Elements of the Immune System and Concepts of Intraocular Inflammatory Disease Pathogenesis






* The author thanks Drs William Paul and Igal Gery for reviewing this chapter. The helpful parts of the chapter are due to their good and wise counsel. The parts that are less so are due to my own shortcomings. RBN




Key concepts





  • T cells play an important role in the pathogenesis of uveitis.



  • The eye is very active immunologically, with ocular resident cells interacting with the immune system.



  • Uveitogenic antigens are found in the eye, and immunization of animals with these antigens induces experimental uveitis, often resembling the human condition.



  • Similar immune responses can be seen in the experimental models of uveitis as in the human condition.



In an ever-changing field, a review of the immune system is the subject of numerous books, courses, and scientific articles. However, certain principles have been established that, in the main, have survived the test of time and rigorous scrutiny. The aim of this chapter is to provide the reader with the essentials needed to follow a discussion on mechanisms proposed for intraocular inflammatory disease; therefore, topics relevant to the understanding of that subject are addressed. In addition, selected themes thought to be important in understanding the unique ocular immune environment and pathogenesis are covered. It is clear to any observer of immunology that a detailed description of immune events would be far beyond the scope of this book, and it would hubris to think otherwise. For those well versed in this field, parts of this chapter may be somewhat superfluous.


The development of the immune system is an extraordinary product of evolution. Its goal is to recognize that which is different from self, so its initial role is to respond to foreign antigens with an innate immune response that is geared to rapidly clear the body of the foreign invader. ‘Innate immunity’ is restricted to the non-antigen-specific immune response involving phagocytic cells that engulf and destroy invaders, humoral factors such as the complement system and receptors on antigen-presenting cells such as phagocytes called ‘toll-like receptors’ that interact with the invaders’ molecules. This activates the antigen-presenting cell to initiate the ‘adaptive’ immune response. Clearly the invader may return, and so the adaptive immune response is in place to respond. The adaptive immune response is antigen specific and deals with the invaders that escaped the innate immune mechanism or have returned. The adaptive immune response consists of both B and T cells, and portions of these populations acquire the properties of memory cells of the secondary immune response. This adaptive immune response connotes an immune memory, hence the development of a complex way in which high-affinity molecules and cell-surface markers can distinguish between the invader and self. A given of this concept is that self antigens are not attacked: that is, an immune tolerance exists. Part of our story deals with the immune system’s appropriate response to outside invaders (such as Toxoplasma ) and the other part deals with understanding (and trying to explain) the response to autoantigens. The dynamic is not as simple as outlined; in fact, it starts as an appropriate response to a foreign antigen and then changes to an abnormal response against the eye. Many mechanisms, such as molecular mimicry, have been proposed.


To achieve this complex but highly specific immune response requires multiple players. Some of these are reviewed in the first part of this chapter. In the second part findings and theories of disease mechanisms relevant to the ocular diseases discussed in later chapters are introduced.




Elements of the immune system


The immune system is the result of several cell types, including lymphocytes (T and B cells), macrophages, and polymorphonuclear cells. However, additional cells, such as dendritic cells in the skin and spleen and ocular resident cells in the eye, also should be included. These components add up to a complex immune circuitry or ‘ballet,’ which in the vast number of individuals responds in a way that is beneficial to the organism.


Macrophages/monocytes


Phagocytic cells originate in the bone marrow. The concept that phagocytosis is important for the immunologic defense of the organism was proposed by Metchnikoff at the end of the nineteenth century. The macrophage, which is relatively large (15 µm), has an abundant smooth and rough endoplasmic reticulum. Lysosomal granules and a well-developed Golgi apparatus are also found. Several functional, histochemical, and morphologic characteristics of these cells can be noted ( Table 1-1 ). In addition to the phagocytic characteristics already alluded to, these cells contain esterases and peroxidases, and bear membrane markers that are typical of their cell line (i.e., OKM1 antigen and F4/80). Other cell-surface markers are also present, such as class II antigens, Fc receptors (for antibody), and receptors for complement. These enzymes and cell markers help to identify this class of cells as well as their state of activation. The presence of esterase is a useful marker to distinguish macrophages from granulocytes and lymphocytes. Monocytes will leave the bloodstream because of either a predetermined maturational process or induced migration into an area as a result of chemotactic substances, often produced during inflammatory events. Once having taken up residence in various tissues, they become macrophages, which are frequently known by other names ( Fig. 1-1 ). Dendritic cells, such as Langerhans’ cells, are found in the skin and cornea, and play an important role in activating naive lymphocytes.



Table 1-1

Macrophage characteristics

















































Histochemical Surface Antigens Receptors Functions
5′-Nucleotidase OKM1 Fc Phagocytosis
Esterase Class II antigens IgM Pinocytosis
Alkaline phosphodiesterase Lymphokine Immune activation
Aminopeptidase Lactoferrin Secretory
Insulin Microbicidal
Cb3 Tumoricidal
Fibrinogen
Lipoprotein



Figure 1-1.


Macrophage differentiation.


Macrophages play at least three major roles within the immune system. The first is to directly destroy foreign pathogens as well as clearing dying or diseased tissue. Killing of invading microbes is in part mediated by a burst of hydrogen peroxide (H 2 O 2 ) activity by the activated macrophage. An example with ocular importance is the engulfment of the toxoplasmosis organism, with the macrophage often being a repository for this parasite if killing is inadequate. The second is to activate the immune system. Macrophages or other cells with similar characteristics are mandatory for antigen-specific activation of T lymphocytes. Internalizing and processing of the antigen by the macrophage are thought to be integral parts of this mechanism, and the macrophage (or dendritic cell) is often described as an antigen-presenting cell (APC). Other cells, such as B cells, can also serve this function. The macrophage and lymphocyte usually need to be in close contact with one another for this transfer to occur. Another requirement is for the cells to have in common a significant portion of their major histocompatibility complex (MHC), genes that express various cell-surface membranes essential for cellular communication and function. Thus this MHC stimulation leads to the initiation of an immune response, ultimately with both T and B cells potentially participating. Other cell-surface markers are needed for activation. This ‘two-signal’ theory has centered on other cell-surface antigens, such as the B7–CD28 complex. The engagement of B7 (on the macrophage side) with CD28 enhances the transcription of cytokine genes. Third, the macrophage is a potent secretory cell. Proteases can be released in abundance, which can degrade vessel surfaces and perivascular areas. Degradation products that result from these reactions are chemotactic and further enhance an immune response. Interleukin (IL)-1, a monokine with a molecular weight of 15 000 Da, is produced by the macrophage (as well as other cells) after interaction with exogenous pathogens or internal stimuli, such as immune complexes or T cells. IL-1 release directly affects T-cell growth and aids this cell in releasing its own secretory products. IL-1 is noted to act directly on the central nervous system, with a by-product being the induction of fever. Still other macrophage products stimulate fibroblast migration and division, all of which have potentially important consequences in the eye.


