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 CONTENTS

Foreword by Rubens Belfort Jr, vi 14 Other Viral Diseases, 169

Preface to the Fifth Edition, vii Scott M. Whitcup
15 Ocular Toxoplasmosis, 179
H. Nida Sen
PART 1 Fundamentals 16 Ocular Histoplasmosis, 197
Elements of the Immune System and Concepts of
1  H. Nida Sen
Intraocular Inflammatory Disease Pathogenesis, 1 17 Ocular Toxocariasis, 206
H. Nida Sen Scott M. Whitcup
18 Onchocerciasis and Other Parasitic Diseases, 215
Alan G. Palestine
PART 2 Diagnosis 19 Postsurgical Uveitis, 229
Alan G. Palestine
Medical History in the Patient with Uveitis, 29
2 
Scott M. Whitcup
Clinical Examination of the Patient with Uveitis, 31
3  PART 5 Noninfectious Uveitic Conditions
Scott M. Whitcup
Development of a Differential Diagnosis, 42
4  20 Anterior Uveitis, 237
Scott M. Whitcup Scott M. Whitcup
Diagnostic Testing and Imaging, 50
5  21 Scleritis, 252
Scott M. Whitcup Scott M. Whitcup
Optical Coherence Tomography in Uveitis, 63
6  22 Intermediate Uveitis, 259
Scott M. Whitcup Scott M. Whitcup
Evidence-Based Medicine in Uveitis, 69
7  23 Sarcoidosis, 269
Scott M. Whitcup Scott M. Whitcup
24 Sympathetic Ophthalmia, 282
H. Nida Sen
PART 3 M
 edical Therapy and Surgical 25 Vogt-Koyanagi-Harada Syndrome, 296
H. Nida Sen
Intervention 26 Birdshot Chorioretinopathy, 310
Approaches to Medical Therapy, 73
8  H. Nida Sen
H. Nida Sen 27 Behçet Disease, 326
Role of Surgery in the Patient with Uveitis, 103
9  H. Nida Sen
H. Nida Sen 28 Retinal Vasculitis, 350
Alan G. Palestine
29 Serpiginous Choroiditis, 368
PART 4 Infectious Uveitic Conditions Scott M. Whitcup
30 White-Dot Syndromes, 378
10 Bacterial and Fungal Diseases, 116 Alan G. Palestine
Scott M. Whitcup 31 Masquerade Syndromes, 395
11 Spirochetal Diseases, 127 Scott M. Whitcup
Scott M. Whitcup
12 Acquired Immunodeficiency Syndrome, 145 Appendix, 408
Scott M. Whitcup Index, 410
13 Acute Retinal Necrosis and Progressive Outer Retinal
Necrosis, 160
Scott M. Whitcup

v
F OR EWOR D

Our knowledge of basic immunology, the human immune response, Part 4 focuses on infectious causes of uveitis. It covers the usual
and how to either augment or inhibit this response has exploded suspects in uveitis like syphilis and tuberculosis (TB), with up to date
over the last decade. These advances have led to changes in the way information on diagnostic tests and antimicrobial therapy. It also cov-
we assess and treat patients with uveitis. In addition to furthering our ers new infectious causes of uveitis like Ebola and Zika.
understanding of the pathogenesis of a large number of uveitis condi- The last section of the book, Part 5, covers noninfectious causes of
tions, we have been challenged with new infectious causes of uveitis uveitis, more specifically, those not caused by an active acute infection.
diseases caused by viruses including the Zika virus, Ebola virus, and In addition, the section includes chapters on anterior uveitis, scleritis,
West Nile virus. Finally, advances in diagnostic testing and imaging and the masquerade syndromes.
have changed the way we diagnose uveitis and monitor our patients’ I met Bob Nussenblatt at the ARVO meeting in 1976 and for over
response to therapy. It is the perfect time for a new edition of Whitcup four decades was able to follow his academic and scientific career as he
and Nussenblatt’s Uveitis: Fundamentals and Clinical Practice. I was flourished at the National Eye Institute and developed an international
very happy to see Dr. Whitcup take on the leadership role for the new reputation as a model for the clinician-scientist. One of his many qual-
5th edition, and for ensuring that this important book continues to be ities was his ability to identify and nurture colleagues both nationally
available to clinicians around the world. and internationally, forming other great leaders in the field of uveitis.
This textbook remains the go-to guide for diagnosing and man- This book is part of Bob’s legacy. Bob and Scott Whitcup had simi-
aging patients with uveitis. It is both comprehensive, covering all the lar backgrounds, both completing residencies in internal medicine in
key topics related to uveitis, but also practical, readable, and useful to addition to ophthalmology. This solid knowledge of medicine, oph-
both ophthalmologists and other clinicians managing patients with thalmology, and basic science contribute to the strong foundation of
inflammatory eye diseases. I have known and worked closely with both the book. Importantly, their high ethical standards, always placing the
Dr. Whitcup and Dr. Nussenblatt for over 40 years, and spent time patient first, gives the book a unique tone and perspective. Two giants
seeing patients with both of them for many years. Their organized and working together under a friendship and professional partnership for
thoughtful approach to diagnosing, treating, and managing patients decades also comes through in reading the book. In spite of working at
with uveitis is clearly elucidated in this book. Although Dr. Nussenblatt one of the most advanced technological medical centers in the world,
was not involved with this edition of the book, having passed away Bob never lost his special and humble way of interacting with patients
suddenly in 2016, his imprint on the book continues. and colleagues in a friendly way. That was Bob. I am sure he would
Part 1 of the book provides an overview of the basic science of ocu- continue to be proud of Scott, Nida, and Alan and the latest edition
lar immunology with specifics on the role of the immune response on of the book. This edition under the leadership of Scott will continue
disease pathogenesis. It also discusses advances in our understanding to help ophthalmologists practice their best medicine, exactly as Bob
of ocular inflammation and how these advances have led to novel ther- did his whole life.
apies for autoimmune and inflammatory disease. I was happy to hear that Dr. Nida Sen had become involved with
Part 2 of the book concentrates on the diagnosis of uveitis. As this edition of the book. Nida is an outstanding clinician-scientist and
expected, the section discusses advances in testing such as the use heads the uveitis program at the National Eye Institute. I was also
of polymerase chain reaction (PCR) in diagnosing infectious causes pleased to see that Alan Palestine has contributed several chapters to
of uveitis. However, an important part of this section is a practi- the book. As with Dr. Whitcup, Dr. Palestine spent time at the National
cal approach to diagnosis by detailing tips on obtaining the medical Eye Institute and has been a major contributor to the uveitis literature
history, examining the uveitis patient, and developing a differential for several decades. In summary, Whitcup and Nussenblatt’s Uveitis is
diagnosis. We as clinicians tend to overlook the benefits and pitfalls both an authoritative review of the subject with a practical approach
of diagnostic testing, and this is beautifully discussed in the chapter on that clinicians will find useful. It is a book that I and others frequently
diagnostic testing. Finally, a new chapter on the use of optical coher- pull off the shelf and that improves the care of our patients.
ence tomography (OCT) in uveitis has been added in this edition.
Part 3 of the book concentrates on the treatment of uveitis: both Rubens Belfort Jr, MD, PhD, MBA
medical and surgical. What is useful in this section is that in addition to Vision Institute, Federal University of Sao Paulo, Brazil
the multitude of new immunotherapies, the book also supplies a phi- President, National Academy of Medicine, Brazil
losophy and approach to treatment decisions in patients with uveitis. 2020

