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Neonatal Immunity

Contemporary ImmunologySeries Editor: Noel R. Rose, MD, PhD

Neonatal ImmunityBy Constantin Bona, 2005

Cytokine Knockouts, Second EditionEdited by Giamila Fantuzzi, 2003

Therapeutic Interventions in the Complement SystemEdited by John D. Lambris and V. Michael Holers, 2000

Chemokines in Disease: Biology and Clinical ResearchEdited by Caroline A. Hébert, 1999

Lupus: Molecular and Cellular PathogenesisEdited by Gary M. Kammer and George C. Tsokos, 1999

Autoimmune ReactionsEdited by Sudhir Paul, 1999

Molecular Biology of B-Cell and T-Cell DevelopmentEdited by John G. Monroe and Ellen V. Rothenberg, 1998

Cytokine KnockoutsEdited by Scott K. Durum and Kathrin Muegge, 1998

Immunosuppression and Human MalignancyEdited by David Naor, 1990

The LymphokinesEdited by John W. Haddon, 1990

Clinical Cellular ImmunologyEdited by Howard H. Weetall, 1990

Neonatal Immunity

by

Constantin Bona, MD, PhD

Mount Sinai School of Medicine,New York, NY

Foreword by

Noel R. Rose, MD, PhD

The Johns Hopkins University Schools of Medicine and Public Health,

Baltimore, MD

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Neonatal immunity / edited by Constantin Bona ; foreword by Noel R. Rose. p. ; cm. -- (Contemporary immunology) Includes bibliographical references and index. ISBN 1-58829-319-X (alk. paper) 1. Infants (Newborn)--Immunology. 2. Maternally acquired immunity. [DNLM: 1. Immunity, Maternally-Acquired. 2. Animals, Newborn--immunology. 3. Antibody-Producing Cells--Infant, Newborn. 4. Autoimmune Diseases--Infant, Newborn. QW 553 N438 2004]I. Bona, Constantin A. II. Series. QR184.5.N46 2004 616.07'9--dc22 2004007841

v

Foreword

Recently, Arthur Silverstein (1), a pioneer in studies of prenatal and neo-natal immunity, published an historical article, entitled “The Most ElegantImmunological Experiment of the XIX Century.” In it, he described someextraordinary experiments carried out by Paul Ehrlich between 1892 and 1894.These experiments can truly be cited as the genesis of today’s developmentaland neonatal immunology. The story, in brief, started after the demonstrationby Behring and Kitasato in 1890 of specific antitoxins circulating in the bloodof animals immunized against diphtheria or tetanus. These experiments werequickly translated into the use of antitoxins to prevent or treat these diseasesin human patients. Intrigued by reports that babies from immune motherswere protected from disease, Paul Ehrlich undertook a series of studies, usingthe plant toxins ricin and abrin as model antigens. He mated ricin- or abrin-immunized male mice with non-immune females and mated immunizedfemales with non-immune males. The offspring of these matings were testedfor protection by a challenge injection of the appropriate toxin. He found thatonly the offspring of immunized females were protected. Protection was spe-cific but only temporary. Pups of immune fathers showed no protection. Thus,for the first time, Ehrlich demonstrated protective passive immunity bymaternal transfer. Realizing that antitoxin transfer could occur across the pla-centa or through milk, Ehrlich next set up experiments in which the offspringof immunized mothers were given to suckle on non-immune foster mothersor, conversely, offspring of non-immune mothers to suckle on immune fostermothers. In the first case, the antitoxic antibody initially present declinedrapidly. In less than 3 wk all of the newborn were susceptible to the toxinchallenge. In contrast, in the offspring of non-immune mothers fed byimmune wet nurses, a high degree of immunity was established, which per-sisted for more than 6 wk. The milk of immune mothers, Ehrlich concluded,is an ideal source of protective antibody for the newborn. Finally, Ehrlichpassively immunized nursing mice with a horse antitoxin and found that theantibodies in milk could traverse the intestinal wall of the newborn.

It can rightly be said that in these simple, elegant experiments, Ehrlichlaid down the basic principles of contemporary neonatal immunology. Hisfundamental experiments have been repeated in many forms and confirmedmany times. For example, Fig. 34 in the present volume nicely recapitulatesEhrlich’s principal findings. It relates, moreover, to some of the urgent prac-tical questions of our time. Group B streptococcal infections are a frequentcause of sepsis and meningitis during the first 2 mo of life. There might be

vi Foreword

great value in immunizing mothers with the appropriate antigen of Group Bstreptococci in order to protect their newborns during this critical, vulnerableperiod. The experiments of Martin, Rioux, and Gagnon described on page163 of this book essentially reproduce Ehrlich’s experiments, using Group Bstreptococcal infection rather than a toxin, and confirm the potential value ofmaternal immunization in protecting the newborn.

Our knowledge of immunology has expanded greatly since the finaldecade of the 19th century and, with it, has come newer understanding ofneonatal and developmental immunology. One half of a century after hefirst proposed it, Ehrlich’s concept that adaptive immunity depends uponspecific receptors represented as side chains on cells was resurrected byBurnet (2) in his clonal selection theory. The basic concept that antigen is aselective rather than an instructive agent still underlies today’s immuno-logic thought. Burnet’s original view was that the interaction of an antigenwith a quiescent, mature, receptor-bearing immunocyte (i.e., lymphocyte)leads to cell proliferation and a positive immune response. A similar interac-tion with an immature immunocyte, on the other hand, provokes death ofthe antigen-specific lymphocyte. Thus exposure to an antigen during fetallife when most lymphocytes are immature is likely to lead to deletion ratherthan proliferation and a positive response. We now recognize that the immu-nologic unresponsiveness of the fetus is relative and time related, and that inmany species a robust immune response is possible in late fetal and neonatalperiods. This volume describes classical studies in which the progressivematuration of the early immune response is clearly described. Yet, we knowthat the newborn human is still unable to manage many infections on its ownand requires passively acquired antibody from its mother. Moreover, pas-sively acquired immunity in the newborn is largely, if not completely, anti-body-born. Cell-mediated protection is not transferred from mother tooffspring. It is, in fact, paradoxical that recent investigations have suggestedthat maternal lymphocytes can pass through the placenta and establish them-selves in the newborn to form immunologic chimeras. There is evidence thatthis chimeric state may result years later in a graft-vs-host reaction that isinterpreted as autoimmune disease.