Macrophages produce IL-12 and IL-18 (once called interferon (IFN)-γ-inducing factor), IL-10, and transforming growth factor (TGF)-β. In a feedback mechanism, IFN-γ can activate macrophages, and the production of IL-12 by the macrophage plays an important role in T-cell activation. The role of macrophages in the eye still needs to be fully explored. One concept (in a disease not usually thought of as being immune driven) is that chronically activated macrophages congregate at the level of the retinal pigment epithelium (RPE), inducing the initial changes that lead to age-related macular degeneration.


Dendritic cells


Although macrophages play an important role, it is conjectured that dendritic cells are important macrophage-like cells in tissue. They are a subset of cells, perhaps of different lineage from macrophages, from which they can be distinguished by a lack of persistent adherence and by the bearing of an antigen, 33D1, on their surface, features that macrophages do not possess. The major role of dendritic cells is to serve as initiators of T-cell responses, for both CD4+ and CD8+ cells. Like macrophages, dendritic cells produce IL-12, an important activator of T-cell responsiveness. They are rich in MHC II intracellular compartments, an important factor in antigen presentation. The MHC class II compartments will move to the surface of the cell when the dendritic cell matures, stimulated by IFN-α and the CD40 ligand. Dendritic cells are special in that they inhabit tissues where foreign antigens may enter. Experiments with painting of the skin brought seminal observations. Antigens painted on the skin are ‘brought’ to the draining lymph nodes by the dendritic cells of the skin (Langerhans’ cells) where T-cell activation can occur. What is interesting is the migratory nature of these cells: they constantly carry important information to peripheral centers of the immune response. Whether dendritic APCs can activate T cells efficiently in the tissues themselves is an open question and is important to our understanding of immune responses in the eye. Dendritic cells are thought to be the APCs (or one of the major players) in corneal graft rejection. Thus the concept of removing dendritic cells from a graft has been proposed and used in experimental models. However, there is an opposing concept that peripheral immune tolerance, induced by antigens that foster programmed cell death (apoptosis), may depend on presentation of antigen bydendritic cells in the tissue.


T cells


T cells are found in large numbers in the systemic circulation. Lymphocytes are broadly divided into two major categories, T cells and B cells (discussed later). These appellations are based on initial observations in chickens, in which a subgroup of lymphocytes homed to the thymus, where they underwent a maturational process leading to the heterogeneous population now recognized as ‘thymus-dependent’ or T cells. The thymus, the first lymphoid organ to develop, has essentially two compartments, the cortex and the medulla. Within the thymus are found epithelial cells, thymocytes (immature lymphocytes), occasional macrophages, and more mature lymphocytes. The highly cellular cortex is the center of mitoses, with large numbers of immature thymocytes and epithelial cells adhering to each other. As the thymocytes mature to T cells they migrate to the medulla and are ultimately released into the systemic circulation. Major alterations occur to the thymocyte during this maturational process. There is the activation of specific genes needed for only this portion of the lifecycle of the cells. In addition, lifelong characteristics are acquired. These include the development of specific receptors that recognize particular antigens, the acquisition of MHC restriction needed for proper immune interactions, and the acquisition of various T-cell functions, such as ‘killing’ and ‘helping’ other cells. These cells are activated by a complex of structures on their surface. The T-cell receptor (specific to the antigen that is being presented to the cell), the CD3 complex, and the antigen cradled in either an MHC class I or II cassette are needed. Other cofactors are also needed for very robust activation.


Some important qualities possessed by these cells are their immunologic recall or anamnestic capacity; this increases the number of specific cells as well as changing them into a ‘memory’ phenotype. They also have the capacity to produce cellular products called cytokines ( Table 1-2 ). A T cell previously sensitized to a particular antigen can retain this immunologic memory (see below) essentially for its lifetime. With a repeat encounter, this memory response leads to an immune response that is more rapid and more pronounced than the first. Such an example is the positive skin response seen after purified protein derivative (PPD) testing.



Table 1-2

Cytokines: An incomplete list








































































































Type Source Target and Effect
Interferon-γ T cells


  • Antiviral effects; promotes expression of MHC II



  • Antigens on cell surfaces; increases MΦ tumor killing; inhibits some T-cell proliferation

Transforming growth factor-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, keratinocyte, endothelial cells, some T and B cells T- and B-cell proliferation; fibroblasts – proliferation, prostaglandin production; CNS – fever; bone 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 – co-mitogen; 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 endothelium; 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 Th1 T 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 T cells but not Th2 or Th0
IL-13 T cells Antiinflammatory activity as IL-4 and IL-10; down regulates IL-12 and IFN-α production and thus favors Th2 T-cell responses; inhibits proliferation of normal and leukemic human B-cell precursors; 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
TGF-β MΦ, lymphocytes Depends on cell interaction
TNF-α 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; MΦ, macrophage; NK, natural killer; RPE, retinal pigment epithelium; TFG, transforming growth factor; TNF, tumor necrosis factor.


The central role of the T cell in the immune system cannot be overemphasized. T cells function as pivotal modulators of the immune response, particularly by helping B-cell production of antibody and augmenting cell-mediated reactions through further recruitment of immunoreactive cells. T cells also may downregulate or prevent immune reactions through active suppression. In addition to these ‘managerial’ types of roles, some T-cell subsets are known to be cytotoxic and are recognized as belonging to the predominant cells in transplantation rejection crises. The accumulated evidence supports the importance of T cells in many aspects of the intraocular inflammatory process – from the propagation of disease to its subsequent downregulation.


Major subsets of T cells


The functions that have been briefly described are now thought to be carried out by at least three major subsets of T cells, with these cells identified either through functional studies or through monoclonal antibodies directed against antigens present on their surface. It was observed early on that T cells (as well as other cells) manifest myriad different molecules on their surface membranes, some of which are expressed uniquely at certain periods of cell activation or function. It was noted that certain monoclonal antibodies directed against these unique proteins bind to specific subsets of cells, thereby permitting a way to identify them ( Table 1-3 ). The antibodies to the CD3 antigen (e.g., OKT3) are directed against an antigen found on all mature human T cells in the circulation; approximately 70–80% of lymphocytes in the systemic circulation bear this marker. Antibodies to the CD4 antigen (e.g., OKT4) define the helper subgroup of human T cells (about 60–80% of the total T cells). These cells are not cytotoxic but rather aid in the regulation of B-cell responses and in cell-mediated reactions. They are the major regulatory cells in the immune system. These CD4+ cells respond to antigens complexed to MHCs of the class II type. The CD4+ subgroup of cells is particularly susceptible to the human immunodeficiency virus (HIV) of the acquired immunodeficiency syndrome (AIDS), with the percentage of this subset decreasing dramatically as this disease progresses. Further, these helper cells are necessary components of the autoimmune response seen in the experimental models of ocular inflammatory disease induced with retinal antigens (see discussion of autoimmunity later in this chapter). There is a subset of CD4+ cells that also bear IL-2 receptors (CD25) on their surface. In rodents, and possibly also in humans, some T-regulatory cells may bear the CD25 receptor (see below).