vi
 P R E FA C E T O T H E F I F T H E D I T I O N

Over the past decade there has been a scientific explosion in both inter- improved the treatment of uveitis. Prompt and accurate diagnosis of
est and research in immunology and the role it plays in disease. Not only uveitis remain crucial to patient outcomes. There have been a couple
have we learned more about how the immune system leads to disease of key technical advances that aid clinicians in the diagnosis of uveitis.
pathogenesis, but we have also advanced our understanding on how Greater availability and accuracy of polymerase chain reaction (PCR)
modulating the immune response can lead to meaningful improve- related tests for a number of infectious agents have improved our diag-
ments in therapeutic outcomes. This research on downregulating the nosis of infectious uveitis, and these are discussed in the chapters that
immune response has led to the development of new immunomodu- discuss viral, bacterial, and fungal causes of disease. Improvements in
latory agents that block the immune system at different sites of action optical coherence tomography (OCT) have increased the utility of the
including T cells, B cells, and cytokines. These new therapies provide instrument in characterizing inflammatory disease in the eye and in
clinicians with a number of additional therapeutic options for treating assessing the response to therapy.
a host of immune-mediated inflammatory diseases including uveitis. There have also been changes in how we deliver drugs locally to
However, from an immunologic perspective, the last 10 years of the eye. The treatment of uveitis has always utilized local therapy of
research are likely to be remembered as the decade of cancer immu- drugs to the eye. This started with topical corticosteroids for anterior
notherapy. Data now show that augmenting the existing antitumor uveitis in the 1950s followed by periocular corticosteroid injections for
immune responses results in the most significant clinical responses intermediate and posterior uveitis. Starting with the use of intravitreal
and the best opportunities for long-term disease remission and cure. injections of ganciclovir and foscarnet, uveitis specialists became aware
These immunotherapies include the checkpoint inhibitors and chi- of both the safety and efficacy of delivering drugs into the vitreous for
meric antigen receptor T-cell therapy (CAR-T). But how do these treating CMV retinitis. Clinical trials of intravitreal injections of the
new immunotherapies relate to uveitis? First, uveitis has occurred as anti-VEGF agents added to the safety and efficacy of this approach.
an adverse effect of some immunotherapies. For example, both ante- Today, intravitreal injections of triamcinolone or sustained-release
rior and posterior uveitis have been reported in patients treated with corticosteroid implants is increasing, while the use of periocular injec-
checkpoint inhibitors. These observations provide further insight into tions of corticosteroids is decreasing.
the immune mechanisms of ocular inflammatory disease and identify As with previous editions of Uveitis: Fundamentals and Clinical
novel potential therapeutic targets for uveitis. Second, it is interesting Practice, our goal remains the same: to provide a comprehensive text
that this new-found success in cancer therapy is based on enhancing that provides both a practical and coherent approach to the diagnosis
the body’s own anticancer immune response. This is analogous to and treatment of the various forms of the disease. The book reviews
advances in treating autoimmune and inflammatory diseases. Despite the basic immunology of uveitis but focuses on the clinical aspects of
development of new immunosuppressive drugs like methotrexate and the underlying science. We also hope that our book will be of value to
cyclosporine for inflammatory diseases, corticosteroids remain the clinicians and scientists outside of eye care, including internists, rheu-
mainstay of treatment for most forms of uveitis today. Hench and col- matologists, and researchers in the field of inflammation.
leagues described the antirheumatic effects of the hormones released The book is divided into five parts. The first part reviews the fun-
by the adrenal gland including adrenocorticotropic hormone (ACTH) damentals of immunology and pathogenesis of ocular inflammatory
in 1949 and corticosteroids were used to treat uveitis shortly thereafter. disease. The second part focuses on diagnosis, including medical his-
Similarly, recent advances in the treatment of autoimmune diseases tory, clinical examination, and diagnostic testing. A chapter on evi-
have resulted not from the synthesis of new anti-inflammatory com- dence-based medicine in uveitis is also included in this section. Part 3
pounds, but by specifically modulating the patient’s immune response, reviews the principles of medical therapy for uveitis and details the
for example, with monoclonal antibodies against key proinflammatory commonly used drugs. A discussion of the role of surgery in the man-
cytokines like tumor necrosis factor (TNF)-alpha, IL-17, and IL-23. agement of uveitis is also included in this part. The infectious causes of
It is clear that studies on the pathogenesis of uveitis have furthered uveitis are included in the chapters in Part 4. These include bacterial,
our understanding of ocular immunology and provided novel targets viral, fungal, and parasitic diseases. This section also includes a chap-
for therapeutic interventions, but this science has been the foundation ter on postsurgical causes of uveitis, because many of these are due to
for examining the role of inflammation in other nonuveitis conditions infection. Finally, the last section includes chapters on uveitis condi-
including age-related macular degeneration (AMD) and diabetic reti- tions that are predominantly caused by noninfectious etiologies: often
nopathy. AMD is the leading cause of central vision loss in developed autoimmune diseases or masquerade syndromes like cancer or toxic-
countries and is a great example of how our understanding of ocular ity. However, it is important to note that some of these conditions, like
immunology has augmented our knowledge about diseases other than white dot syndromes, may have infectious causes.
uveitis. Genetic data from genome wide association studies and rare There have been a number of important additions and updates to
variant analyses pointed to involvement of the complement system in this edition of the book. Because of the increase in use of OCT in both
AMD. Other studies have supported the role of inflammation in the the evaluation and management of the uveitis patient, a new chapter
development or severity of AMD including inflammasome activation on OCT has been added (Chapter 6). With advances in our under-
and dysregulated para-inflammation. Para-inflammation was defined standing of immunology and the development of a number of new
by Medzhitov as a low level of inflammation that can exist between medications for the treatment of uveitis, the chapters on fundamentals
basal homeostasis and a progressive and more active inflammatory (Chapter 1) and medical therapy (Chapter 8) have been extensively
response. These data have supported the development of immune updated. Chapters in the section on infectious causes of uveitis have
strategies for the treatment of atrophic AMD, including drugs to block also been extensively revised, incorporating information on new diag-
the complement system. nostic testing such as PCR. Furthermore, there have been a number
In addition to progress in our understanding of the immune of new infectious causes of uveitis, like Ebola virus, and most recently
response, there have been a number of other advances that have severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), that

vii
viii PREFACE TO THE FIFTH EDITION

have been added to this section. Importantly, we have added many new She is an accomplished researcher and clinician and is an integral part
figures to better illustrate clinical features of uveitis and added new to this fifth edition. It is also great to have Alan involved in this 5th
case histories where useful. edition of the book. Dr. Palestine is currently the Chief of the Uveitis
This edition of Whitcup and Nussenblatt’s Uveitis: Fundamentals and Ocular Immunology Section and Director of Resident Research
and Clinical Practice is the first I have written without my colleague at the University of Colorado School of Medicine. Alan was involved
and friend Bob Nussenblatt. Bob passed away in 2016. To those of us with the early editions of the book and brings a wealth of practical
who know Bob, we have lost a friend who inspired us every day. The experience to some key chapters of this edition. I also want to thank
entire clinical immunology community has lost a great researcher, Chi-Chao Chan, Igal Gery, and Rachel Caspi, whose knowledge,
teacher, and clinician. Bob arrived at the National Institutes of Health inquisitiveness, and insight contributed to the evolution of this text-
(NIH) in 1977 as a clinical associate and held key leadership posi- book. As a friend and colleague, I greatly appreciate that Dr. Rubens
tions there, including chief of the National Eye Institute Laboratory of Belfort has both contributed many clinical photographs and written
Immunology, Clinical Director, Scientific Director, Deputy Director the foreword to the book. This edition has a new chapter on the use of
for Intramural Research, and an NIH Distinguished Investigator. He OCT in uveitis, and I thank Elias Reichel for contributing a number
was a mentor who loved teaching and a clinician-scientist who focused of OCTs for the chapter and reviewing the text. I also must thank the
on the bench to bedside path every day. At its heart, the book stems staff of the NEI, including the technicians and photographers. Most
from patient discussions with Bob at the National Eye Institute (NEI). importantly, I would like to thank our patients who have participated
My hope is that the heart of the book remains. in clinical research and valued the opportunity to contribute to our
I would like to thank a number of people who are important to understanding of uveitis in an attempt to help others.
this edition of the book. First, I would like to thank the other two Finally, I would like to thank family and friends for their support
contributors to the book, Dr. H. Nida Sen and Dr. Alan Palestine. of this latest edition and again to Bob for his continued inspiration.
Nida is the director of the Uveitis Fellowship Program at the NEI and
Professor at the George Washington University School of Medicine. Scott M. Whitcup, MD
 ACKNOWLED GMENTS

We would like to thank our colleagues for their input over the years and for supplying many of the outstand-
ing images that help bring our text to life. We especially want to thank Igal Gery and Chi-Chao Chan for
their assistance with the basic science of the book, Elias Reichel for his review of the new chapter on ocular
coherence tomography, and to Rubens Belfort Jr. for authoring the Foreword for our book. We also thank
the photographers and ophthalmic technicians of the National Eye Institute for their assistance in obtaining
photographs, angiograms, OCT images, and other materials for the book. We also would like to thank Julie
Taylor, Louise Cook, and the staff at Elsevier for their expertise and assistance in putting the book together.
Importantly, we thank our patients for their willingness to participate in clinical trials and contribute to our
understanding of uveitis and its treatment in the quest to help others.

ix
 We dedicate this fifth edition of the book to
Robert B. Nussenblatt.
Bob was a founding leader in ocular immunology, an outstanding scientist,
a caring physician, and a dedicated teacher. He treasured learning, valued
­scientific collaboration, and loved his family. Bob saw the importance of a
­uveitis textbook that combines the basic science of immunology and ocular
inflammatory disease with clinical discussions focused on the patient.
We hope that this edition of the book continues to educate
and help improve the lives of our patients.
To our families, friends, colleagues, and patients.
 PART 1 Fundamentals

1
Elements of the Immune System and
Concepts of Intraocular Inflammatory
Disease Pathogenesis
H. Nida Sen

KEY CONCEPTS
• T cells play an important role in the pathogenesis of uveitis. • Uveitogenic antigens are expressed in the eye, and immunization
• The eye is very active immunologically, with ocular resident cells of animals with these antigens induces experimental uveitis, which
interacting with the immune system. resembles human uveitis in many aspects.
  