Burnet’s original premise that contact of an immature lymphocyte withits corresponding antigen would lead to cell elimination rather than prolifera-tion is represented in our present view of central tolerance of self. Expressionof self-antigens in the thymus during early stages of T-lymphocyte generationleads to negative selection and deletion of self-reactive clones. The ability toexpress self-antigens in the thymic environment is genetically controlled andat least one of the genes responsible has recently been identified in humansand mice as belonging to the >AIRE= family of genes. This is certainly only

Foreword vii

the first of many genes with similar functions and is of the greatest impor-tance in understanding the construction of the neonatal immune response.

We now realize that clonal deletion of T cells in the thymus is anincomplete process and self-reactive T cells are often present in peripherallocations. Post-thymic mechanisms are required to keep such self-reactive Tcells in check. A number of regulatory mechanisms are engaged in avoidingthe onset of pathogenic autoimmune responses and maintaining physiologichomeostasis.

With that thought in mind, a number of years ago, we (3) proposed thatclonal balance rather than clonal selection is a more appropriate term fordescribing the development of the immune response. The term clonal balancesuggests that the immune response depends on the ratio of factors promotingor retarding the immune response. The balance between positive and negativevectors is very delicate and the outcome is determined by the genetics of thehost as well as the manner of antigen presentation. In practical terms, it is thecontext of the presentation of an antigen that dictates the quality and quantityof the immune response. It depends upon the signals passed by the cells pre-senting antigen to the initial T cells and by the T cells interacting with othercells. These signals depend, among other factors, on the degree of maturity ofthe host.

If we accept the notion of clonal balance, developmental and neonatalimmunology is the study of the context in which antigen is presented toimmunocompetent cells. The context is a dynamic one and depends on thematurity of the many cells that direct the immune response. It determines thecellular basis of both the adaptive immune response and of the earlier innateimmune response.

The present volume sets out the issues of neonatal immunology in termsof our contemporary appreciation of the changing dynamics of the clonal bal-ance. Many enigmatic phenomena of the past now become more understand-able; yet, the basic principles laid down by Ehrlich in 1892 are still applicable.

REFERENCES

1. Silverstein AM. The most elegant immunological experiment of the XIX cen-tury. Nat Immunol 2000;1:93–94.

2. Burnet FM. The Clonal Selection Theory of Acquired Immunity. Cambridge,Cambridge University Press, 1959.

3. Rose NR. Current concepts of autoimmune disease. Transplant Proc 1988; 20(Suppl 4):3–10.

Noel R. Rose, MD, PhD

The Johns Hopkins University Schools of Medicine and Public Health

ix

Preface

The field of classical neonatal immunology has been dominated overthe past 50 years by two theories. The first theory is that lymphocytes spe-cific for self-antigens are deleted during fetal development. Current researchcarried out in transgenic mice supports this hypothesis, with continual evi-dence of the deletion of self-reactive clones during fetal development. Thesecond long-held theory is that the immune system of neonates is highly sus-ceptible to developing immune tolerance, leading to unresponsiveness to im-munization. This theory became a paradigm based on an experiment performedin mice showing that the injection of newborns with allogeneic bone marrowcells prevented the rejection of an allogeneic skin graft.

Commonly, a paradigm built on a widely accepted finding becomes aneternal truth; an established paradigm generally leads to its temporal domi-nance and an apparent intellectual rigor. Scientists frequently differentiateinto several groups with respect to a given paradigm. One group of scientistssearches for new findings aimed at strengthening the paradigm, and thesefindings sometimes result in a beneficial practical application. For example,the paradigm of neonatal tolerance was instrumental in advancing our knowl-edge of the mechanism of tolerance. Currently, the reconstitution of the im-mune system of immune-deficient patients with cord blood stem cells isconsidered less harmful than reconstitution with bone marrow. A second groupof scientists continues to look for new findings that cannot always be ex-plained by the current paradigm. The accumulation of new findings that donot fit within the currently accepted paradigm leads to an intellectual crisis.This is a critical point in the evolution of science in which the competitionbetween old and new findings may then lead to the formulation of a newparadigm. The cycle paradigm–crisis–paradigm is repeated until a new theoryor a universal model is formulated or accepted.

In the last several decades there has been remarkable progress in ourunderstanding of neonatal immunity. Advances in cellular immunology, mo-lecular biology, recombinant DNA and proteins, and the function of cytokinesand chemokines have changed our understanding of the immunity of neo-nates and infants. New discoveries have revolutionized the study of neonatalimmune responsiveness, facilitating the development of efficient vaccines fornewborns, a greater understanding of impaired immune response in neonatesafflicted by immunodeficiency or genetic defects, and a clearer knowledge ofthe developing immune system. New experimental findings and clinical ob-servations have shown that neonates, and in some species fetuses, respond to

foreign antigens in a manner consistent with the existence of both quantita-tive and phenotypic differences between newborn and adult lymphocytes.

The aim of Neonatal Immunology is to present classical and currentinformation and discuss cutting-edge discoveries that will hopefully lead tonew horizons in biological research and result in scientific progress in suchareas as vaccination of infants, stem cells, gene therapy, and transplantation.

I wish to express my thanks for the support of my wife, Alexandra, andmy daughter Monique for their help in editing some of the chapters. Thanksalso to Cristina Stoica for her help in gathering references and composingfigures, to Dr. T. Brumeanu for valuable information and discussion regard-ing Chapter 9, and to Dr. Sunil Thomas for proofreading the manuscript.

Constantin Bona, MD, PhD

x Preface

Contents

xi

Foreword ........................................................................................................... v

Preface ............................................................................................................. ix

1. Structure and Function of the Immune System in Vertebrates ....... 1

2. Ontogeny and the Development of Cells Mediating Innateand Adaptive Immunity .......................................................... 57

3. Phenotypic Characteristics of Neonatal B Cells ............................ 91

4. Molecular Characteristics of Neonatal B-Cell Repertoire ..........107

5. Genetically Programmed Temporal Ordered Activationof Neonatal B-Cell Clones ...................................................131

6. Antibody Response in Fetuses and Newborns .............................145

7. Effect of Maternal Antibodieson Neonatal B-Cell Response ..............................................159

8. Neonatal Autoimmune Diseases Causedby Maternal Pathogenic Autoantibodies .............................179

9. Phenotypic Characteristics of Neonatal T Cells ..........................193

10. Expression of MHC Molecules in Neonates ................................219

11. Neonatal Cytokine Network .........................................................227

12. Cell-Mediated Immune Responses in Neonates ..........................241

13. Fetal and Neonatal Tolerance .......................................................253

14. Development of Efficient Prophylactic Vaccinesand Theracines for Newborns and Infants ...........................273

References ................................................................................................303

Index ..............................................................................................................373

Structure and Function of the Immune System in Vertebrates 1

1

From: Contemporary Immunology: Neonatal ImmunityBy: Constantin Bona © Humana Press Inc., Totowa, NJ

1Structure and Function of the Immune Systemin Vertebrates

1. INTRODUCTION

During evolution, vertebrates developed an immune system able to copewith the universe of foreign antigen represented by approx 1 billion differentstructurally defined antigenic determinants (epitopes). The word “immune”(from the Latin immunis meaning except or free from taxes) defines the func-tion of the immune system, which is endowed with intricate defense mecha-nisms to neutralize aggressive foreign molecules or to overcome microbialinfectious agents. The immune system has a limited reactivity to self-antigenby virtue of its ability to discriminate self from nonself.