Table 1-3

Selected human leukocyte differentiation antigens (Incomplete list)


























































































Cluster Designation Main Cellular Distribution Associated Functions
CD3 T cells, thymocytes Signal transduction
CD4 Helper T cells MHC class II coreceptor
CD8 Suppressor T cells, cytotoxic T cells MHC class I receptor
CD11a Leukocytes LFA-1, adhesion molecule
CD11b Granulocytes, MΦ Mac-1 adhesion molecule
CD11c Granulocytes, MΦ, T cells, B cells α-Integrin, adhesion molecule
CD19 B cells B-cell activation
CD20 B cells B-cell activation
CD22 B cells B-cell regulatory
CD25 T cells, B cells α chain of IL-2 receptor (Tac) activation
CD28 T cells Co-stimulatory T-cell marker
CD45 Leukocytes Maturation
CD54 Endothelial, dendritic, and epithelial cells; activated T and B cells ICAM-1, adhesion molecule; ligand of LFA-1 and Mac-1
CD56 NK cells N-CAM, adhesion molecule
CD68 Macrophages
CD69 NK cells, lymphocytes Signal transmission receptor
CX3CR1 Monocytes Chemoattractant
CXCR3 T cells Cell maturation
CCR7 T cells Migration to inflammation
CCR5 T cells Chemokine receptor
CD8 – Co-receptor TRC during antigen stimulation with cytotoxic T-cells

ICAM, intercellular adhesion molecule; IL, interleukin; LFA, lymphocyte function-associated molecule; MHC, major histocompatibility complex; N-CAM, neural cell adhesion molecule.


Antibodies to the CD8 antigen (i.e., OKT8) distinguish a population that includes cytotoxic T cells, making up about 20–30% of the total number of T cells. (In the older literature it was thought to harbor suppressor cells, but this is no longer thought to be the case). Antibodies directed against the CD8 antigen block class I histocompatibility-associated reactions.


Cytokines


Intercellular communication is in large part mediated by cytokines and chemokines (see below). Cytokines are produced by lymphocytes and macrophages, as well as by other cells. They are hormone-like proteins capable of amplifying an immune response as well as suppressing it. With the activation of a T lymphocyte, the production and release of various lymphokines will occur. One of the most important is IL-2, with a molecular weight of 15 000 Da in humans. The release of this lymphokine can stimulate lymphocyte growth and amplify or augment specific immune responses. Another lymphokine is IFN-γ, an important immunoregulator with the potent capacity to induce class II antigen expression on cells. TGF-β is a ubiquitous protein produced by many cells, including platelets and T cells; it appears to have the distinct ability to downregulate immune responses, and to play an important role in anterior chamber-acquired immune deviation (ACAID) and oral tolerance. The number of lymphokines that have been purified and for which effects have been described (see Table 1-2 for a partial list) continues to grow rapidly.


T-cell subsets


Helper T cells have been further subdivided, based on their functional characteristics, into several groups ( Fig. 1-2 ). The first is the Th1 cell ( Fig. 1-3 ). These cells show a cytokine profile of IFN-γ production. The cytokine profile of Th2 cells comprises IL-4, IL-5, IL-13 and perhaps TGF-β, and IL-10. In many animal models of human disease Th1 cells are associated with the initiation of disease, whereas Th2 cells are related to disease downregulation and allergy initiation, or are involved in parasitic diseases. But this story is still unclear. We know from experimental models of uveitis (see below), in which the autoaggressive cells that induce disease are the Th1 cells, that under certain conditions one can induce disease with Th2 cells (nature did not read the textbooks!). Indeed, yet another subset of cells that has been the center of great interest recently is that of the Th17 cell. These cells produce proinflammatory cytokines including IL-17 (hence the name), IL-21 and 22. These cells develop in different environments depending on whether we look in the mouse or the human. In humans, IL-1, IL-6, and IL-23 appear to promote these cells. The cells play a role in host defense mechanisms against fungi and bacteria, and also in autoimmune disease. We have reported the presence of Th17 cells in the blood of sarcoidosis patients with uveitis. Additionally, another human T-cell subset, NKT cells, also produce IL-17 and bear IL-23 receptors on their surface.




Figure 1-2.


Helper T-cell subsets now recognized.

(From: Zhi Chen, O’Shea JJ. Th17 cells: a new fate for differentiating helper T cells. Immunol Res 2008; 41: 87, with permission.)



Figure 1-3.


Development of three types of T cell participating in the immune response. Other T-cell types also exist, but are not shown.

(With kind permission from Springer Science & Business Media: From Th17 cells: a new fate for differentiating helper T cells. Zhi Chen – John J. O’Shea. Immunol Res (2008) 41:87–102.)


One concept is that Th1 cells may initiate an immune response but the Th17 cells are involved in more chronic activity. Anti-IL-17 will almost certainly be an area of intense investigation in the coming years. An interesting question is whether Th1 cells and IL-17 are distinct cells, or are they rather a function of the immune environment, so that under certain circumstances they produce IL-17 and under others a Th1 repertoire? One still cannot answer that question in the human setting, but under experimental conditions it has been seen that Th17 cells may switch to a Th1 character, but that Th1 cells maintain that phenotype and do not change. Also under experimental conditions in animals, when comparing these cells the nature of the intraocular inflammatory response was seen to be different. Th17 did not induce a large lymphoid expansion and splenomegaly, as did Th1 cells; Th1 cells infiltrating the eye dissipate rapidly, whereas IL-17 cells remain; and markers on the surface of these infiltrating cells are different.