In an ever-­changing field, a review of the immune system is the subject system’s appropriate response to outside invaders (e.g., Toxoplasma)
of numerous books, courses, and scientific articles. However, certain and the other part deals with understanding (and trying to explain) the
principles have been established, and in the main, these have survived response to autoantigens. The dynamic is not as simple as outlined; in
the test of time and rigorous scrutiny. The aim of this chapter is to pro- fact, it starts as an appropriate response to a foreign antigen and then
vide the reader with the essentials needed to follow a discussion on the changes to an abnormal response against the eye. Many mechanisms,
mechanisms proposed for intraocular inflammatory disease; therefore such as molecular mimicry, have been proposed.
topics relevant to the understanding of that subject are addressed. In Achieving this complex but highly specific immune response
addition, selected themes thought to be important in understanding requires multiple players. Some of these are reviewed in the first part of
the unique ocular immune environment and pathogenesis are covered. this chapter. In the second part, findings and theories of disease mech-
The development of the immune system is an extraordinary prod- anisms relevant to ocular diseases are introduced and discussed.
uct of evolution. Its goal is to recognize what is different from self, so
its initial role is to respond to foreign antigens with an innate immune
ELEMENTS OF THE IMMUNE SYSTEM
response that is geared to rapidly clear the body of the foreign invader.
“Innate immunity” is restricted to the non–antigen-­specific immune The immune system consists of several cell types, including macro-
response, including phagocytic cells that engulf and destroy invaders phages, dendritic cells, polymorphonuclear cells, and a variety of lym-
(macrophages and polymorphonuclear cells [PMNs]); populations phocyte lines with specific functions. These components add up to a
of lymphocytes that include innate lymphoid cells [ILCs], i.e., natu- complex immune circuitry or “ballet,” which, in the vast number of
ral killer [NK] cells and natural killer T cells (NKTCs); and humoral individuals, responds in a way that is beneficial to the organism. The
factors, such as the complement system, and the receptors on antigen-­ interactions among these cells and their movements are mediated by
presenting cells, such as phagocytes, called toll-­like receptors (TLRs), several families of molecules, including cytokines, chemokines, and
which interact with the invaders’ molecules. This activates the antigen-­ adhesion molecules.
presenting cell to initiate the “adaptive” immune response. Clearly, the
invader may return, in which case the adaptive immune response is in Macrophages/Monocytes
place to respond. The adaptive immune response is antigen specific Phagocytic cells originate in bone marrow. The concept that phago-
and deals with the invaders that escaped the innate immune mech- cytosis is important for the immunologic defense of the organism
anism or have returned. The adaptive immune response consists of was proposed by Metchnikoff at the end of the nineteenth century.
both B and T cells, and portions of these populations acquire the prop- The macrophage, which is relatively large (15 μm), has an abundant
erties of the memory cells of the secondary immune response. This smooth and rough endoplasmic reticulum. Lysosomal granules and
adaptive immune response connotes an immune memory, hence the a well-­developed Golgi apparatus are also found. Several functional,
development of a complex way in which high-­affinity molecules and histochemical, and morphologic characteristics of these cells can be
cell-­surface markers can distinguish between the invader and self. A noted (Table 1.1). In addition to the phagocytic characteristics already
given of this concept is that self-­antigens are not attacked; that is, an alluded to, these cells contain esterases and peroxidases and bear mem-
immune tolerance exists. Part of our story deals with the immune brane markers that are typical of their cell line (i.e., OKM1 antigen

1
2 PART 1 Fundamentals

participating. Other cell-­surface markers are needed for activation. This

TABLE 1.1 Macrophage Characteristics
“two signal” theory has centered on other cell-­surface antigens, such
Surface as the B7–CD28 complex. The engagement of B7 (on the macrophage
Histochemical Antigens Receptors Functions side) with CD28 enhances the transcription of cytokine genes. Third,
5´-­Nucleotidase OKM1 Fc Phagocytosis the macrophage is a potent secretory cell. Proteases can be released in
abundance, and this can degrade vessel surfaces and perivascular areas.
Esterase Class II antigens Immunoglobulin Pinocytosis
Degradation products that result from these reactions are chemotactic
M (IgM)
and further enhance an immune response. Interleukin (IL)-­1, a mono-
Alkaline phosphodies- Lymphokine Immune activation kine with a molecular weight of 15 kilodaltons (kDa), is produced by the
terase macrophage (and other cells) after interaction with exogenous pathogens
Aminopeptidase Lactoferrin Secretory or internal stimuli, such as immune complexes or T cells. IL-­1 release
Insulin Microbicidal directly affects T-­cell growth and aids this cell in releasing its own secre-
tory products. IL-­1 is noted to act directly on the central nervous system
Cb3 Tumoricidal
(CNS), with a by-­product being induction of fever. Still other macro-
Fibrinogen phage products stimulate fibroblast migration and division, all of which
Lipoprotein have potentially important consequences in the eye.
Macrophages also produce IL-­12, IL-­18, IL-­10, and transforming
growth factor (TGF)-­β. In a feedback mechanism, interferon (IFN)-­γ
can activate macrophages, and the production of IL-­12 by the macro-
6WHPFHOO
phage plays an important role in T-­cell activation. The role of macro-
phages in the eye remains to be fully explored.
3URPRQRF\WH
Dendritic Cells
0RQRF\WH 0DFURSKDJH
Although macrophages play an important role, it is conjectured that
dendritic cells are important macrophage-­like cells in tissues. They
2UJDQVSHFLILF 6LQXVOLQLQJ "3RVVLEOH are a subset of cells, perhaps of different lineage from macrophages,
$OYHRODU 6SOHQLF /DQJHUKDQV¶ VNLQ and they can be distinguished by lack of persistent adhesion and by
3HULWRQHDO /\PSKQRGH 0LFURJOLD &16 the bearing of an antigen, 33D1, on their surface, features that macro-
0HVDQJLDO NLGQH\ %RQHPDUURZ 'HQGULWLF O\PSKRLG phages do not possess. The major role of dendritic cells is to serve as
.XSIIHUFHOOV OLYHU 2VWHRFODVWV APCs for both CD4+ and CD8+ cells. Like macrophages, dendritic cells
Fig. 1.1 Macrophage differentiation. produce IL-­12, an important activator of T-­cell responsiveness. They
are rich in MHC II intracellular compartments, an important factor
and F4/80). Other cell-­surface markers are also present, such as class II in antigen presentation. The MHC class II compartments move to the
antigens, Fc receptors (for antibody), and receptors (for complement). surface of the cell when the dendritic cell matures, stimulated by IFN-­γ
These enzymes and cell markers help identify this class of cells and their and the CD40 ligand. Dendritic cells are special in that they inhabit
state of activation. The presence of esterase is a useful marker to dis- tissues where foreign antigens may enter. Experiments with painting
tinguish macrophages from granulocytes and lymphocytes. Monocytes of the skin brought seminal observations. Antigens painted on the skin
will leave the bloodstream because of either a predetermined matura- are “brought” to the draining lymph nodes by the dendritic cells of
tional process or induced migration into an area as a result of chemo- the skin (Langerhans’ cells) where T-­cell activation can occur. What
tactic substances, often produced during inflammatory events. After is interesting is the migratory nature of these cells: They constantly
taking up residence in various tissues, they become macrophages, carry important information to the peripheral centers of the immune
which are frequently known by other names (Fig. 1.1). Dendritic cells, response. Whether dendritic APCs can activate T cells efficiently in the
such as Langerhans’ cells, are found in the skin and the cornea and play tissues themselves is an open question, and the answer is important to
an important role in presenting antigens to lymphocytes. our understanding of immune responses in the eye. Dendritic cells are
Macrophages play at least three major roles within the immune sys- thought to be the APCs (or one of the major players) in corneal graft
tem. The first is to directly destroy foreign pathogens and clear dying or rejection. Thus the concept of removing dendritic cells from a graft
diseased tissue. Killing of invading microbes is, in part, mediated by a has been proposed and used in experimental models. However, there
burst of hydrogen peroxide (H2O2) activity by the activated macrophage. is an opposing concept that peripheral immune tolerance, induced by
An example with ocular importance is the engulfment of the toxoplas- antigens that foster programmed cell death (apoptosis), may depend
mosis organism, with the macrophage often being a repository for this on presentation of antigen by dendritic cells in the tissue.
parasite if killing is inadequate. The second is to activate the immune
system. Macrophages, dendritic cells, or other cells with similar char- T cells
acteristics are mandatory for antigen-­specific activation of T lympho- T-­cell responses to antigens provide the basis for a large part of the
cytes. Internalizing and processing of the antigen by the macrophage are inflammatory process. They are generated in bone marrow and
thought to be integral parts of this mechanism, and the macrophage or mature in the thymus, the first lymphoid organ to develop. The thy-
the dendritic cell is named antigen-­presenting cell (APC). Other cells, such mus consists of two compartments, the cortex and the medulla. In
as B cells, can also serve this function. The macrophage and the lympho- the cortex, immature thymocytes develop through a complex process;
cyte usually need to be in close contact with each other for this transfer to their T-­cell receptors (TCRs; see later) then interact with thymic epi-
occur. Another requirement is for the cells to have in common a signif- thelium, a process that determines their becoming CD4 (“helping”)
icant portion of their major histocompatibility complex (MHC), genes or CD8 (“cytotoxic”) T cells. Thymocytes that fail this process die
that express various cell-­surface membranes essential for cellular commu- by apoptosis (“positive selection”), whereas thymocytes that succeed
nication and function. Thus this MHC stimulation leads to the initiation in this selection migrate to the thymic medulla, where epithelial and
of an immune response, ultimately with both T and B cells potentially dendritic cells express all the body’s major autoantigens. Thymocytes
 CHAPTER 1 Elements of the Immune System 3