The vertebrate immune system comprises two major arms: innate immunity(natural, nonspecific) and adaptive immunity (specific, acquired). Cells of theimmune system and soluble factors mediate the interaction between these twoarms. Germline genes selected by evolutionary forces encode the function ofthe immune system, and the replicative genetic material is inherited by off-spring. Although the information contained in the genetic material that en-codes innate immunity is quite stable, that which encodes the adaptiveimmunity can be modified throughout life by subtle changes in germ line generepertoire and in various vertebrate species by changes during evolutionthrough Darwinian selection pressure.

2. INNATE IMMUNITY

Innate immunity is mediated by two defense reactions: constitutive and in-ducible by self-altered cells or by macromolecules and microbes.

The constitutive mechanisms include physical barriers (e.g., skin and mu-cosal epithelia) and chemical factors endowed with antibacterial propertiespresent within the skin or in secretion of mucosal tissues (saliva, tears), such aslysozyme and defensins. These molecules exhibit destructive properties againstbacteria, and fungal cell walls are not active against a host’s cells or self-mol-ecules. Similarly, macromolecules endowed with antimicrobial properties arepresent in various cells or tissues and are recruited in the defense reactions.

2 Neonatal Immunity

One of the effector molecules of innate immunity is the complement. Theliver constitutes the primary site of complement synthesis during ontogeneticdevelopment and in adults. Complement is an effector system of host defensereactions, which acts against pathogens contributing to phagocytosis of bacte-ria and the release of inflammatory mediators at the site of inflammation. Otherfactors include lysins, agglutinins, antibacterial peptides, and acute phase pro-teins. All of these molecules that mediate innate immunity are germline-geneencoded, and their presence and activity do not depend on stimulation by for-eign antigen.

The cells involved in innate immune defense reaction are the polymorpho-nuclear leukocytes (PMNs) (neutrophils, eosinophils, basophils), monocytemacrophages, dendritic cells, natural killer (NKs), and / T cells. These cellsare able to clear the microbes or the toxins from the body and secrete cytokines,which form a functional network that enables crosstalk between the cells thatmediate innate and adaptive immunity.

Innate immunity in vertebrates has an ancestral origin. This concept origi-nated from the discovery by Metchnikoff of phagocytosis in invertebrates, in-cluding the transparent larvae of starfish and the water flea Daphnia (1). Soonafter Metchnikoff’s discovery, the phagocytosis of bacteria was demonstratedin other invertebrates (insects and flies) and in vertebrates. In vertebrate spe-cies, the neutrophils, eosinophils, mast cells, macrophages, and dendritic cellsare able to phagocytize not only bacteria and foreign cells but also aged orapoptotic self cells (2). We demonstrated that the neutrophils and macrophagesare also able to internalize both exotoxins (Dick toxin, diphtheria toxin, andEscherichia coli neurotoxin) and endotoxins (3–5) by fluid phase pinocytosis.

There is a large body of information demonstrating the presence of compo-nents that are characteristic of innate immunity in invertebrates (reviewed inref. 6; see Table 1). Some of these molecules are constitutively expressed andothers are induced subsequent to infection with bacteria (6).

The ancestral origin of innate immunity in vertebrates is also illustrated bythe fact that Toll-like receptors that are expressed in the effector cells of verte-brates were found in Drosophila (7,8). The Drosophila Toll proteins play animportant role in flies’ defense reactions against infection caused by fungi (8).

There are various target molecules that are recognized by the effector cellsthat mediate innate immunity. The target molecules could be classified intotwo major categories: (a) molecules involved in the phagocytosis process, and(b) molecules that are recognized by receptors, which trigger signaling path-ways that result in the activation of genes that are involved in the immuneprocesses.


2.1. Molecules Recognized by Receptors Mediating Internalization

The effectors of innate immunity—neutrophils, macrophages, and dendriticcells—can recognize and internalize microbes and altered self-macromoleculesand cells.

In early studies, we showed that the pinocytosis of human immunoglobulinsand sheep red blood cells in the absence or presence of specific serum was sig-nificantly reduced after treatment of guinea pig neutrophils with periodic acidand neuraminidase (Table 2). These results suggest that the glycoprotein mol-ecules associated with the surface of neutrophils are involved in the internaliza-tion process (9). These results also showed that the phagocytic process and theinternalization of immune complexes is significantly enhanced by opsonins, be-cause the internalization via FcR (10) is more efficient than the phagocytosismediated by nonspecific opsonins such as histones or other proteins (11).

Scavenger receptors, which are associated with the membranes of endothelialcells and macrophages located in liver (Kupfer cells) and spleen, are involved inrecognizing numerous physiological waste macromolecules and clearing themfrom the blood. Such molecules are self-polysaccharides and proteins that resultfrom hydrolysis of extracellular matrix glycoproteins, altered serum proteins, orforeign macromolecules caused by the degradation of bacterial and fungal pro-teins or colloidal dyes. Seternes et al. (12) showed that the presence of scavenger

Table 1Molecules Characteristic of Innate Immunity in Invertebrates

MoleculeSpecies Acute phase proteins

Insects Pentraxin, complement, macroglobulinCrustaceans C-reactive protein, LPS binding protein

Antimicrobial peptides

Tunicates Clavanin, stylelin, lumbricinDrosophila Defensin (cecropin, transferrin)Mytilus Defensin (mytilin, myticin)

Lysines

Earthworm (Eisenia fetida) Fetidin, lysenin, eiseniapore, CCF-1

Complement-like proteins

Sea urchin C3

Abbreviations: LPS, lipopoly saccharide.