IL-22 is part of the IL-17 group of cytokines produced during an inflammatory response. Albeit made by lymphocytes, its receptors are present on epithelial cells. Thus it has been suggested that one of it major roles is to be the cross-talk lymphokine between resident tissue cells and infiltrating inflammatory cells, particularly T cells. This proinflammatory cytokine is found in the synovia of patients with rheumatoid arthritis and is upregulated in both Crohn’s disease and ulcerative colitis. ,


T-regulatory cells


It is clear that just as the immune system needs cells to initiate a response it needs cells to suppress or modify an immune response. One of the ways that need is met is with T-regulatory (Tr) cells. , It is hypothesized that these derive from a naive T cell under the influence of cytokines different from those of either Th1 or Th2 cells (see Fig. 1-3 ). T regs can be found in the thymus (u T regs) or in the peripheral circulation which can be induced (i T regs). Of interest is a report by Kemper and co-workers of stimulating CD4+ cells with CD3 and CD46 (a complement regulator) and inducing Tr cells, that is, producing large amounts of IL-10, moderate amounts of TGF-β, and little IL-2. The literature is replete with information about different types of Tr cell and they have been reported in several organs, such as the gut, where peripheral immune tolerance needs to be induced. Certain characteristics of many of these cells have been described ( Table 1-4 ), and the underlying feature is their ability to produce IL-10 and TGF-β. They are capable of downregulating both CD4- and CD8-mediated inflammatory responses, requiring cell-to-cell contact. There are probably many types because nature usually provides redundancies. Of great interest are those that bear CD25 (the IL-2 receptor) on their cell surface. Much interest has centered on cells that have large numbers of these receptors on their surface (‘bright cells’), with work suggesting that they are indeed ‘negative regulatory’ cells – that is, suppressor cells that can modify an immune response. Although the evidence is much clearer in mouse models, this area still is unfolding in human immunology, and it is not clear what the best markers for these cells are. Such an example is forkhead/winged helix transcription factor, or FoxP3, thought to be a reliable marker in mice for the development and function of naturally occurring T-regulatory cells, but its expression has been seen in T-effector cells (cells that induce inflammation) and so its value has been called into question, at least in humans. When we evaluated the T cells of patients with ocular inflammatory disease, we found that the FoxP3 marker varied tremendously between patients and was not a very good indicator of poor T-regulatory function.



Table 1-4

Cytokine repertoire of various CD4+ T cells

Based on findings in Roncarolo MG, Bacchetta R, Bordignon C, et al. Type 1 T regulatory cells. Immunol Rev 2001; 181: 68–71.



































































Cytokine Tr1 Th0 Th1 Th2 Th17
IL-2 ± 3+ 3+ ±
IFN-γ 2+ 2+ 3+ ±
IL-4 2+ ± 3+
IL-5 2+ 2+ ± 3+
IL-10 3+ 1+ 1+ 2+
TGF-β 3+ 2+ 2+ 2+
IL-17 3+
IL-22 2+


An interesting observation is the increase in a subset of NK cells (so called CD56 ‘bright’) after daclizumab therapy was noted; this subset makes large amounts of IL-10. The implication of this increase in this cell population is that a regulatory cell is to be found there. The increase is seen when patients’ disease is well controlled, and it has also been seen in multiple sclerosis patients receiving daclizumab therapy.


T-cell receptor


Much interest has centered on the T-cell receptor (TCR) ( Fig. 1-4 ). T cells need to produce the TCR on their cell surface to recognize the MHC; this is part of the system that permits information transmitted to it by peptides presented on the APC. This complex interaction involves the MHC antigen on the APC surface, the peptide, either the CD4 or the CD8 antigen, and the TCR. The TCR is similar in structure to an immunoglobulin, having both an α and a β chain. The more distal ends of these chains are variable, and the hypervariable regions are termed V (variable) and J (joining) on the α chain and V, and D (diversity) regions on the β chain. Compared with the number of immunoglobulin genes, there are fewer V genes and more J genes in the TCR repertoire. It is logically assumed that the peptide, which has a special shape and therefore fits specifically in a lock-and-key fashion into the groove between the MHC and the TCR, would be the ‘cement’ of this union. In general that would be true, but ‘superantigens,’ which can bind to the sides of these molecules, can also bring them together and, under the right circumstances, initiate cellular responses. These superantigens are glycoproteins and can be bacterial products such as enterotoxins or viral products. It has been suggested that of all the possible combinations of gene arrangements that could possibly produce the variable region believed to cradle the peptide, certain genes within a family seem to be noted more frequently in autoimmune disease. One such group is the Vα family, with Vβ8.2 receiving much attention. A very small number of cells have a TCR made up not of α and β chains but rather γ and δ chains. These cells are usually CD4 and CD8 , and their ability to interact with APCs is not great. They appear to be highly reactive to heat-shock proteins.




Figure 1-4.


T-cell receptor in three dimensions to give an idea of the complexity of interaction. A, TCR is on top with various chains shown in different colors. Major histocompatibility antigen is below. B, Close-up of TCR MHC interphase. C, Molecular surfaces of interacting TCR, peptide, and MHC.

(From Garcia KC, Degano M, Pease LR, et al: Structural basis of plasticity in T cell receptor recognition of a self peptide-MHC antigen, Science 279:1166–1172 (20 Feb), 1998. Reprinted with permission from American Association for the Advancement of Science.)


A state of suspended animation can be induced in T cells which is termed anergy. For T cells to be activated several signals need to be given: one through the TCR and the other through co-stimulatory receptors such as CD28; the third is the co-stimulant B7 linking to CD28 (which is on the T cell). If the TCR is activated but the co-stimulant is not, one sees a growth arrest in these cells: they simply stop functioning but do not die. A second way this can occur is when a weakly adherent peptide is linked to the TCR, even if co-stimulation occurs. It would seem to be a mechanism to prevent unwanted or nuisance immune responses. The full response takes place only if all the appropriate interactions have occurred.


Chemokines


This family of chemoattractant cytokines is characterized by its ability to induce directional migration of white blood cells. They will direct cell adhesion, homing, and angiogenesis. There are four major subfamilies of chemokines: CXC (nine of which are found on chromosome 4), CC (11 of which are found on chromosome 17), C (only one well-defined member, lymphotactin, on chromosome 11), and CX3C (fractalkine, on chromosome 16). The nomenclature is based on cysteine molecules. The CC chemokines have two adjacent cysteines at their amino terminus; the CXC chemokines have their N terminal cysteines separated by one amino acid; the C chemokines have only two cysteines, one at the terminal end and one downstream; the CX 3 C chemokines have three amino acids between their two N terminal cysteines. Each chemokine family has special functions that affect different types of cell. An example of this fine specificity is seen within the CXC family. Those CXC chemokines with a Glu–Leu–Arg sequence near the end of the N terminus bind well to the CXCR2 on neutrophils. CXC chemokines not possessing that sequence are chemotactic for monocytes and lymphocytes. IL-8 can bind with either CXCR1 or CXCR2 (i.e., the chemokine receptors). Organisms have adapted to these chemokines as well. HIV gp120 will bind to CCR5 and CCR3, aiding its entry into the lymphocyte. This area is still evolving. Clearly, cell homing has importance in ocular inflammatory disease but probably in other conditions as well, such as diabetes and age-related macular degeneration, in which the immune components of the disease are just being explored but which may be important areas for therapeutic interventions.


