiomatc. The patient in question has an underlying complement deficiency, which has compromised her ability to deal with thisThe Complement System | 85 infectious agent. Complement deficiencies are associated with a breakdown in host resistance to microorganisms. Complement is involved in host defense mechanisms as {an opsonin (C3b), a chemotaxin (C5a),aIytic system (C5-C8), and in altering vascular permeability (C3a, C4a, C5a), Deficiencies in the C’proteins ofthe membrane attack complex are associated with increased frequency and severity of infections by encapsulated bacteria, particularly members ofthe genus Neisseria (ive, the gonococcus and meningococcus species) tis of interest that deficiencies of C5 through CB are involved in this association; C9 defi- ciency does not seem to predispose to neisseral disease. Deficiencies in other C proteins are associated with recurrent pyogenic infections (par- ticularly C3) and autoimmune (immune complex) diseases. A deficiency in the Ci esterase inhibitor regulatory protein isthe most common C protein deficiency known and is asso- ciated with hereditary angioedema,86 [crop 4 STUDY QUESTIONS DIRECTIONS: Each of the numbered items or incomplete statements in this section is followed by answers or by completions of the statement. Select the ONE lettered answer ‘or completion that is BEST in each case 1.A patient is admitted to the hospital with a history of multiple bacterial infections. Lab- oratory results indicate that he is devoid of the third complement component. Which of the following complement-associated events ‘would stil be present in such a patient? (A) Bacteriolysis (B) Bacterial opsonization (C) Chemotaxis (D) Hemolysis (E) Hereditary angioneurotic edema (HANE) 2. Anaphylatoxin inactivator is a serum en- zyme that destroys the biologic activity of (A) Cla (8) Ca (C) C2b (0) C3b (E) C5a 3. C3 nephritic factor (C3NeF) has which of the following properties? Its (A) a pathologic component of the alternative pathway (8) important in neutralizing virus activity (C) essential for release of neutrophils from bone marrow (D) an antibody against C3a (E) an antibody against C3b 4. Complement regulatory proteins that are active in the cell membrane include (A) Clq (B) CT esterase inhibitor (CT INH) (C) Cé binding protein (C4b8P) (D) Decay-accelerating factor (DAP) (E} Ge DIRECTIONS: Each of the numbered items or incomplete statements in this section is, negatively phrased, as indicated by a capitalized word such as NOT, LEAST, or EXCEPT, Select the ONE lettered answer or completion that is BEST in each case. 5. All of the following are serine proteases EXCEPT (a) Cir (B) Cis (C) Cab () C3b (E) Bb 6. All of the following complement compo- rents are required in the alternative pathway EXCEPT (A) C1, C2, C4 (B) C5, C6, C7 ) (0) C8, ca (E) ProperdinThe Complement Sytem | 87 he set of matching questions in this section consists of a list of lettered ‘options (some of which may be in figures) followed by several numbered items. For each ‘numbered item, select the ONE lettered option that is most closely associated with it ‘To avoid spending too much time on matching sets with large numbers of options, its generally advisable to begin each set by reading the lst of options, Then, for each item in the set try t0 generate the correct answer and locate it in the option list, rather than evaluating each option individually. Each lettered option may be selected once, more than once, or not at all Questions 7-11 For each characteristic of a complement com- ponent listed below, select the component that is most closely associated with i. (A) C2 (B) C4 (©) C3BBB (D) Ge (E) Cb (F) Ga @ c9 (H) C5B6785 7. Neutralizes virus activities 8. Stabilized by properdin 9. Associated with symptoms seen in heredi- tary angioedema 10. Provokes release of neutrophils from bone marrow 11, Promotes opsonization8B [chapter 4 BBRANSWERS AND EXPLANATIONS 1. The answer is E [ILA 1; IV B 1-2; VB 1b] The third complement component (C3) is the junction for both the classical and the alterna- tive pathways of complement activation. Most ofthe biologic activites are expressed after this point inthe cascade. Thus, lytic phenom ena, such as bacteriolysis and hemolysis, are the result of membrane damage caused by the terminal membrane attack complex ofthe cas- cade. The major opsonin, C3b, would obvi- ously be absent in a C3-deficient individual, as would the inability to generate the chemo- taxins C3a and Ca. There might be slight chemotactic activity because of C4a, but this would be minimal at best. The propensity to Gevelop immune complex diseases is accentu- ated in individuals who lack the early compo: nents Cl, C2, and C4, Hereditary angioneu- rotic edema (HANE) occurs in individuals who are deficient in the inhibitor of C1 esterase. This disease is characterized by swelling of the face, neck, genitalia, and extremities, accompanied by abdominal cramps and vomiting, Laryngeal swelling can be life- threatening. Attacks occur aiter surgical trauma and severe stress. C1 esterase is acti= vated and splits C2, releasing the C2a peptide that causes increased capillary permeability with resultant edema. Because C1, C4, and C2 all participate in the Complement cascade be- fore the entry of C3, a deficiency inthe latter molecule would not interfere with the devel- lopment of ANE 2. The answer is E [V A 2]. C5a is destroyed by anaphylatoxin inactivator. C5a, and also C3a and Ca, are referred to as anaphylatox- ins. They cause the release of vasoactive amines from mast cells and basophils; the ‘amines, in turn, cause smooth muscle contrac- tion and increased vascular permeability. Ana~ phylatoxin inactivator (serum carboxypeptid- ‘ase N) removes the carboxy-terminal arginine residue from the anaphylatoxins, destroying their biologic activity 3. The answer is A {IV B 3 cl. C3 nephrit iactor (C3NeF) is a pathologic component of the alternative complement pathway that is found in the circulation of patients with me- sangiocapillay glomerulonephrits. C3NeF is an antibody against the C3b8b compiex and leads to a marked hypocomplementemia. 