4 Neonatal Immunity

receptors are common to cells from seven different vertebrate species: lamprey,fish, frogs, lizards, chickens, mice, and rats. The scavenger function that inducesthe clearing of physiological waste molecules is mediated by at least five typesof receptors: (a) hyaluronan receptors that recognize hyaluronan and condroidinsulfate (13); (b) the collagen receptor for several collagen chains (14) and N-terminal peptides of procollagen (14); (c) the mannose receptor for C-terminalpeptides derived from collagen (15); (d) the receptor for modified lipoproteins(16); and (e) FcR for immune complexes (10).

The C-type lectins play a role in the phagocytosis of apoptotic cells, such asrodent thymocytes induced to undergo apoptosis by glucocorticoids. The C-lectins probably recognize galactose-rich glycoproteins because the phagocy-tosis of apoptotic cells can be inhibited by preincubation of macrophages withN-acetyl galactosamine or galactose (17).

The recognition of altered self is poorly understood but could be related to thealteration in the surface glycoproteins or the phospholipid component of themembrane (17). This hypothesis is strongly supported by our previous studies,which demonstrate an increased phagocytosis of guinea pig macrophages of au-tologous red blood cells treated with periodic acid or lecithinase (18). The re-sults presented in Table 3 show that the rate of phagocytosis of autologous redblood cells (RBC) is negligible. However, it was significantly increased afterartificial alteration of glycoproteins subsequent to oxidation (with periodic acid)of hydroxy or -amino alcohol groups of polysaccharides or the hydrolysis oflecithin, which is a major component of the RBC membrane. The internalizationof the altered cells may be mediated by the phosphatidyl serine receptor. Thisreceptor binds red-cell ghosts and sickled red cells, which have lost normal asym-metry of membrane phospholipid leaflets by unmasking anionic phospholipidssuch as phosphotidylserine contained in the inner monolayer (17).

Table 2Alteration of the Uptake of Foreign Matter Subsequent to Treatmentof Guinea Pig Neutrophils With Chemical or Enzymatic Agents

Phagocytosis index of SRBC%Pinocytosis index of Igs % + Anti-SRBC

Agents Nil + Anti-Ig serum Nil serum

Control 34.6±2.48 38.9±4.54 1.1± 0.38 12.4±1.08Periodic acid 11.2 ±2.71 9.9± 2.62 1.2±0.58 1.6±1.01Neuraminidase 21± 2.3 24.77±3.1 0.25±0.13 1.72±0.48Trypsin 34.5± 6.24 34.77±4.27 0.71±0.25 2.69±0.7.8

Data published from ref. 9.Abbreviations: SRBC, sheep red blood cells.


2.2. Molecules Recognized by Toll Receptors Involved in SignalingPathways and the Activation of Various Genes

The molecules recognized by Toll-like receptors (TLRs) that trigger signalingpathways are refereed to pathogen-associated molecule patterns (PAMPs) (17).

Toll proteins that were originally described in Drosophila were consideredto play a role in controlling the development of the dorsal patterning of the flyembryo and antifungal defense reaction (8,19). In Drosophila, four membersof the TLR family have been identified. Structural studies demonstrated thatthe cytoplasm tail domain of the Drosophila Toll protein exhibits a high ho-mology with the mammalian interleukin (IL)-1 receptor (20). Activation ofToll receptors plays a role in innate immune reaction and has been demon-strated in transfection experiments. Transfection of Schneider insect cells withplasmid-containing Drosophila Toll protein activated the transcription of genesthat encode antimicrobial peptides such as diptericin, attacin, and defensin (21).Genetic complementation studies have suggested that the secreted proteinSpatzle is the natural ligand for TLR in Drosophila (22).

Later, homologs of Drosophilla Toll proteins were rapidly identified in hu-mans (23,24). The effector cells mediating innate immunity (e.g., monocytes;macrophages; and intestinal, dendritic, and NK cells) express TLRs.

In vertebrates, numerous ligands for the nine TLRs were identified (Table4). The major characteristic of the binding of ligands recognized by cells thatmediate innate immunity is that the recognition process is not clonal restrictedsince the ligands bind to TLRs expressed on a variety of cells.

The TLR recognized PAMPs, conserved molecular macromolecules, essen-tial for the survival of bacteria.

The IL-1 receptor/Toll-like receptor represents a superfamily of receptorsfound in many vertebrate species. This family includes IL-1R, IL-1R Acp, IL-18R, IL-18R Acp, and the orphan receptor. Although IL-1 and IL-18 are the

Table 3Effect of Enzymatic Pretreatment of Phagocytosisof Autologous RBC by Guinea Pig Macrophages

Pretreatment of RBC with Mean rate of phagocytosis

Nil 0.3±0.1Cathepsin-C 0.93±0.2Periodic acid 21.6±8.45Lecithinase 22.1±1.61

Data published from ref. 19.Abbreviations: RBC, red blood cells.

6 Neonatal Immunity

ligands of IL-1 and IL-18 receptors, the ligand for the orphan receptor isunknown. The TLRs signal pathways that lead to the activation of multiplegenes, which encode proteins involved in host response to infectious agents orinjuries (25).

TLR 2 recognizes mainly bacterial, mycoplasmal lipoproteins and glycolip-ids (reviewed in ref. 26). Lipoproteins bind to TLR by their lipoylated N-ter-mini. Most lipoproteins are triacylated at the N-termini, with the exception ofmycoplasma lipoprotein, which is diacylated. TLR 2 also recognizes mycobac-terium cell wall glycolipids such as lipoarabinomannan (LAM), which containsarabinofuranosyl sidechains. Interestingly, the receptor can distinguish subtlestructural differences in LAM. For example, it does not recognize AraLAMfrom M. tuberculosis or bacillus Calmette-Guerin (BCG), which contain LAMthat is terminally capped with mannose. Macrophages produce proinflammatorycytokines in response to zymosan, which binds to TLR 2. It was proposed thatTLR 2 subsequent to the binding of various ligands is recruited in phagosomesand participates in receptor-mediated phagocytosis and, therefore, may discrimi-nate between structurally different ligands. This concept is supported by cyto-logical data, which demonstrate that after phagocytosis of zymosan bymacrophages the TLR 2 is enriched around the phagosome (27).