Thymic expression and central immune tolerance


T-cell responses to an antigen are the basis of a large part of the ocular inflammatory process. For a T cell to ‘recognize’ an antigen it needs to bear on its surface a receptor that will combine with the antigen. The development of the T-cell receptor is a complex mechanism that involves the random recombination of at least three distinct gene segments that control the expression of the T-cell receptor. These T cells go through a selection process in the thymus. Immature cells from the bone marrow find their way into the thymus, rearranging their T-cell receptor components and at the same time expressing CD4 and CD8 co-receptor molecules. These cells move to a portion of the thymic cortex where they interact with stromal cells or dendritic cells bearing on their surface MHC molecules and self peptides. Thymocytes that fail to recognize the MHC complex are induced to die (apoptose). The T cells that have been selected will then migrate further into the thymus, coming into contact with dendritic cells expressing MHC molecules and self peptides. Here the cells that bind tightly to the MHC complex on dendritic cells are negatively selected and undergo programmed cell death (apoptosis). Only a very small fraction (3–5%) of the T-cell precursors that come into the thymus will emerge as mature T cells. The system is not perfect, and some autoresponsive cells escape the negative selection process, finding their way into the mature immune system. It is believed that they form the nidus of autoimmune responses. We can perhaps see evidence of this when we observe T-cell immune memory responses from normal individuals to the uveitogenic antigens from the back of the eye. The way the body deals with these cells falls under the rubric of peripheral tolerance. However, with regard to the thymus and how these observations affect the ocular immune response, we know that the thymus can often express organ-specific molecules such as insulin. Egwuagu and co-workers have shown interesting findings in the thymus. It has been noted for some time that the susceptibility of some animal strains to uveitis after immunization with uveitogenic antigens depended on whether they expressed these antigens in the thymus. An example can be seen in Figure 1-5 .




Figure 1-5.


Transcription of S-antigen and IRBP genes (uveitogenic antigens) in eyes and thymuses of mouse strains. S-antigen and IRBP are abundant in the eyes of all animals and S-antigen is found in the thymuses of all four strains tested. However, IRBP was seen only in thymuses of two strains – BALBk and AKWJ – and not in those of B10.A or B10 RIII. The last two animals are susceptible to induction of uveitis with IRBP.

(From Egwuagu CE, Charukamnoetkanok P, Gery I: Thymic expression of autoantigens correlates with resistance to autoimmune disease, J Immunol 159:3109–3112, 1997.)


Four inbred strains of mice were evaluated for the expression in their thymus of two uveitogenic antigens (see below): interphotoreceptor retinoid-binding protein (IRBP) and S-antigen (arrestin). All four strains were resistant to the induction of uveitis when arrestin was used as the immunizing antigen, and all four expressed arrestin in their thymus. However, two of the four strains, B10.A and B10.RIII, were susceptible to uveitis induction when IRBP was used as the immunizing antigen. Of great interest was the fact that no IRBP mRNA could be detected using quantitative PCR assays in their thymus glands. These observations now include other rodents and primates. In the Lewis rat, which is susceptible to both antigens, neither message is found in the thymus. For the rhesus monkey, which is susceptible to both S-antigen (S-Ag) and IRBP, no message is seen for IRBP and for S-Ag it is variable. These observations may provide an insight into the propensity for the disease in humans; thymuses removed from patients for various indications were investigated to see if these observations hold. Takase et al. evaluated 18 human thymus samples taken from patients undergoing surgery for congenital heart disease. They found that there was indeed expression of the four antigens that can induce experimental uveitis (S-antigen, recoverin, RPE65 and interphotoreceptor retinoid-binding protein) in the thymi of the patients tested (none had uveitis). However, the expression of the various antigens was very variable, with some thymus samples showing strong expression whereas others did not. Many of the patients had peripheral T cells that responded to the S-antigen, but much less so to other antigens. The implication of these studies is that expression of these antigens in the thymus is very variable in humans, similar to what is seen in the differences between various rodent strains. Further, whereas the low expression and ‘avidity’ of the T cells to the antigen in the thymus may explain to some degree the finding of T cells in the blood that respond to the S-antigen, it clearly suggests that other mechanisms are also at work.


Recent work has identified the AIRE gene, the protein produced by which is expressed in a subset of medullary thymic epithelial cells. These cells are involved in the negative selection performed by thymic cells. AIRE appears to permit the expression of organ-specific autoantigens, thereby helping in the removal of autoaggressive cells. Loss of the AIRE gene leads to autoimmunity. This is known to occur in humans and leads to autoimmune polyglandular syndrome (APS) type I, an autoimmune disease that is inherited in an autosomal recessive fashion. In addition to the adrenal insufficiency, mucocutaneous infections, and hypoparathyoridism, these patients can manifest diabetes, Sjögren’s syndrome, vitiligo, and uveitis.


B cells


B cells make up the second broad arm of the lymphocyte immune response. Originating from the same pluripotential stem cell in the bone marrow as the T cell, the maturational process and role of the B cell are quite different. The term B cell originates from observations obtained from work with chickens, in which it was noted that antibody-producing cells would not develop if the bursa of Fabricius, a uniquely avian structure, was removed. The human equivalent appears to be the bone marrow. The B cell, under proper conditions, will develop into a plasma cell that is capable of secreting immunoglobulin. Therefore, its role is to function as the effector cell in humoral immunity. The unique characteristic of these cells is the presence of surface immunoglobulin on their cell membranes.


B-cells begin as a group of cells originating from stem cells designated as pro- or pre-B cells. The maturation process leading to a B cell is complex and not fully understood. What is clear is that various gene regions that control the B-cell’s main product, immunoglobulins, are not physically next to each other. Through a process of translocation these genes align themselves next to each other, excising intervening genes. IL-7 is an important factor in the maturation process. B cells can be activated by their interaction with CD4+ T cells that express on their surface class II MHC antigens and CD40 ligand. B-cell activation will cause these cells to divide, usually in the context of T-cell interaction and cytokines elaborated by the T cell, including IL-4, IL-5, IL-6, IL-17 and IL-2.


Subgroups of B cells have been described. Naive, conventional (B2) B cells are found. Another type, memory B cells, live for long periods, are readily activated, and will produce immunoglobulin (Ig) isotypes other than IgM (see next section). These cells presumably play an important role in the anamnestic response of the organism. This is the very rapid antigen-specific immune response that occurs when the immune system encounters an antigen to which it has already been sensitized. Another subgroup consists of B1 (CD5+) lymphocytes, whose characteristics overlap with those of other B cells but which appear to be derived from a separate lineage and are very long-lived. These cells produce IL-10 and have been associated with autoantibody production. Chronic lymphocytic leukemias often derive from B1 cells.