4. The answer is D [V C]. Decay-accelerating factor (DAF) is found on the surface of many cells of the body. tt prevents the assembly of C3 convertases by promoting dissociation of the enzymatically active fragments C2 and Bb from their membrane-associated ligands, C4b and C3b, respectively. DAF is not an integral ‘membrane protein but is anchored to the mem- brane by attachment to phosphatidylinosito. 5. The answer is D [Table 4-3] Five serine proteases became activated in the comple- ‘ment cascade: Clr, Cls, C2b, and factors D and B (as Bb). These serine proteases are pro- teolytic enzymes that cleave the next compo- nents in the cascade. C3b is a potent opsonin that sensitizes bacteria and other foreign parti- les to phagocytosis. Neutrophils and mac- rophages have on their membranes a receptor that reacts with C3b and holds the microbe in close proximity to the phagocytic cell, thus enhancing the enguliment process. 6. The answer is A [Il D]. The alternative path- way of complement activation does not re- quire C1, C4, and C2. The altemative pathy way, also referred to as the properdin pathway, is considered to be a primitive de- fense system, a mechanism that bypasses components C1, C4, and C2. The initial re- ‘quirement for the alternative pathway is the presence of C3, specifically C3b. 7-11, The answers are: 7-8 {IVC 11, 8-€ [ll D 2d, el, 9A (IV DI], 10-D [IV B 3 al, 11-E (IV B 2b. The binding of C1 and C4 by a virus antibody complex can neutralize virus activ- ity; itis probable that the C4 prevents viral attachment to target cells. Inthe alternative pathway, the C3bBb complex is cleaved by factor D. The Ba fragment is released, and the C3DBB complex, stabilized by properdin, be- ‘comes a C3 convertase. C2 is thought to be involved in the symptoms of hereditary angio- neurotic edema, a disease caused by uncon- trolled C1 esterase activity. C2 cleavage has been reported to be linked to the production (of a kinin-like molecule (C2a) that increases vascular permeability and contracts smooth muscle. C3e provokes a release of neutrophils from the bone marrow, causing prompt leuko cytosis, C3e is derived by proteolytic cleavage from C3c, which, in tur, is derived from C3b. C3b plays an important role in opsonizationChapter 5 The Immune Response System An acutely ill 3-year-old boy was brought to the emergency room by his aunt. His breathing was extremely la- bored, and he had hemoptysis (i.e., blood-tinged, rust-colored sputum). Grams stain of the sputum revealed humerous polymorphonuclear neutro- phils and gram-positive cocci in ‘arape-like clusters. A tentative diagno- sis of staphylococcal pneumonia was made, and the child was hospitalized and placed on intravenous oxacillin, ‘The aunt revealed that the child had ‘experienced several similar episodes. She has been the child’s daytime care- taker and had been able to control his previous infections successfully with antibiotics that she had in her medi- Cine cabinet. The child also had prob- lems with skin infections, such as boils, and had recently recovered from poison ivy. He contracted measles approximately 6 months before being ‘admitted to the hospital and had re- ‘covered normally. 1. On the basis of the information ob- ined from the aunt, the physician ‘was able to exclude what type of immune deficiency diseases from the differential diagnosis? Laboratory data: Leukocytes 14,800/mm? Platelets 200,000/mm? Hemoglobin 12 gidl Hematocrit 49% Neutrophils 69% Lymphocytes 21 Monocytes 5 Eosinophils 4 Basophils 1 2. Which, if any, of the above values seem abnormal? Leukocyte function studies were also performed. Chemotaxis, phagocytosis, and intracellular killing activities were all within normal limits. An immuno- globulin profile was ordered. The child had no detectable immunoglob- ulin A oF M (IgA or IgM) in his serum, A small amount (30 medi) of IgG was detected when the assay was repeated with low-level radial immunodiffusion plates. 3, From these additional laboratory data, what immunodeficiency do you suspect? 4, What prophylactic measures would the physician be likely to recom mend to prolong the life expectancy (of this child? 8990 | chapter 5A EXPOSURE TO AN ANTIGENIC SUBSTANCE, either by injection or infection, unfurls a complex series of events (Figure 5-1). A] An antigen presning cl sly a macrophage) process the antigen and presen i Sethe cece mre oc 1, For a successful immune response to occur, the processed antigen (specifically, its epitope) must be presented to the lymphocytes in association with a glycoprotein en- ‘coded by genes of the major histocompatibility complex (MHC) a. This requirement for effective cel interaction is called MHC restriction. b. Specific lymphoid cells recognize this particular epitope and react with i 2. The result is the activation of antigen-specific B and T cells, which causes them to pro- liferate and mature. 3. The consequences of the initial interaction between lymphocytes and their homologous epitopes are far-reaching, a. Subsequent exposure to antigen induces some B lymphocytes (memory B cells) t0 proliferate and differentiate into antibody-secreting plasma cells. (1) These active plasma cells release their specific antibody in large amounts when, they contact antigen a second time, a phenomenon known as anammnesis (an anamnestic response). (2) The secreted antibody reacts specifically with the antigen that originally in- duced the B cell to proliferate. The potential exists to produce an extremely large (> 100,000) variety of difierent, specifically reactive antibodies. FIGURE 5-1. Cellular interactions in the humoral immune response. CD3, CD4 = cluster of differentiation antigens 3 and 4; 1-1, -2 = interleukins 1 and 2; MHC'Nl = ‘major histocompatibility Complex class I! antigen; TCR = T-cell receptor for antigen.