BALB/c B10.A B10.RIII AKR/J (2) the retina being isolated from circulating cells by the blood–retina
E T E T E T E T barrier. The normal presence of T cells is indicated by the finding of T
cells specific to retinal antigens in healthy individuals with no eye disease.
S-Ag 595 bp
One important quality possessed by T cells is their immunologic
IRBP 658 bp recall or anamnestic capacity after re-­exposure to their specific target
antigen. The exposure to the antigen increases the number of specific
-actin 650 bp cells and changes them into a “memory” phenotype. A memory T
cell to a particular antigen can retain this immunologic memory (see
Fig. 1.2 Transcription of S-­ antigen (S-­Ag) and interphotoreceptor
retinoid-­
binding protein (IRBP) genes (uveitogenic antigens) in eyes
later) essentially for its lifetime. With a repeat encounter, this memory
and thymuses of mouse strains. S-­Ag and IRBP are abundant in the response leads to an immune response that is more rapid and more
eyes of all animals and S-­Ag is found in the thymuses of all four strains pronounced than the first. An example is the positive skin response
tested. However, IRBP was seen only in thymuses of two strains – seen after purified protein derivative (PPD) testing.
BALB/c and AKR/J – and not in those of B10.A or B10.RIII. The last The central role of the T cell in the immune system cannot be overem-
two animals are susceptible to induction of uveitis with IRBP. (From phasized. T cells function as pivotal modulators of the immune response
Egwuagu CE, Charukamnoetkanok P, Gery I. Thymic expression of auto- by helping production of antibody by B cells and augmenting cell-­
antigens correlates with resistance to autoimmune disease. J Immunol. mediated reactions through the release of molecules, named cytokines,
1997;159:3109–3112. Copyright 1997, The American Association of which activate immune-­related and other cells. T cells also may downreg-
Immunologists, Inc.)
ulate or prevent immune reactions through active suppression. (i.e., Treg
cells). The cytotoxic (CD8) T-­cell subset plays a major role in transplan-
expressing TCRs with strong affinity to autoantigens are deleted (“neg- tation rejection crises. Accumulated evidence supports the importance of
ative selection”), and the remaining T cells enter the lymphoid system. T cells in many aspects of the intraocular inflammatory process – from
Importantly, the negative selection is incomplete and T cells specific to the propagation of disease to its subsequent downregulation.
autoantigens do escape the negative selection (see later). A state of suspended animation can be induced in T cells; this is
A major component of the negative selection system is the auto- termed anergy. For T cells to be activated, several signals need to be
immune regulator (AIRE), a protein that is produced by medullary given: one through the TCR and the other through costimulatory
cells and that controls the expression of organ-­specific antigens. Loss receptors, such as CD28; the third is the costimulant B7 linking to
of the AIRE gene leads to autoimmunity,1 which is known to occur CD28 (which is on the T cell). If the TCR is activated but the costim-
in humans who develop autoimmune polyglandular syndrome (APS) ulant is not, a growth arrest can be seen in these cells: They simply
type I, an autoimmune disease that is inherited in an autosomal reces- stop functioning but do not die. A second way this can occur is when
sive fashion. In addition to adrenal insufficiency, mucocutaneous a weakly adherent peptide is linked to the TCR, even if costimulation
infections, and hypoparathyroidism, these patients can have diabetes, occurs. It would seem to be a mechanism to prevent unwanted or nui-
Sjögren syndrome, vitiligo, and uveitis.2 sance immune responses. The full response takes place only if all the
The expression of ocular self-­antigens in the thymus was investi- appropriate interactions have occurred.
gated in both mice and humans. Egwuagu et al.3 have shown in differ-
ent mouse strains an inverted relationship between thymic expression T-­cell Receptor
of ocular-­specific retinal antigens and the susceptibility to induction Much interest has focused on the TCR. T cells need to produce the TCR
of experimental autoimmune uveitis (EAU): Thymic expression of on their cell surface to recognize the target immunogenic peptide on the
retinal antigens causes lack of responsiveness to these antigens. An MHCs of APCs. This complex interaction involves either CD4 or CD8
example of this phenomenon is shown in Fig. 1.2. Four inbred strains and their TCRs. The TCR is similar in structure to an immunoglobulin,
of mice were evaluated for the expression in their thymus of two uve- having both α and β chains. The more distal ends of these chains are
itogenic antigens, interphotoreceptor retinoid-­binding protein (IRBP) variable, and the hypervariable regions are termed V (variable) and J
and S-­antigen (S-­Ag). All four strains were resistant to the induction (joining) on the α chain and V and D (diversity) regions on the β chain.
of uveitis when S-­Ag was used as the immunizing antigen, and all four Compared with the number of immunoglobulin genes, there are fewer
expressed S-­Ag in their thymus. However, two of the four strains, V genes and more J genes in the TCR repertoire. It is logically assumed
B10.A and B10.RIII, did not express IRBP in their thymuses and were that the target peptide, which has a special shape and therefore fits spe-
susceptible to uveitis induction when IRBP was used as the immu- cifically in a lock-­and-­key fashion into the groove between the MHC
nizing antigen. These observations were extended to include other and the TCR, would be the “cement” of this union. It has been suggested
rodents and primates.4 In the Lewis rat, which is susceptible to both that of all the possible combinations of gene arrangements that could
antigens, neither message was found in the thymus. These observations possibly produce the variable region believed to cradle the peptide, cer-
may provide an insight into the propensity for the disease in humans. tain genes within a family seem to be noted more frequently in autoim-
Takase et al.5 evaluated 18 human thymus samples taken from patients mune disease. One such group is the Vα family, with Vβ8.2 receiving
undergoing surgery for congenital heart disease. They found that there much attention. A small number of cells have a TCR made up not of α
was expression of the four antigens that can induce experimental uve- and β chains but, rather, of γ and δ chains (detailed later). In addition
itis (S-­Ag, recoverin, RPE65, and IRBP) in the thymuses of the tested to these physiologic mechanisms, T cells may also be activated by “supe-
patients. However, the expression of the various antigens was very rantigens,” which are bacterial products, such as enterotoxins, or plant
variable, with some thymus samples showing strong expression and products, such as phytohemagglutinin. In addition, T cells may be acti-
others not. The implication of the findings from these studies is that vated by antibodies to certain surface antigens, mostly CD3 and CD28.
expression of these antigens in the thymus is very variable in humans,
similar to what is seen in the differences among various rodent strains. Major Populations of T Cells
T cells with specificity to ocular self-­antigens that escape the nega- The functions that have been briefly described are carried out by sev-
tive selection are found in the circulation, but do not induce uveitis. This eral subsets of CD4 T cells, identified by their products and functions.
observation is explained by two mechanisms: (1) the inhibitory effect of It was observed early on that T cells (and other cells) manifest myriad
T-­regulatory (Treg) cells, which are normally present in the body; and different molecules on their surface membranes, some of which are
4 PART 1 Fundamentals