B cells initially express surface IgM and IgD simultaneously, with differentiation occurring only after appropriate activation. Five major classes of immunoglobulin are identified on the basis of the structure of their heavy chains: α, γ, µ, δ, and ϵ, corresponding to IgA, IgG, IgM, IgD, and IgE ( Table 1-5 ). The structure of the immunoglobulin demonstrates a symmetry, with two heavy and two light chains uniformly seen in all classes except IgM and IgA ( Fig. 1-6 ). The production of immunoglobulin usually requires T-cell participation. Many ‘relevant’ antigens are T-cell dependent, meaning that the addition of antigen to a culture of pure B cells will not induce immunoglobulin production. However, polyclonal B-cell activators, such as lipopolysaccharide, pokeweed mitogen, dextran, and the Epstein–Barr virus (as well as other viruses), have the capacity to directly induce B-cell proliferation and immunoglobulin production. For a primary immune response B cells will produce IgM, which binds complement. With time – and if they encounter these antigens again – B cells will switch immunoglobulin production to IgG, usually during the primary response. This immunoglobulin class switching, which requires a gene rearrangement, is inherent in the B cell and is partly controlled by lymphokines. IL-4 has been associated with a switch to express IgG (in mouse IgG 1 , in human IgG 4 ) and IgE, whereas IFN-γ controls a switch to IgG 2a and TGF-β to IgA.



Table 1-5

Characteristics of human immunoglobulins

From Allansmith M. Unpublished data 1987. Used with permission.



























































































































IgG IgA IgM IgE IgD
Molecular weight (10 3 ) 150 150–300 900 190 180
Heavy chain γ α µ δ
Subclass 1,2,3,4 1,2 1,2
J chain + +
Crosses placenta +
Serum half-life (days) 21 6 5 2 3
Complement activation + +
Serum concentration (mg/dL) 110 25 10 0.001 0.3
IN EYE
Conjunctiva Rich Rich Varies Varies Varies
Cornea Moderate Moderate 0 ? 0
Aqueous Low Low Low ? 0
Iris Low Low Low Varies Varies
Choroid Rich Rich Rich Varies Varies
Retina Low Low Low 0 0
Vitreous



Figure 1-6.


Structure of human IgG molecule.


Classes of Immunoglobulin


More IgA is made than any other immunoglobulin, much 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 (G 1 –G 4 ). G 1 and G 3 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 is being made. When doing tests in the serum or the chambers of the 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. 1-7 ). Only about one-fifteenth as much IgM as IgG is produced. Because of its size, it generally stays within the systemic circulation and, unlike IgG, will not cross the blood–brain barrier or the placenta. This antibody is expressed early on the surface of B cells. Therefore, initial antibody responses to exogenous pathogens, such as Toxoplasma gondii , are of this class. The observation of an IgM-specific antibody response helps to confirm a newly acquired infection. IgM has a complement-binding site and can mediate phagocytosis by fixing C3b, a component of the complement system.




Figure 1-7.


IgM pentamer with J chain.


One major role of both IgG and IgM is to interact with both effector cells and the complement system to limit the invasion of exogenous organisms. These immunoglobulins aid effector cells through opsonization, which occurs by the antibody coating an invading organism and assisting the phagocytic process. The Fc portion of the antibody molecule then can readily interact with effector cells, such as macrophages, thereby helping effectively resolve the infection. Persons with deficiencies in IgG and IgM are particularly prone to infection by pyogenic organisms such as Streptococcus and Neisseria species. In addition, both of these antibodies will activate the complement pathway, inducing cell lysis by that mechanism as well.


IgA is the major extravascular immunoglobulin, although it comprises only about 10–15% of the intravascular total. Two isotypes of IgA are noted: IgA 1 is more commonly seen intravascularly, whereas IgA 2 is somewhat more prevalent in the extravascular space. The IgA-secreting plasma cells are found in the subepithelial spaces of the gut, respiratory tract, tonsils, and salivary and lacrimal glands. IgA is an important component to the defense mechanism of the ocular surface, being found in a dimer linked by a J chain, a polypeptide needed for polymerization. In addition, a secretory component, a unique protein with parts of its molecule having no homology to other proteins, is needed for the IgA to appear in the gut and outside vessels. The secretory component is produced locally by epithelial cells that then form a complex with the IgA dimer/J chain ( Fig. 1-8 ). This new complex is internalized by mucosal cells and then released on the apical surface of the cell through a proteolytic process. The amount of IgA within the eye is quite small. IgA can fix complement through the alternate pathway, and can serve as an opsonin for phagocytosis. IgA appears to exert its major role by preventing entry of pathogens into the internal environment of the organism by binding with the infectious agent. It may also impede the absorption of potential toxins and allergens into the body. Further, it can induce eosinophil degranulation.




Figure 1-8.


IgA dimer with J chain and secretory piece.


IgE is slightly heavier than IgG because its heavy chain has an additional constant domain. Mast cells and basophils have Fc receptors for IgE, and IgE is thought to be one of the major mediators of the allergic or anaphylactoid reaction (see next section). It appears to be an important defense mechanism against parasites: one way IgE accomplishes this is to prime basophils and mast cells. Although its role in ocular surface disease has been well recognized, this has not been the case for intraocular inflammation.


IgD is found in minute quantities in the serum (0.5% of serum Ig). It is found simultaneously with IgM on B cells before specific stimulation. Little more is known about this antibody other than it is a major B-cell membrane receptor for antigen.


Antibodies directed toward specific antigens, particularly cell-surface antigens of the immune system, have provided the clinical and basic investigator with a powerful tool with which to identify various components of the immune system, as was described in the section on the T cell. The development of monoclonal antibodies using hybridoma technology has permitted the production of these immune probes in almost unlimited quantity. 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 isolated and grown, yielding essentially an unlimited source of the antibody derived from one clone of cells and directed against one specific determinant. Monoclonal antibodies have been raised against cell markers of virtually all cellular components of the immune system. Antibodies can now be ‘humanized’ so that only small parts of the variable end remains of mouse origin. The advantage to this is the reduced probability of an immune response to a foreign protein.


Other cells


Mast Cells


This large (15–20 µm) cell is intimately involved with type I hypersensitivity 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 cells are suggestive of mucosal mast cells found in the mouse. Mast cells contain a large number of biologically active agents, including histamine, serotonin, prostaglandins, leukotrienes, and chemotactic factors of anaphylaxis as well as cytokines and chemokines. Histamine is stored within the mast-cell granules. Once released into the environment, histamine can cause smooth muscle to contract and can increase small vessel permeability, giving the typical ‘wheal and flare’ response noted in skin tests. Serotonin, in humans, appears to have a major effect on vasoconstriction and blood pressure, whereas in rodents it may also affect vascular permeability. Prostaglandins, a family of lipids, are capable of stimulating a variety of biologic activities, including vasoconstriction and vasodilation. Leukotrienes are compounds produced de novo with antigen stimulation. Leukotriene B 4 is a potent chemotactic factor for both neutrophils and eosinophils, whereas leukotrienes C 4 and D 4 , for example, enhance vascular permeability. At least two chemotactic factors of anaphylaxis attract eosinophils to a site of mast-cell degranulation, whereas other factors attract and immobilize neutrophils.