TLR 3 recognizes viral double-stranded RNA (dsRNA) that is synthesizedduring the replication process by various viruses. The recognition of dsRNA

Table 4Toll Receptors and Their Ligands

Toll receptor Ligand

TLR IL-I IL-1, IL-18, LPSTLR 2 Clindrical E. coli LPS, yeast zymosan

Lipoproteins (mycoplasma, mycobacteria, OspA Borrelia burgdoferi)

Lipopeptide (Treponema pallidum)Peptidoglycan (Staphylococcus aureus, B.subtilis

Streptoccocus)Arabinofuranos (Mycobacteria)

TLR 3 Viral dsRNATLR 4 Conical Pseudomones gingivalis, leptospira LPS,

Lipoteichoic acid (S. aureus), HSP60TLR 5 FlagellinTLR 6 Neisseria LPSTLR 7 ImiquimodTLR 9 CpG motif

Abbreviations: TLR, toll receptor; IL, interleukin; LPS, lipopolysaccharide, dsRNA,double-stranded ribonucleic acid.


by TLR 3 was elegantly demonstrated in an experiment showing that althoughpoly (I:C) stimulates human cells expressing TLR 3, it failed to activate pro-duction of IL-6, IL-12 and tumor necrosis factor (TNF) by macrophages fromTLR 3–/– mice (28).

TLR 4 mainly recognizes LPS from various gram-negative bacteria,lipoteichoic acid from Streptococcus aureus, and HSP60, which is an endog-enous protein. The role of TLR 4 in the recognition of LPS is strongly sup-ported by two sets of observations: (a) the presence of an mis-sense pointmutation within the TLR 4 gene-encoding cytoplasm tail of the receptor inC3H/HeJ mice, which are unresponsive to LPS (29), and (b) lipopolysaccha-ride (LPS) hyporesponsiveness of TLR4–/–mice resembles the defective LPSresponse of C3H/HeJ mice (30). The recognition of HSP60 endogenous pro-tein by TLR 4 was demonstrated by the inability of macrophages from C3H/HeJ—but not C3H/HeN—mice to induce the synthesis of TNF- and nitricoxides after exposure to HSP60 protein (31).

TLR 5 recognizes flagellin, the major component of the flagella, whichextends from the outer membrane of gram-negative and gram-positive bacteria(32). This was demonstrated by studying the ability to stimulate cells incu-bated with flagellated and nonflagellated Listeria moncytogenis of E. coli anda mutant Salmonella typhimurium strain bearing the deleted flagellin gene.

TLR 9 recognizes CpG-rich bacterial DNA. This was demonstrated by alack of production of cytokines by macrophages and the maturation of den-dritic cells in TLR9-/- mice following exposure to CpG containing DNA (33)(Table 4).

Studies carried out in Drosophila showed that the signaling via Toll proteinsinvolves an adaptor protein tube that transmits the signal to Pele, a serine-threonine kinase, which, in turn, signals the dorsal-Cactus complex equivalentto mammalian NF- B (34–36). The signaling pathways through mammalianTLR 1,2,3, and 4 are quite common and resemble those described in Droso-phila, suggesting a highly phylogenetically conserved system. The intracellu-lar domain (TIR) of TLR 2,3, and 4 is similar to IL-1R domain. In the case ofIL-1R, the ligand engagement recruits the MyD88 adaptor protein, equivalentof Drosophila tube protein, that associates to IL-1R protein by direct TIR–TIRdomain interaction. The death domain of MyD88 recruits IL-1R-associatedprotein kinases, (IRAK4) that is related to the Pele kinase of Drosophila. Afterphosphorylation, IRAK 4 dissociates from the receptor and associates with theTNF receptor activation factor 6 (TRAF6). TRAF6 then activates NF- B, p38mitogen-activating protein kinase (MAPK), and Jien kinase (JNK), leading toactivation of transcription factors c-Jun and AP-1 (37). ECSIT protein identi-fied both in Drosophila and humans appears to bridge TRAF6 to MAPK acti-vation via MEKK1 (21).

8 Neonatal Immunity

There are some subtle differences in the signaling pathways triggered byTLRs. Thus, in the case of TLR 4, a MyD88 independent pathway exists inwhich the binding of LPS to receptor recruits Mal-TIRAP, which then acti-vates IRAK4 (38). The MyD88 independent signaling pathway was demon-strated in a study showing that the LPS induced NF- B activation inMyD88-deficient cells (reviewed in ref. 38).

Signaling pathways mediated by TLR 2 and 4 leads to the activation of thep38-MAPK pathway and of AP-1, NF- B, and c-Jun transcription factors (38).The signaling pathway via TLR 3 by dsRNA induces MyD66-dependent acti-vation of IL production, whereas the maturation of dendritic cells and the acti-vation of NF- B, c-Jun, and p38MAPK are induced via MyD88-independentpathway (28).

2.3. NK Cells and Innate Immunity

NK cells play an important role in innate immunity, particularly in earlierstages of the invasion of the body by infectious agents or aberrantly differenti-ated cells subsequent to oncogenic transformation. Like the response of mono-cytes and dendritic cells to pathogens, the NK response is not clonallyrestricted. This results from the monomorphic nature of NK receptors. Onetype of receptor represented by Fc receptor mediates Antibody-dependent cell-mediated cytotoxiated (ADCMC) reaction, a process in which the antibodyspecific for a target antigen bridges the target cell with NK cells. A secondgroup of receptors is involved in lysing virus-infected and tumor cells that lackmajor histocompatibility complex (MHC) molecules. The second group of re-ceptors can be divided into three families. The first family contains killer-cellinhibitory receptors (KIR) expressed on human NK cells. The second familyconsists of receptors that display high homology to the C-type lectin receptorsexpressed on the surface of other effector cells involved in innate immunity.This receptor is a heterodimer composed of CD94/NKG2 that is expressed inboth human and murine NK cells. In humans, these receptors recognize HLA-E, a minor MHC antigen and in mouse Qa-1b, a nonclassical MHC antigen(39). In mice, whereas CD94 is monomorphic, the NKG2 is polymorphic. Thethird family of inhibitory receptors, Ly49, is expressed only in some murinestrains. This receptor family is polymorphic, because it consists of 14 highlyrelated genes (Ly49 A–N). The inhibitory effect results from the binding ofLy49 to the 1/ 2 domain of MHC class I molecules (40).

In humans, NK cells constitute 10–15% of peripheral blood lymphocytes(PBL) and mediate cytotoxic activity against cells infected with viruses oragainst tumor cells irrespective of their antigen specificity. Similarly to mice,human NK cells express three types of receptors: (a) the Fc receptors thatmediate ADCMC; (b) the inhibitory receptors that belong either to the Ig


superfamily (KIR and ILTC) or to the C-type lectin family (NKG2A/CD94);and (c) the activating receptor group composed of several molecules, includingNKp30, NKp44, and NKp46 (41).