TABLE 1.2 Selected human leukocyte expressed uniquely at certain periods of cell activation or function. It
was noted that certain monoclonal antibodies directed against these
Differentiation antigens (Incomplete list)
unique proteins bind to specific subsets of cells, thereby permitting a
Cluster Main Cellular Associated way to identify them (Table 1.2). The antibodies to the CD3 antigen in
Designation Distribution Functions humans (e.g., OKT3) are directed against an antigen found on all mature
CD3 T cells, thymocytes Signal transduction
human T cells in the circulation; approximately 70% to 80% of lympho-
cytes in the systemic circulation bear this marker. Antibodies to the CD4
CD4 Helper T (Th) cells MHC class II coreceptor
antigen (e.g., OKT4) define the helper subgroup of human T cells (Th
CD8 Suppressor T cells, MHC class I receptor cells; about 60%–80% of the total T cells). These CD4+ cells respond
cytotoxic T cells to antigens complexed to MHCs of the class II type. The CD4 cells are
CD11a Leukocytes LFA-1, adhesion molecule particularly susceptible to human immunodeficiency virus (HIV) that
CD11b Granulocytes, MΦ Mac-1, adhesion molecule causes acquired immunodeficiency syndrome (AIDS), with the per-
centage of this subset decreasing dramatically as this disease progresses.
CD11c Granulocytes, MΦ, α integrin, adhesion
Furthermore, these helper cells are necessary components of the autoim-
T cells, B cells molecule
mune response seen in the experimental models of ocular inflammatory
CD19 B cells B-­cell activation disease induced by retinal antigens (see discussion of autoimmunity later
CD20 B cells B-­cell activation in this chapter). Antibodies to the CD8 antigen (i.e., OKT8) distinguish
CD22 B cells B-­cell regulatory a population that includes cytotoxic T cells, making up about 20% to
CD25 T cells, B cells —α chain of IL-­2 receptor 30% of the total number of T cells. Antibodies directed against the CD8
(Tac) activation antigen block class I histocompatibility-­associated reactions.
The two major populations of T cells (CD4 and CD8) are further
CD28 T cells Costimulatory T-­cell marker
divided into subpopulations that are detailed later. These subpopula-
CD45 Leukocytes Maturation tions are generated by combinations of cytokines, which are products
CD54 Endothelial, dendritic, ICAM-1, adhesion molecule; of other cells, and affect the immune system by the specific cytokines
and epithelial cells; ligand of LFA-1 and Mac-­1 they produce.
­activated T and B cells
CD56 NK cells N-­CAM, adhesion molecule
T-­cell Subsets
The population of Th cells has been further subdivided on the basis of
CD68 Macrophages
their functional characteristics into several subsets. The major subsets
CD69 NK cells, lymphocytes Signal transmission receptor are named Th1, Th2, and Th17. In normal conditions, Th1 cells defend
CX3CR1 Monocytes Chemoattractant against intracellular pathogens, Th2 cells defend against extracellular par-
CXCR3 T cells Cell maturation asites and mediate antibody production, and Th17 cells defend against
CCR7 T cells Migration to inflammation extracellular pathogens. Pathologically, Th1 and Th17 cells are respon-
sible for initiation of “cell-­mediated” immune responses, such as foreign
CCR5 T cells Chemokine receptor
tissue rejection and pathogenic autoimmune processes, whereas Th2
ICAM, intercellular adhesion molecule; IL, interleukin; LFA, lymphocyte cells are involved in allergic responses and in immunoregulation. Th1
function-­associated molecule; MHC, major histocompatibility complex; cells (Fig. 1.3) produce mostly IFN-­γ, IL-­2, and tumor necrosis factor-­α
N-­CAM, neural cell adhesion molecule; NK, natural killer.

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entiating helper T cells. Immunol Res. 2008;41:87, with permission.)
 CHAPTER 1 Elements of the Immune System 5

(TNF-­α). The cytokine profile of Th2 cells consists of IL-­4, IL-­5, IL-­10, IL-­ Innate Lymphoid Cells
13, and perhaps TGF-­β , and the major cytokines produced by Th17 cells The recently discovered ILCs are lymphocytes that lack antigen speci-
are IL-­17, IL-­21, and IL-­22. In many animal models of human diseases, ficity and are involved in the immune response by releasing cytokines
Th1 and Th17 cells are associated with initiation of disease, whereas Th2 or carry cytotoxic capacity (NK cells; see later). ILCs are mainly tissue
cells are associated with disease downregulation and allergy initiation. resident and play major roles in keeping the homeostasis in these tis-
Importantly, under experimental conditions, Th17 cells may switch sues.20 Unlike T or B cells, ILCs react promptly to stimulations, such
to a Th1 phenotype, but Th1 cells maintain their phenotype and do as pathogen invasion. ILCs are separated into three major groups
not change.6 (ILC1, ILC2, and ILC3) that selectively collaborate with Th1, Th2, and
IL-­22 is part of the IL-­17 group of cytokines produced during an Th17 lymphocytes in the defense against intracellular pathogens and
inflammatory response.7 Albeit made by lymphocytes, its receptors are tumors, large extracellular pathogens and allergens, and extracellular
present on epithelial cells. Thus it has been suggested that one of its pathogens, respectively.20
major roles is to be the cross-­talk lymphokine between resident tis-
sue cells and infiltrating inflammatory cells, particularly T cells. This Natural Killer and Invariant Nature Killer T Cells
proinflammatory cytokine is found in the synovia of patients with NK cells and invariant natural killer T (iNKT) cells are major com-
rheumatoid arthritis and is upregulated in both Crohn disease and ponents of the innate immune response, whose main function is to
ulcerative colitis8,9 and in both the serum and intraocular fluids of carry out the rejection by cytolytic activity of both tumors and virally
patients with uveitis.10,11 infected cells. The main difference between NK cells and iNKT cells
is their morphology, with NK cells being large granular lymphocytes,
Gamma Delta (γδ) T Cells whereas iNKT cells express highly conserved TCRs. Both populations
γδ Τ cells, which constitute a small fraction of peripheral T cells, play release large amounts of cytokines upon activation. NK cells seem to be
important roles in inflammatory processes, such as EAU.12 Of par- involved in the pathogenesis of EAU because the disease was found to
ticular importance is the involvement of γδ Τ cells in mucosal tissue be diminished in mice with no NK cells.21 Grajewski et al.22 showed that
inflammation in the conjunctiva, as shown by St Leger et al.,13 where similar to NK cells, iNKT cells ameliorate the EAU process. However,
an inflammatory response to a commensal bacterium is mediated to a iNKT cells were also found to have a dual effect in the pathogenic pro-
large extent by IL-­17 produced by γδ cells. cess of EAU: The disease was enhanced in iNKT-­deficient mice, but
activation of these cells also exacerbated the pathologic process.23
T-­regulatory Cells
It is now clear that just as the immune system needs cells to initiate a Cytokines
response, it needs cells to suppress or modify immune responses. One Intercellular communication, which is crucial for active immune
of the ways this need is met is with Treg cells.14 These cells derive from a response, is mediated by cytokines, chemokines, and adhesion mol-
naive T-­cell population under the influence of cytokines that are differ- ecules. Cytokines are produced by lymphocytes, macrophages, and
ent from those involved in Th1, Th17, or Th2 cell development (see Fig. other cells. They are hormone-­like proteins capable of amplifying an
1.3). Treg cells can be found in the thymus or in the peripheral circula- immune response and of suppressing it. With activation of a T lym-
tion, where a large portion is “induced” (iTregs). An interesting report phocyte, the production and release of various lymphokines will occur.
by Kemper et al.15 described stimulating CD4+ cells with CD3 and One of the most important cytokines is IL-­2, with a molecular weight
CD46 (a complement regulator) and inducing Treg cells, that is, pro- of 15 kDa in humans. The release of this lymphokine stimulates lym-
ducing large amounts of IL-­10, moderate amounts of TGF-­β, and little phocyte growth and augments specific immune responses, including
IL-­2. The literature is replete with information about different types of stimulation of Treg cells. Of particular interest are cytokines involved
Treg cells, and these cells have been reported in several organs, such in the inflammatory process. They include proinflammatory cytokines,
as the gut, where peripheral immune tolerance needs to be induced.16 IFN-­γ, IL-­1, IL-­17, and TNF-­α and the anti-­inflammatory IL-­4, IL-­5,
Certain Treg cells are characterized by their ability to produce IL-­10 IL-­10, and TGF-­β. Of interest, IFN-­γ and TGF-­β are active in both
and TGF-­β. They are capable of downregulating both CD4-­and CD8-­ activation and suppression of immune responses. The number of
mediated inflammatory responses and apparently require cell-­to-­cell lymphokines that have been purified and for which effects have been
contact. The majority of Treg cells bear CD25 (the IL-­2 receptor) on described continues to grow rapidly. An incomplete list is shown in
their cell surface and express the transcription factor forkhead/winged Table 1.3 and a more recent list has been provided by Akdis et al.24
helix (FoxP3),17 which is a reliable marker for the development and
function of naturally occurring Treg cells.18 When we evaluated the T Chemokines
cells of patients with ocular inflammatory disease, we found that the This family of chemoattractant cytokines is characterized by its ability
FoxP3 marker varied considerably among patients and was not a very to induce directional migration of movable cells. They direct cell adhe-
good indicator of poor Treg cell function.19 An interesting observation sion, homing, and angiogenesis. There are four major subfamilies of
is the noted increase in a subset of NK cells (so-called CD56 “bright”) chemokines: CXC (nine of which are found on chromosome 4); CC
after daclizumab therapy; this subset makes large amounts of IL-­10, (11 of which are found on chromosome 17); C (only one well-­defined
indicating the regulatory nature of these cells. The increase is seen member, lymphotactin, is found on chromosome 11); and CX3C (frac-
when the patient’s disease is well controlled, and it has also been seen talkine is found on chromosome 16). The nomenclature is based on
in patients with multiple sclerosis receiving daclizumab therapy. the cysteine molecules. The CC chemokines have two adjacent cysteines
at their amino terminus; the CXC chemokines have their N-­terminal
Lymphocytes of Innate Immune System cysteines separated by one amino acid; the C chemokines have only
In addition to adaptive immunity cells and molecules, mentioned ear- two cysteines, one at the terminal end and one downstream; the CX3C
lier, the protection against invasion of pathogenic agents is carried out chemokines have three amino acids between their two N-­terminal cys-
by components of “innate” immunity that lack antigenic specificity but teines. Each chemokine family has special functions that affect different
are capable of providing immunity. The cell populations of the innate types of cells. An example of this fine specificity is seen with the CXC
immunity are discussed in the following sections. family. These chemokines, with a Glu–Leu–Arg sequence near the end
6 PART 1 Fundamentals