Mast-cell involvement in several external ocular conditions has been established. However, it is not yet clear what role this cell may play in intraocular inflammatory disorders. Mast cells are present in abundance in the choroid, and appear to be related to the susceptibility of at least one experimental model for uveitis (see discussion on autoimmunity). Human work supports the hypothesis that many cytokine-dependent processes are implicated in IgE-associated disorders. Many different cytokines and chemokines have been seen in mast cells. These include IL-4, IL-6, IL-8, tumor necrosis factor (TNF)-α, vascular endothelial growth factor (VEGF), and macrophage inflammatory protein (MIP)-1α.


All of these findings link the mast cell to a whole variety of immune processes. It can be speculated that when a mast cell degranulates in the choroid it also releases chemokines and lymphokines, which may be the initiating factor of what we describe as a T-cell-mediated disorder.


Eosinophils


These bilobed nucleated cells are about 10–15 µm in size and are thought to be terminally differentiated granulocytes. Their most morphologically unique characteristic is the approximately 200 granules that are highly acidophilic (taking up eosin in standard staining procedures) and which are found in the cytoplasm. They are almost entirely made up of major basic protein (molecular weight 9000 Da), but other toxic cationic granules include eosinophil-derived neurotoxia, eosinophil cationic protein, and eosinophil peroxidase. A minor percentage of these cells (5–25%) have IgG receptors, and about half may have complement receptors on their surface membranes, although it is not clear whether receptors for IgE are present. Eosinophils contain an abundant number of enzymes, which are quite similar in nature to those contained in neutrophils. Both cells contain a peroxidase and catalase, both of which can be antimicrobial, but eosinophils lack lysozymes and neutrophils lack the major basic protein. Eosinophils also contain several anti-inflammatory enzymes such as kininase, arylsulfatase, and histaminase. In addition, eosinophils produce growth factors such as IL-3 and IL-5, chemokines such as RANTES and MIP-1, cytokines such as TGF-α and TGF-β, VEGF, TNF-α, IL-1α, IL-6, and IL-8.


The eosinophil arises in the bone marrow from a myeloid progenitor, perhaps from a separate stem cell than neutrophils. The time spent in the systemic circulation is probably quite short, and the number seen on a routine blood smear is usually very low (1% or less of nucleated cells). These cells can be attracted to an area in the body by the release of mast-cell products and, once localized to an inflammatory site, are capable of performing several functions. The eosinophil may play an immunomodulatory role in the presence of mast-cell and basophil activation.


As mentioned, the cell contains the anti-inflammatory agents histaminase and arylsulfatase, capable of neutralizing the effect of histamine release and slow-reacting substance, both products of mast cells. Further, basophil function may be inhibited by prostaglandins E 1 and E 2 , both produced by eosinophils. An additional immunomodulatory mechanism is the capacity of the eosinophil to ingest immunoreactive granules released by mast cells. An extremely important role played by these cells is in the response of the immune system to parasitic organisms. Eosinophils are seen in high numbers at the site of a parasitic infiltration and are known to bind tightly to the organism through receptors. Further, the release of the major basic protein granules or an eosinophil-produced peroxidase complexed with H 2 O 2 and deposited on the parasite’s surface membrane will lead to the death of the invading organism. Major basic protein may play a role in corneal ulceration in severe cases of allergy.


Neutrophils


Neutrophils are the most abundant type of white blood cell and it is clear that they play an important role in acute inflammation. They do not live as long as monocytes or lymphocytes, and are attracted to inflammatory sites by IL-8, interferon-γ, and C5a. One of their main functions is phagocytosis, in particular killing microbes using reactive oxygen species and hydrolytic enzymes. Whereas their role in innate immunity seemed clear, very provocative findings suggest a relationship with IL-17. IL-17 is made by not only by T cells and macrophages, but also by neutrophils. Further, IL-17 appears to mobilize lung neutrophils following a bacterial challenge. This would therefore suggest that neutrophils are responding to immune responses from both the innate and the acquired side of the immune process.


Resident Ocular Cells


The interaction of the resident ocular cells with those of the immune system is a most provocative concept. It is clear that several cells of the eye, including RPE and Müller cells, either have functions similar to cells within the immune system or can be induced to bear markers that potentially permit them to participate in immune-mediated events. There are microglia in the retina that are of hematopoietic origin. One can speculate (but there is no in vivo proof) that the initial priming of the immune system may occur through this interchange, or that the continued recruitment of immune cells may be mediated through these mechanisms. The effects of immune cells and their products may also be important for certain ocular conditions, inasmuch as macrophages as well as T-cell products have a profound effect on fibrocyte growth and division, and the RPE and Müller cells may respond in like fashion. RPE, when activated, can act as efficient APCs. Numerous lymphokines are found in the eye, many of which are produced by ocular resident cells. As mentioned above, it is not clear whether there can be antigen presentation in the eye, but in experimental models these cells do modulate this process. We also know that resident ocular cells do modulate the ocular environment by eliciting molecules that alter the immune process (ACAID).


Complement system


The complement system is a cascade of soluble proteins that ‘complement’ the function of antibodies in the immune system. Each complement protein is a proteolytic enzyme that acts as a substrate for the enzymes that precede it in the cascade, and which then acts as a part of a proteolytic complex for the next protein in the cascade. The classic complement pathway begins when C1q, C1r, and C1s (parts of the first component of complement) interact with membrane-bound antigen–antibody complexes to form an enzyme that cleaves C4 into C4a and C4b. C4b binds to the cell membrane, followed by C2, which is then split by C1s to yield a complex called C4b,2a. This complex splits C3 into C3a and C3b, which then joins the complex to make C4b,2a,3b. This complex cleaves C5 into C5a and C5b. C5b then binds to the cell membrane, and C6, C7, and C8 bind to it. The resulting C5b,6,7,8 complex then leads to C9 polymerization into the membrane.


The alternate pathway of complement does not require antibody but can be activated directly by bacterial cell walls and is therefore a nonspecific defense mechanism. In this pathway a small amount of pre-existing C3b cleaves factor B into Ba and Bb. The bacterial cell wall or other membranes assist in this step. The resulting C3b,Bb complex then cleaves more C3, forming a C3b,Bb,3b complex which can then cleave C5, and the pathway proceeds as already described.