3. ADAPTIVE IMMUNITY

Adaptive immunity represents the specific immune response of individualswho have been exposed to foreign antigen or, in some pathological conditions,to self-antigen. The effectors of adaptive immunity include B lymphocytes,which are derived from bone marrow or bursa of Fabricius, and T lympho-cytes, which are derived from the thymus.

B lymphocytes mediate humoral immune response by virtue of their ability toproduce antibody identical to the Ig receptor that is associated with their mem-brane. The Ig receptor is able to recognize epitopes on the surface of native orpartially degraded antigenic macromolecules. T cells mediate cellular immunereactions. In contrast to B cells, T cells, via their T-cell receptor (TCR), do notrecognize epitopes on the surface of native antigen. They are able to recognizepeptides of various lengths resulting from the degradation of macromoleculestaken up by antigen-presenting cells (monocytes, macrophages, dendritic cells,and B cells). They can recognize the peptides only in association with MHCclass I molecules, such as a subset of T cells that display CD8 marker or MHCclass II and CD1 molecules by T cells that display the CD4 marker.

Adaptive immunity can be divided into two major categories: (a) activeadaptive immunity induced by natural exposure to foreign antigen or subse-quent to vaccination, and (b) passive immunity resulting from passive transferof antibodies from mother to newborn (natural, passive immunity) subsequentto parenteral administration of specific antibodies (artificial, passive immu-nity) or infusion of lymphocytes (adoptive transfer immunity).

Adaptive immunity is characterized by three major features. First, the im-mune response is specific for 1 billion structurally different antigens, borne byprotein, glycoprotein, polysaccharide, lipoprotein, and glycolipid macromol-ecules. The receptor of the lymphocyte recognizes a single epitope of a macro-molecule even if a given macromolecule bears a multitude of epitopes. Eachlymphocyte recognizes a given epitope by its receptor: Ig receptor in the caseof B cells and TCR in the case of T cells. Antigen-specific lymphocytes existin the body before exposure to antigen, and the specificity of their receptorswas selected during species history by evolutionary Darwinian forces, namely,antigen-selection pressure.

The second main feature of adaptive immunity is the ability of lymphocytesto discriminate self from nonself. This remarkable property is related to theelimination (deletion) or functional inactivation (anergy) of lymphocytes ex-pressing receptors that are specific for self-antigen during fetal development.

10 Neonatal Immunity

The third feature is immunological memory. Subsequent to the exposure toantigen, the pre-existing lymphocytes are activated and differentiate either intoeffector cells or memory cells. After a new exposure to antigen, the memorycells are rapidly activated, proliferate, and make a prompt specific response,enabling the body to respond specifically and rapidly to antigen. Therefore, thememory cells are lymphocytes that were previously activated by antigen. Thesecells have a long half-life. Obviously, the prompt response of memory cells toa new exposure to antigen (challenge) represents a beneficial mechanism ofadaptive immunity in defense reactions against infectious agents. It is note-worthy that in certain conditions this can be deleterious when the tolerance oflymphocytes to self-antigen is broken down and leads to the occurrence ofautoimmune diseases.

The concept of adaptive immunity was derived from three revolutionarydiscoveries: the discovery of the anti-smallpox vaccine by Jenner, the discov-ery of the anti-anthrax and rabies vaccines by Pasteur, and the demonstrationof passive acquired immunity through injection of anti-diphtheria toxin anti-bodies by von Behring and Kitasato (42).

The demonstration that antitoxin antibodies prevent diphtheria led to theconcept of humoral theory of adaptive immunity, namely, that substancespresent in body fluids mediate the immune response. Ehrlich (43) proposedthat these substances actually represented receptors expressed on the surfaceof cells that are able to interact with antigen based on a chemicalcomplementarity.

The discovery of phagocytosis by Metchnikoff (1) provided the framework forcellular theory of the immune response. This theory emerged from the observa-tion that the cells that are able to phagocytize microbes are the major players inthe defense reactions. Both theories were proposed in a period when the immu-nologists ignored the function of lymphocytes. Gowans (44) demonstrated laterthat the lymphocytes are the effectors of specific adaptive immune responses.

However, these theories failed to explain two major features of the adaptiveimmunity: specificity and tolerance.

The properties of adaptive immunity were explained by Burnet, who pro-posed the clonal theory (45).

The clonal theory is based on the concept that each lymphocyte is geneti-cally programmed to recognize a single antigenic specificity. Thus, the im-mune system represents a collection of clones derived from precursors that areable to recognize a single epitope by its receptor. Evolutionary forces duringphylogeny selected the genes that encode the specificity of the lymphocytereceptors. The antigen represents the Darwinian selective pressure, and thediversity of the immune response results from a random rearrangement of genesthat encode the lymphocyte receptor, which diversify may be amplified furtherby the somatic hypermutation process throughout life. Therefore, once the


antigen enters into the body, it selects the clone that bears a complementaryreceptor for a given epitope.

A unifying element of clonal theory is the ability of lymphocyte clones todiscriminate self from nonself. Burnet proposed that the clones that are able torecognize self-antigen are deleted during ontogeny. Thus, clonal theory pro-posed that both the randomization of specificity for foreign antigen and toler-ance susceptibility for self-antigen are limited to the ontogenetic and perinatalperiod of the development of the immune system.

Clonal theory became a paradigm for the immunologists since a large bodyof experimental evidence supported it. Thus, the following was demonstrated:

(a) The lymphocytes belonging to the same clone bind a single antigen and lympho-cytes could be eliminated if the antigen is labeled with a radioactive element.

(b) In a heterozygous animal, a B cell makes antibodies by carrying a single allotypeand idiotype genetic marker. The allotypes are antigenic markers of heavy orlight chains of Ig molecules that are shared by all individuals of a genotypicallyidentical, inbred strain or by groups of individuals of outbred species. Theidiotypes are phenotypic markers of V genes that encode the Ig receptor of Bcells or TCR receptor of T cells.

(c) All the progenitors of a clone share identical genes that encode the specificity oftheir receptor (ref. 49).