TABLE 1.3 Cytokines: An incomplete list

Type Source Target and Effect
IFN-­γ T cells Antiviral effects; promotes expression of MHC II
Antigens on cell surfaces; increases MΦ tumor killing; inhibits some T-­cell proliferation
TGF-­β T cells, resident ocular cells Suppresses generation of certain T cells; involved in ACAID and oral tolerance
Interleukin
IL-­1 Many nucleated cells, high levels in MΦ, kerati- T-­and B-­cell proliferation; fibroblasts – proliferation, prostaglandin production; CNS – fever; bone
nocyte, endothelial cells, some T and B cells and cartilage resorption; adhesion-­molecule expression on endothelium
IL-­2 Activated T cells Activates T cells, B cells, MΦ, NK cells
IL-­3 T cells Affects hemopoietic lineage that is nonlymphoid eosinophil regulator; similar function to IL-­5 GM-­CSF
IL-­4 T cells Regulates many aspects of B-­cell development; affects T cells, mast cells, and MΦ
IL-­5 T cells, eosinophils Affects hemopoietic lineage that is nonlymphoid, eosinophil regulator: similar function to IL-3 GM-­
CSF; induces B-­cell differentiation into IgG-­and IgM-­secreting plasma cells
IL-­6 MΦ T cells fibroblasts; endothelial cells, RPE B cells – cofactor for Ig production; T cells – comitogen; proinflammatory in eye
IL-­7 Stromal cells in bone marrow and thymus Stimulates early B-­cell progenitors; affects immature T cells
IL-­8 NK cells, T cells Chemoattractant of neutrophils, basophils, and some T cells; aids in neutrophils adhering to endo-
thelium; induced by IL-­1, TNF-­α, and endotoxin
IL-­9 T cells Supports growth of helper T cells; may be enhancing factor for hematopoiesis in presence of other
cytokines
IL-­10 T cells, B cells, stimulated MΦ Inhibits production of lymphokines by T helper 1 (Th1) cells
IL-­11 Bone marrow stromal cells (fibroblasts) Stimulates cells of myeloid, lymphoid, erythroid, and megakaryocytic lines; induces osteoclast
formation; enhances erythrocytopoiesis, antigen-­specific antibodies, acute-­phase proteins, fever
IL-­12 B cells, T cells Induces IFN-­γ synthesis; augments T-­cell cytotoxic activity with IL-2; is chemotactic for NK cells and
stimulates interaction with vascular endothelium; promotes lytic activity of NK cells; antitumor
effects regulate proliferation of Th1 cells but not Th2 or Th0 cells
IL-­13 T cells Anti-­inflammatory activity as IL-4 and IL-10; downregulates IL-12 and IFN-­α production and thus
favors Th2 T-­cell responses; inhibits proliferation of normal ανδ leukemic human B-­cell precur-
sors; monocyte chemoattractant
IL-­14 T cells Induces B-­cell proliferation, malignant B cells; inhibits immunoglobulin secretion
IL-­15 Variety of cells Stimulates proliferation of T cells; shares bioactivity of IL-­2 and uses components of IL-­2 receptor
IFN-­α Variety of cells Antiviral
IFN-­β Variety of cells Antiviral
IFN-­γ T and NK cells Inflammation, activates MΦ
TNF-­α MΦ Inflammation, tumor killing
TNF-­β T cells Inflammation, tumor killing, enhanced phagocytosis
ACAID, anterior chamber acquired immune deviation; CNS, central nervous system; GM-­CSF, granulocyte macrophage–colony-­stimulating factor;
IFN, interferon; Ig, immunoglobulin; MΦ, macrophage; MHC, major histocompatibility complex; NK, natural killer; RPE, retinal pigment epithelium;
TGF, transforming growth factor; TNF, tumor necrosis factor.

of the N terminus, bind well to the CXCR2 on neutrophils. CXC chemo- development of the inflammatory response. These adhesion molecules
kines not possessing that sequence are chemotactic for monocytes and are especially important for directing leukocytes to areas of inflam-
lymphocytes. IL-­8 can bind with either CXCR1 or CXCR2 chemokine mation. The upregulation of CAM expression on the vascular endo-
receptors. Organisms have adapted to these chemokines as well. HIV thelium and surrounding area allows inflammatory cells to home to
gp120 binds to CCR5 and CCR3, aiding its entry into the lymphocyte. inflamed tissues.25 CAMs are also involved in the interaction of lym-
This area of knowledge is still evolving. Clearly, cell homing has impor- phocytes and APCs, important for lymphocyte stimulation.
tance in ocular inflammatory disease but probably in other conditions CAMs are divided into three structural groups: selectins, integrins,
as well, such as diabetes and age-­related macular degeneration (AMD), and the immunoglobulin gene superfamily. The selectins are a group
in which the immune components of the disease are just being explored of CAMs that appear to mediate the initial adhesion of inflammatory
but which may be important areas for therapeutic interventions. cells to the vascular endothelium, leading to a rolling of the cells along
the vascular wall.26 The integrins and members of the immunoglobulin
Cell-Adhesion Molecules and Their Role in Lymphocyte supergene family then interact to form a more firm adherence between
Homing and in Disease the leukocytes and the vascular endothelium, leading to transendothe-
Cell-­adhesion molecules (CAMs) are cell-­ surface glycoproteins lial migration of the cells into the inflamed tissue.26
important for the interaction between cells and for the interaction of E-­selectin, also known as endothelial leukocyte adhesion molecule-­1
cells with the extracellular matrix. CAMs play an integral role in the (ELAM-­1, CD62E), mediates the attachment of polymorphonuclear
 CHAPTER 1 Elements of the Immune System 7

leukocytes to endothelial cells in vitro and appears to be important As indicated by animal data, clinical trials in 18 patients who received
in the recruitment of neutrophils in a local endotoxin response in cadaver donor renal allografts showed that immunosuppression with
the skin.27 We investigated the expression of E-­selectin in eyes with anti-­ICAM-­1 antibody resulted in significantly less rejection.45 These
endotoxin-­induced uveitis (EIU), a useful animal model for the study data showed not only that CAMs are involved in the pathogenesis of
of acute ocular inflammation,28 which is characterized by iris hyper- inflammation but also that treatment with drugs to block these adhe-
emia, miosis, increased aqueous humor protein, and inflammatory sion molecules should provide effective therapy for inflammatory dis-
cell infiltration into the anterior uvea and anterior chamber.29,30 ease. We used efalizumab (Raptiva), a CD11a antibody that inhibits
Inflammatory cells first enter the eye 6 hours after endotoxin injection, binding of LFA-­1 to ICAM-­1, in the treatment of patients with uveitis
and the resultant uveitis peaks within 24 hours. EIU is thought to result in a small pilot study, with positive therapeutic effects .46
from mediators released by activated cells, including macrophages, but
the exact mechanism causing infiltration into the eye is not clearly B Cells
defined. Recent data suggest that CAMs play an important role in the B cells make up the second broad arm of the lymphocyte immune
pathogenesis of this animal model of disease and that CAM expression response. Originating in mammals from the same pluripotent stem
is important for the recruitment of leukocytes into eyes with EIU. cells in bone marrow as T cells, the maturational process and role of
ICAM-­1 binds not only to Mac-­1, but also to lymphocyte function-­ B cells are quite different. The term B cell originates from the find-
associated molecule-­1 (LFA-­1, CD11a/CD18), a second β2-­integrin ing that in chickens, antibody-­producing cells mature in the bursa of
expressed on all leukocytes predominantly involved in lymphocyte Fabricius, a uniquely avian structure. The mammal equivalent appears
trafficking. A number of groups have studied how ICAM-­1 and LFA-­1 to be bone marrow. The role of the B cell is to function as the effec-
affect the development of EIU. In eyes with EIU in C3H/HeN mice, tor cell in humoral immunity. The unique characteristic of these cells
ICAM-­1 is first expressed on the ciliary body epithelium 6 hours after is the presence of surface immunoglobulin on their cell membranes.
endotoxin injection and, later, on the vascular endothelium of the There are two major subgroups of B cells. Innate-­like B1 cells orig-
ciliary body and iris and on the corneal endothelium.31 Elner et al.32 inate in the fetal liver, are long lived, and self-­renew. They produce
demonstrated the expression of ICAM-­1 (CD54) on the corneal endo- natural antibodies, mostly immunoglobulin M (IgM), in the absence
thelium, and the expression of this cell adhesion molecule also appears of antigen stimulation. In contrast, B2 cells are crucial for adaptive
to be important to the development of keratic precipitates. In experi- immunity. They derive from bone marrow and produce high-­affinity
ments on Lewis rats, we have seen that EIU can be prevented by treating antibodies in response to exogenous stimuli. They produce immu-
the animals with anti-­ICAM-­1 or anti-­LFA-­1 antibody at the time of noglobulins other than IgM (see next section). B2 cells also mediate
endotoxin injection,33 even when administered 6 hours after endotoxin the anamnestic, rapid, high-­affinity antibody response to previously
injection when the eyes are already clinically inflamed. Rosenbaum sensitizing antigens. When activated for antibody production B cells
and Boney34 also showed that antibody to LFA-­1 significantly reduced undergo morphologic change and are named “plasma cells” that are
the cellular infiltrate associated with rabbit models of uveitis but that typical by having a round, eccentric nucleus with coarse clumps of het-
vascular permeability was less affected. An ICAM-­neutralizing anti- erochromatin and euchromatin.
body can inhibit viral infection of the RPE by HTVL-­1.35 The maturation process of B cells is complex and not fully under-
The secretion of cytokines, particularly by infiltrating T lympho- stood. What is clear is that various gene regions that control the B
cytes, appears to regulate adhesion molecule expression. IFN-­γ, IL-­1, cell’s main product, that is, immunoglobulins, are not physically next
and TNF induce strong ICAM-­1 expression at a transcriptional level, to each other. Through a process of translocation, these genes align
although the response to cytokines varies among cell types.36-­38 In vitro themselves next to each other, excising intervening genes. IL-­7 is an
studies have shown that ICAM-­1 expression on the cornea and the important factor in this maturation process. B cells can be activated
RPE is upregulated by cytokines, such as IL-­1.39,40 It is clear that one of through their interaction with CD4+ T cells, which express class II
the major effects of cytokines in the pathogenesis of EIU involves the MHC antigens and CD40 ligand on their surface. This process is pro-
upregulation of adhesion molecule expression. moted by T-­cell cytokines, including IL-­2, IL-­4, IL-­5, IL-­6, and IL-­17.
CAMs have also been shown to play a critical role in the pathogene- B cells initially express surface IgM and IgD simultaneously, with
sis of EAU. We studied the expression of ICAM-­1 and LFA-­1 in B10.A differentiation occurring only after appropriate activation. Five major
mice with EAU.41 ICAM-­1 was first expressed on the vascular endo- classes of immunoglobulins are identified on the basis of the struc-
thelium of the retina and ciliary body by 7 days after immunization, ture of their heavy chains: α, γ, μ, δ, and ε, corresponding to IgA, IgG,
whereas infiltrating leukocytes expressing LFA-­1 were not observed IgM, IgD, and IgE (Table 1.4). The structure of the immunoglobulin
until 9 days after immunization, and clear histologic evidence of ocular demonstrates symmetry, with two heavy chains and two light chains
inflammation did not occur until 11 days after immunization. uniformly seen in all classes except IgM and IgA (Fig. 1.4). The produc-
Treatment with monoclonal antibodies against ICAM-­1 and LFA-­1 tion of immunoglobulins usually requires T-­cell participation. Many
inhibited the development of EAU, suggesting that antiadhesion mol- “relevant” antigens are T cell dependent; that is, the addition of antigen
ecule antibodies could inhibit EAU by interfering with immunization to a culture of pure B cells will not induce immunoglobulin produc-
and antigen sensitization and/or by blocking leukocyte homing and tion. However, polyclonal B-­cell activators, such as lipopolysaccharide,
migration into the eye. These data indicate that antibodies against pokeweed mitogen, dextran, and certain viruses, such as Epstein-­Barr
ICAM-­1 and LFA-­1 inhibit EAU by interfering with both the induc- virus, have the capacity to directly induce B-­cell proliferation and
tion and the effector phases of the disease. Adhesion molecules are immunoglobulin production. For a primary immune response, B cells
also involved in the pathogenesis of lens-­induced uveitis. Till et al.42 will produce IgM, which binds complement. With time – and if they
showed that antibodies against adhesion molecules reduced ocular encounter these antigens again – B cells will switch immunoglobulin
inflammation in lens-­induced uveitis. production to IgG, usually during the primary response. This immu-
Recent studies in humans have shown that cell-­adhesion mole- noglobulin class switching, which requires a gene rearrangement, is
cules are important in the development of ocular inflammation. We inherent in the B cell and is partly controlled by lymphokines. IL-­4
have shown that ICAM-­1 is expressed in the retina and choroid of has been associated with a switch to express IgG (in mouse IgG1, in
human eyes with posterior uveitis.43 In addition, we demonstrated human IgG4) and IgE, whereas IFN-­γ controls a switch to IgG2a and
increased expression of ICAM-­1 in corneas with allograft rejection.44 TGF-­β to IgA.
8 PART 1 Fundamentals

+\SHUYDULDEOHUHJLRQ
TABLE 1.4 Characteristics of human DQWLJHQELQGLQJ
immunoglobulins
9+
IgG IgA IgM IgE IgD
Molecular weight 150 150–300 900 190 180 9/
(103) +HDY\
Heavy chain γ α μ – δ /LJKW FKDLQ
FKDLQ
Subclass 1,2,3,4 1,2 1,2 – –
&+
J chain – + + – –
Crosses placenta + – – – – &/
Serum half-­life (days) 21 6 5 2 3
Complement acti- + – + – –
+LQJHUHJLRQ
vation &RPSOHPHQWELQGLQJ
Serum concentration 110 25 10 0.001 0.3 UHJLRQ
(mg/dL) &+

IN EYE
Conjunctiva Rich Rich Varies Varies Varies
Cornea Moderate Moderate 0 ? 0
&+
Aqueous Low Low Low ? 0
Iris Low Low Low Varies Varies )FSRUWLRQ
Choroid Rich Rich Rich Varies Varies &HOOXODUDWWDFKPHQW
Retina Low Low Low 0 0 Fig. 1.4 Structure of human immunoglobulin G (IgG) molecule.
Vitreous – – – – –
From Allansmith M. Unpublished data 1987. Used with permission.