Burnet (45) postulated that unresponsiveness to self-antigen results fromthe process of deletion of self-reactive clones during ontogeny (clonal dele-tion), which leads to central tolerance. Central tolerance occurs as a result ofnegative selection in central lymphoid organs—in bone marrow where B cellsare generated and in the thymus where T cells develop. The concept of nega-tive selection of B cells is well supported by studies carried out in transgenicmice. Thus, in mice, it has been shown that B cells that express VH and VL

genes, which encode antibodies specific for allogeneic MHC class I molecules(46) or hen egg lysozyme (HEL), have been deleted (47).

Clonal theory views the immune system as disconnected clones that arewaiting to encounter an antigen, which induces their expansion and differen-tiation into effector cells.

Jerne (48) proposed a theory that pictured the immune system as a networkin which the clones speak to each other using idiotype dictionary. The idiotypeexpressed on the receptor of a clone can be recognized by the receptor of adifferent clone, and this type of recognition of self-structures ensures the con-nectivity of clones that make up the immune system.

The network theory is based on the following three major postulates:

(a) The idiotypes expressed on the receptors of lymphocytes function as links betweenclones exhibiting various specificities, and as a target for regulatory mechanisms,which control expansion of clones in various stages of the ontogenetic develop-ment of the immune system and in various stages of the immune response.


(b) Idiotypes are internal images of antigen and, therefore, represent links betweenthe external universe of foreign antigen and the inner immune system.

(c) Through a self-recognition process, idiotypes can activate a cascade of comple-mentary clones (operationally called anti-idiotypes), which display regulatoryfunctions. The activation of clones by anti-idiotypic antibodies can replace thestimulation by foreign antigen. The idiotype-mediated suppression shall preventoverwhelming of the immune system by a clone that is expanded subsequent toantigen stimulation (reviewed in ref. 49).

4. CELLS OF THE IMMUNE SYSTEM

4.1. Phenotypic Characteristics of Mature Cells Involved in InnateImmunity

The cells mediating innate immunity, an important first line of defense, arethe granulocytes (neutrophils, eosinophils, and basophils), monocyte-macroph-ages, and dendritic and NK cells. Each type of cells exhibits different pheno-types based on cell structure, the expression of different membranecytodifferentiation antigen and receptors, enzyme content, and the display ofdifferent functions.

4.1.1. Neutrophils

Neutrophils represent approx 60% of total white blood cells (WBCs). Thesecells are 10–15 μm in diameter, with slightly basophilic cytoplasm and in-dented nuclei, which characterize the young (band) cells, and with polylobatednuclei, which are characteristic for older (segmented) cells. Within the cyto-plasm, three types of granules can be observed: (a) primary, or azurophilic,granules, which stain purple with Giemsa; (b) secondary granules, which stainfaintly pink with Giemsa; and (c) peroxisomes. Primary and secondary gran-ules are typical lysosomes, which fuse with endosomes containing phagocy-tized particles that are degraded by the enzymes contained in these granules(Fig. 1).

Primary granules contain various types of hydrolyzes that have an optimalactivity at pH 5.0, including lysozyme and cationic proteins. Secondary gran-ules contain lysozyme, collagenase, cytochrome, vitamin B12-binding protein,and lactoferrin. The peroxisomes contain myeloperoxidase, which reduces oxy-gen and hydrogen peroxide (Table 5).

The neutrophils express some cytodifferentiation antigen, which are sharedwith other WBCs and various receptors. Some of these receptors bind to growthfactors that are essential for the differentiation of myeloid cells. Another set ofreceptors is involved in the internalization of immune complexes and bacteriacovered with IgG opsonins and complement. The neutrophils express recep-tors that bind to chemokines or to chemoatractans formyl peptides (FPR,mFPR1, mFPR2). These receptors play an important role in the migration of


neutrophils from the blood to tissue and, in particular, toward inflammatoryfoci caused by pathogens. In general, the movements of neutrophils are ran-dom. However, when the local medium contains formyl peptides derived frombacteria, the neutrophils move toward the source of peptides, a phenomenoncalled chemotaxis. The chemotaxis allows for the accumulation of leukocytesat the site of microbial infection and inflammation. The FPRs expressed inhuman neutrophils have an important role in innate immunity. For example,dysfunctional FPR alleles were associated with juvenile peridontitis (50). There

Fig. 1. Electron micrograph and light microscopy of a neutrophil. Multilobatednucleus with predominant heterochromatin. The cytoplasm is packed with an abun-dant variety of granules: lysosome (L); phagolysosomes (PL), which are mostly clear;peroxisome (P); and azurophilic granules, which are the darkest.


are also receptors for various cytokines, such as CD118 binding to IFN- and- and CD132 binding to IL-2 and receptors for IL-4, IL-7, and IL-14 cytokines(Table 6).

The main function of neutrophils is to internalize particulate antigen by ph-agocytosis and soluble macromolecules by pinocytosis. Figures 2 and 3 showneutrophils phagocytizing autologous red blood cells (RBCs) treated with leci-thinase and the pinocytosis of fluoresceine-labeled Dick erythrotoxin.

The opsonin antibodies that bind complement enhance phagocytosis. Thiswas clearly demonstrated in the phenomenon of immunoadherance (51). Fig-ure 4 shows the kinetics of phagocytosis using neutrophils of Staphylococcithat are bound to RBC covered with antibacterial opsonins and complement.Recent studies showed that endosomes bearing vesicle-associated protein 3(VAMP3) fuse with the plasma membrane at the site where phagocytosis isinitiated, and subsequently, the phago-endosome is internalized. This processcontributes to the preservation of the cell membrane during phagocytosis,avoiding loss and preserving the integrity of the membrane (52). Phagocytosisinduces the activation of Ras-ERK MAPK module in neutrophils. In contrast,phagocytosis that is initiated by Fc R recruits Syk (reviewed in ref. 53).