Surprisingly, recent studies have revealed that B cells also function

as immunoregulatory cells. Wang et al.47 showed that the cytokine
IL-­35 induces B cells with immunoregulatory capacity (“Breg” cells),
which release the immunosuppressive cytokines IL-­ 10 and IL-­ 35.
Furthermore, Bregs were found to inhibit the development of EAU
by inhibiting pathogenic Th1 and Th17 and promoting the expansion
on Treg cells. Adoptive transfer of Breg cells was found to inhibit the
development of EAU.48

Classes of Immunoglobulins
More IgA is made than any other immunoglobulin, most of it in
the gut. IgG is the major circulating immunoglobulin class found in
humans; it is synthesized at a very high rate and makes up about 75%
of the total serum immunoglobulins. Plasma cells that produce IgG are
found mainly in the spleen and the lymph nodes. Four subclasses of IgG
have been identified in humans (G1–G4). G1 and G3 fix complement
readily and can be transmitted to the fetus. The production of these
subclasses is not random but reflects the antigen to which the antibody -FKDLQ
is being made. When doing tests in the serum or the chambers of the
Fig. 1.5 Immunoglobulin M (IgM) pentamer with J chain.
eye (aqueous or vitreous), we usually look at IgG production.
IgM is a pentamer made up of the typical antibody structure linked
by disulfide bonds and J chains (Fig. 1.5). In conventional responses of exogenous organisms. These immunoglobulins help effector cells
B2 cells, IgM is produced in minute amounts. Because of its size, IgM through opsonization, which occurs by the antibody coating an invad-
generally stays within the systemic circulation and, unlike IgG, will not ing organism and assisting the phagocytic process. The Fc portion of
cross the blood–brain barrier or the placenta. This antibody is expressed the antibody molecule then can readily interact with effector cells,
early on the surface of B cells. Therefore initial antibody responses to such as macrophages, thereby helping effectively resolve the infection.
exogenous pathogens, such as Toxoplasma gondii, are of this class. The Persons with deficiencies in IgG and IgM are particularly prone to
observation of an IgM-­specific antibody response helps confirm a newly infections by pyogenic organisms, such as Streptococcus and Neisseria
acquired infection. IgM has a complement-­binding site and can mediate species. In addition, both these antibodies will activate the comple-
phagocytosis by fixing C3b, a component of the complement system. ment pathway, inducing cell lysis by that mechanism as well.
One major role of both IgG and IgM is to interact with both IgA is the major extravascular immunoglobulin, although it comprises
effector cells and the complement system to limit the invasion of only about 10% to 15% of the intravascular total. Two isotypes of IgA are
 CHAPTER 1 Elements of the Immune System 9

almost unlimited quantities. Immortalized myeloma cells can be fused

with a B cell committed to the production of an antibody directed
toward a relevant antigen. This is usually accomplished with the use of
polyethylene glycol, which promotes cell membrane fusion. By careful
screening, clones of these fused cells (i.e., hybrid cells or hybridomas)
can be identified as producing the antibody needed. These can be iso-
lated and grown, yielding essentially an unlimited source of the antibody
derived from one clone of cells and directed against one specific deter-
minant. Monoclonal antibodies have been raised against cell markers of
-FKDLQ virtually all cellular components of the immune system. Antibodies can
now be “humanized” so that only small parts of the variable end remain
of mouse origin. The advantage of this is the reduced probability of an
immune response against the foreign protein.

6HFUHWRU\ Other Cells

SLHFH
Mast Cells
This large (15–20 μm) cell is intimately involved in type I hypersensi-
tivity reactions (see next section). Its most characteristic feature is the
presence of large granules in the cytoplasm. It is clear that there are
subtypes of mast cells. In humans, mast cells are characterized by the
presence or absence of the granule-­associated protease chymase. It has
been suggested that tryptase-­positive, chymase-­negative human mast
Fig. 1.6 Immunoglobulin A (IgA) dimer with J chain and secretory cells are suggestive of mucosal mast cells found in the mouse. Mast
piece. cells contain a large number of biologically active agents, including his-
tamine, serotonin, prostaglandins, leukotrienes, chemotactic factors of
anaphylaxis, and cytokines and chemokines. Histamine is stored within
noted: IgA1 is more commonly seen intravascularly, whereas IgA2 is some- the mast-­cell granules. Once released into the environment, histamine
what more prevalent in the extravascular space. The IgA-­secreting plasma can cause smooth muscle to contract and can increase small vessel per-
cells are found in the subepithelial spaces of the gut, respiratory tract, meability, giving the typical “wheal and flare” response noted in skin
tonsils, and salivary and lacrimal glands. IgA is an important component tests. Serotonin, in humans, appears to have a major effect on vaso-
to the defense mechanism of the ocular surface, being found in a dimer constriction and blood pressure, whereas in rodents, it may also affect
linked by a J chain, a polypeptide needed for polymerization. In addition, vascular permeability. Prostaglandins, a family of lipids, are capable of
a secretory component, a unique protein with parts of its molecule having stimulating a variety of biologic activities, including vasoconstriction
no homology to other proteins, is needed for the IgA to appear in the gut and vasodilation. Leukotrienes are compounds produced de novo with
and outside vessels. The secretory component is produced locally by epi- antigen stimulation. Leukotriene B4 is a potent chemotactic factor for
thelial cells, which then form a complex with the IgA dimer/J chain (Fig. both neutrophils and eosinophils, whereas leukotrienes C4 and D4, for
1.6). This new complex is internalized by mucosal cells and then released example, enhance vascular permeability. At least two chemotactic fac-
on the apical surface of the cell through a proteolytic process. The amount tors of anaphylaxis attract eosinophils to a site of mast-­cell degranula-
of IgA within the eye is quite small. IgA can fix complement through tion, whereas other factors attract and immobilize neutrophils.
the alternate pathway and can serve as an opsonin for phagocytosis. IgA Mast-­cell involvement in several external ocular conditions has been
appears to exert its major role by preventing entry of pathogens into the established. However, it is not yet clear what role this cell may play in
internal environment of the organism by binding with the infectious intraocular inflammatory disorders. Mast cells are present in abundance
agent. It may also impede the absorption of potential toxins and allergens in the choroid and appear to be related to the susceptibility of at least
into the body. Furthermore, it can induce eosinophil degranulation. one experimental model for uveitis (see discussion on autoimmunity).
IgE is slightly heavier than IgG because its heavy chain has an Findings from human studies have supported the hypothesis that many
additional constant domain. Mast cells and basophils have Fc recep- cytokine-­dependent processes are implicated in IgE-­associated disor-
tors for IgE, and IgE is thought to be one of the major mediators of ders. Many different cytokines and chemokines have been seen in mast
the allergic or anaphylactoid reaction. It appears to be an important cells. These include IL-­4, IL-­6, IL-­8, TNF-­α, vascular endothelial growth
defense mechanism against parasites: one way IgE accomplishes this is factor (VEGF), and macrophage inflammatory protein (MIP)-­1α.
to prime basophils and mast cells. Although its role in ocular surface All of these findings link the mast cell to a whole variety of immune
disease has been well recognized, this has not been the case for intra- processes. It can be speculated that when a mast cell degranulates in the
ocular inflammation. choroid, it also releases chemokines and lymphokines, which may be
IgD is found in minute quantities in the serum (0.5% of serum the initiating factors of what we describe as a T-cell–mediated disorder.
immunoglobulin). It is found simultaneously with IgM on B cells The role of mast cells in the pathogenesis of EAU has been noted in
before specific stimulation. Little else is known about this antibody early studies. Mochizuki et al.49 have noted that rat strain susceptibility
other than that it is a major B-­cell membrane receptor for antigen. to EAU induced with S-­Ag was dramatically associated with the num-
Antibodies directed toward specific antigens, particularly cell-­ ber of mast cells in the choroid, and de Kozak et al.50 have shown that
surface antigens of the immune system, have provided clinical and basic mast cells in the choroid degranulate just before the influx of T cells
investigators with a powerful tool with which to identify various com- into the eye, suggesting that these cells “open the door” into the eye
ponents of the immune system, as was described in the section on the for the T cells. This concept is especially provocative because Askenase
T cell. The development of monoclonal antibodies by using hybridoma et al.51 showed that mast-­cell degranulation can be induced not only by
technology has allowed for the production of these immune probes in IgE antibodies but also by T cells.