Table 5Proteins and Enzymes Contained Within NeutrophilsGranules Primary, Azurophilic Granules

Enzymes

PRIMARY GRANULESCathepsins, collagenases, proteinas-3, elastases, lysosyme,

-manno-sidase, -galactosidase, -glucuronidase,-glycerphosphatase N-acetyl- -glucoseaminidase,

meyloperoxidase lipase, -amylase, phosphlipases,nucleases

Proteins

Cationic proteins, -defensin

SECONDARY GRANULES Enzymes

Collagenases, lysozyme

Proteins

Lactoferrin, cytochrome b, vitamin B12-BPPeroxisomes

Enzymes

Peroxydases


Table 6CD Antigens and Receptors Associated With Neutrophil’s Membrane

CD antigens

CD65 (poly-N-acetyllactose amine), CD69CD165 (sialomucine), CD94 (C-type lectin)

Receptor Ligand

-integrin family

CD11a ICAM-1.3CD11b ICAM-1CD11c Fibrinogen, iC3b

-integrin family

CD18 ICAM-1-3, fibrinogen, iC3bCD29 Collagen, Laminin, fibronectin

Complement receptors

CD35 C3b, C4bCD46 C3b,C4bCD48 Cb2CD88 C5a

Fc receptors

CD23 Ig FcCD64 (Fc RI, CD32 Fc RII) Ig Fc

Growth factor receptors

CD114 G-CSFCD116 GM-CSF

Interleukin receptors

CD118 IFN andCD132 IL-2, IL-4, IL-7, IL-14, IL-15CD153 TNF-

Chemokine receptors

CD128 IL-8CXCR2 CXCL2,CXCL3, CXCL5CXCR1 CXCL7, CXCL8, CXCL9, CCl24

Chemotactic receptors

FFR, mFR1, mFPR2, FPRL1 Formyl-peptides

Abbreviations: ICAM, intercellular adhesion molecule; C, complement; FCR, Fcreceptor; G-CSF, granulocyte-colony stimulating factor; GM-SCF, granulocytemonocyte-colony stimulating factor; FPR, formyl-peptide receptor.


Fig. 2. Phagocytosis of autologous red cells was following treatment with periodicacid. May-Grunwald- Giemsa staining of guinea pig neutrophils (×1, 250). (From BonaC et al. Nature [London] 1967; 213:824–825.)

By virtue of the broad specificity of hydrolyses contained in lysosomes, thephagocytized or pinocytized materials are completely degraded and, therefore,the neutrophils are unable to present antigen to T cells, which suggests thattheir major function is in innate immunity. Adaptive immunity via opsonins,antibodies specific for particulate antigen, enhances the function of neutrophilin the innate immune processes.

A major link between innate and adaptive immunity exists in the cytokinenetwork. The cytokines produced by cells that mediate innate immunity dis-play immunomodulatory properties on lymphocytes, and the cytokines that aresecreted by lymphocytes modulate the function of cells involved in the innateimmunity. In spite of the fact that the neutrophils are terminally differentiated,short-lived cells, they produce some cytokines or chemokines subsequent toexposure to antigen, such as LPS, which binds to Toll receptors, or C5a, whichbinds to complement receptors. Thus, LPS induces the synthesis of thecytokines, such as IL-1 , TNF- , TGF- , or IL-8 and MIP-1 chemokines (53).

4.1.2. Eosinophils

Eosinophils constitute 3–5% of total WBCs. Structurally, they are cells of10–15 μm in diameter, with bilobated nuclei and cytoplasm that is full of gran-ules that stain bright red with eosin. The granules contain unique crystalloid


Fig. 3. Pinocytosis of fluorescein-labeled Salmonella typiymurium endotoxin byguinea pig neutrophil. The pinocytosis was observed in fluorescent microscopy 45min after incubation of cells with labeled endotoxin.

core structures (Charcot–Leyden) that are composed of several basic proteins(Fig. 5).

In contrast to neutrophil granules, eosinophil granules contain fewer hydro-lytic enzymes (arylsulfatses, acid phosphatase, ribonuclease, cathepsins, andlysophospholipases) but are richer in major basic proteins, which are toxic foreukaryotic cells, and cationic proteins, which are toxic for prokaryotic and eu-karyotic cells.

The eosinophils express receptors for Fc fragments of IgG, IgA, IgE, recep-tors for complement (C1q, C5a), and growth factors (G-CSF and GM-CSF).

Eosinophils can internalize macromolecules and, in certain conditions, gen-erate peptides from the processing of internalized macromolecules. Antigen-presenting property was shown by the ability of murine and human eosinophilsto present peptides derived from ovalbumin (OVA) to activate OVA-specific Tcells (54) and by the ability of human eosinophils to present peptides derivedfrom foreign proteins (55). The antigenic-presenting property is related to the


ability of eosinophils activated by GM-CSF to induce higher expression ofclass II molecules, which present the peptides to CD4 T cells (55).

The major function of eosinophils in innate immunity is related to defensereaction against higher-ordered eukaryotic parasites such as helminths. Theyalso play a role in allergic diseases. In both instances, the number of eosino-phils in the blood is increased (eosinophilia).

4.1.3. Basophils

Basophils represent 1% of WBCs in humans but are found in all vertebrate—and some invertebrate—species. Frederich von Recklinghausen reported thefirst description of basophils in 1863, and, in 1879, Ehrlich described the samecells in various tissues and called them Mastzellen: the mast cells. These cellswere named basophils because of strong metachromatic staining of granules

Fig. 4. Phenomenon of immunoadherence. Red blood cells coated with specificantibodies were incubated with complement and bacteria and then with guinea pigperitoneal cells. The figure represents four different steps analyzed by microcin-ematography. It is noteworthy that in the last figures, the majorities of bacteria werephagocytized and are present in vacuole (V) .


with Toluidine blue, which is because their contents are rich in anionic sub-stances (Fig. 6). The granules of basophils and mast cells contain proteoglycan,biogenic amines, leukotrines, and hydrolytic enzymes (Table 7). Severalreceptors and glycoproteins are associated with the membranes of basophilsand mast cells (Table 8).

Fig. 5. Electron micrograph and light microscopy of an eosinophil. The cytoplasmof this guinea pig eosinophil contains many granules. The granules contain a uniquecrystalloid core structure (Charcot–Leyden) composed of several basic proteins (L).


The binding of ligands to some receptors caused degranulation of basophils,leading to the release of a broad variety of proteins and biogenic amines, whichplay an important role in innate immunity, inflammatory processes, and aller-gic diseases. Among these receptors, one may cite Fc R, which is capable ofbinding IgE-antigen complexes. The activation of basophils via the IgE recep-tor plays an important role in inflammation and adaptive immunity but not ininnate immunity because IgE activation is totally dependent on Th2 cells. IgE-mediated degranulation leads to the release of histamine, heparin, -glucu-ronidase, and eosinophilic chemotactic factor. All these factors are importantmediators of acute inflammatory processes.

Fig. 6. Electron micrograph and light microscopy of a mastocyte. The cytoplasm ispacked with large granules strongly metachromatically stained with Toluidine bluebecause their contents are rich in anionic substances. The granules of basophils andmast cells contain proteoglycan, biogenic amines, leukotrines, and hydrolytic enzymes.

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