The result is the generation of chemotactic protein fragments (C5a), protein fragments that cause smooth muscle contraction (C3a and C5a), protein fragments that cause mast-cell degranulation (C5a), molecules that assist in neutrophil phagocytosis (C3b), and molecules that are capable of promoting cell lysis (C5b,6,7,8,9). The complement system is therefore involved in many of the effectors of the inflammatory response.


Complement has become an area of special focus because of its possible role in the pathogenesis of age-related macular degeneration (AMD). Complement factors have been found in the drusen of AMD eyes, suggesting that an immune response may have occurred after the activation of the complement cascade. Several reports have appeared showing an association between a complement factor H variant and AMD. These observations are most provocative and still need to be defined functionally. However, we have felt that it may be part of a larger series of mechanisms that collectively we have called the ‘downregulatory immune environment’ of the eye. Indeed, this concept is now supported by the report that the CFH variant is associated with multifocal choroiditis, hence an alteration not unique to AMD.




Cellular interactions: hypersensitivity reactions


Figure 1-9 is a simplified version of the myriad interactions that have been identified in the immune system’s repertoire in the eye. Although many exceptions and alternative mechanisms (sometimes contradictory) have been proposed or partially demonstrated, certain useful basic concepts can be of help to the observer. The initiation of a response leading to immune memory requires antigen to be presented to T cells. Classically this is performed by dendritic cells (and perhaps macrophage cell lines) bearing the same class II (HLA-DR) antigens as the T cells. Other cells, however, may also be equally competent in performing this task. Potential candidates in the eye include the vascular endothelium, RPE, and Müller cells. Macrophages release factors such as IL-1 that are essential for the activation of the T cell. IL-1 also may be necessary as a cell-membrane component for antigen presentation to occur.




Figure 1-9.


Schematic representation of (1) numerous interactions in the eye of cells of the immune system, and (2) cells resident in the eye.

(Courtesy Rachel Caspi, PhD.)


The subsets of T cells, discussed earlier, cover a wide range of functions, from aiding B cells to produce antibody, to cell-mediated killing, to modulation of the immune response. A point worth bearing in mind is that T-cell recruitment is very much dependent on the release of factors (cytokines) that will help recruit and activate other initially uncommitted T cells. This seems to be a basic underlying mechanism for T-cell function.


Other cells also have a major impact on this T-cell–B-cell–macrophage axis. Mast-cell degranulation may assist the egress of immune cells into an organ, and the eosinophils, as well as neutrophils, will aid in killing and/or preparing pathogens for disposal by other parts of the immune system. T cells have a direct effect on mast-cell maturation in the bone marrow by the release of IL-3, whereas the T cell and other immune components have similar effects on other cells of the nonlymphoid series by the release of colony-stimulating factors.


Classic immune hypersensitivity reactions


Although it is not rare for any inflammatory response to involve several arms of the immune repertoire, it frequently appears that one arm of the system predominates. Inflammatory reactions were originally classified into four types or ‘hypersensitivity reactions’ by the British immunologists Philip Gell and Robin Coombs, with some recent additions.


Type I


This inflammatory reaction is mediated by antibodies, especially IgE. The binding of this antibody to mast cells or basophils results in the degranulation of these cells and the release of pharmacologically active products, as already mentioned. An ocular example of this reaction is hay fever. Typically a large amount of edema without structural damage is noted. The role for this immune mechanism in intraocular inflammatory disease is still unclear. It is not inconceivable that mast cells could play an ancillary role in some cases, but hard evidence is still lacking.


Type II


This type of reaction is mediated by cytotoxic antibodies and is thought to mediate hemolytic disorders, such as blood mismatch reactions and the scarring seen in ocular pemphigoid. It is clear that in ocular pemphigoid antibodies directed to the basement membrane of mucosal surfaces are present and may indeed be cytotoxic. One might consider the antibody effect of carcinoma or melanoma associated retinopathy to be a type II reaction. Intravitreal injections of human MAR IgG has been shown to alter retinal signaling. Another ocular example may be the rare disorder acute anular outer retinopathy. However, T cells can be noted to be infiltrating into the lesion in this disease. Some have suggested including in this category reactions termed antibody-dependent cell-mediated cytotoxicity, thereby making this category one that has a mixed mechanism.


Type III


This reaction is frequently referred to as an immune complex-mediated inflammatory response. The binding of antibody to an antigen – either fixed in tissue or free floating, that then deposits as a complex – can initiate the complement cascade, which in turn attracts cells capable of causing tissue damage. An example is the Arthus reaction, seen about 4 hours after the injection of antigen into the skin of a sensitized person or animal having substantial levels of circulating antibody directed to the antigen being injected locally. This hypersensitivity reaction had been suggested as being one of the major immune mechanisms leading to intraocular inflammatory disease, such as Behçet’s disease. However, more recent evidence suggests that its role in the uveitic process is more limited. Phacoanaphylaxis is a disorder that appears to be immune complex driven, at least in part.


Type IV


This category of immune response is for those mediated solely by T cells. It is therefore termed a cell-mediated immune mechanism, rather than a humoral mechanism, as was the case for the other three types of hypersensitivity reactions. The positive skin test reaction noted 48 hours after a PPD test is placed in the skin is an example of a type IV hypersensitivity reaction. Granulomatous responses as seen in sarcoid are mediated by this mechanism, as well as sympathetic ophthalmia. In all of these cases the humoral arm of the immune system is thought not to play a significant role in the inflammatory reaction. To date, the evidence suggests that T-cell dysregulation or T cell-controlled inflammatory responses are an extremely important – perhaps even essential – mechanism for intraocular inflammatory disease.


Type V


This reaction has been added to the original four. In this reaction an antibody can act as a stimulant to a target cell or organ. An example is long-acting thyroid stimulator (LATS) antibody, a feature of Graves’ disease. The LATS antibody is directed toward a portion of the TSH receptor in the thyroid and mimics the function of thyroid-stimulating hormone.




Concepts of disease pathogenesis


The potential mechanisms by which tissue damage is mediated by the immune system pose a question that has been hotly debated for some time. The debates are particularly vociferous because most arguments are difficult to support. However, recently these potential mechanisms have opened some of their secrets to observers, and the arguments of a previous generation are no longer acceptable. With our increased understanding of immune mechanisms comes the realization of the network’s complexity: that the system has many alternative choices and that there is an extraordinary intertwining of events that appears to be necessary for the immune system to respond appropriately, as well as inappropriately. It still is conceptually valid to simplify these potential mechanisms, and in the following pages we attempt to do that – to provide the reader with concepts rather than numerous specific details. The understanding of these mechanisms is certainly an intellectually stimulating undertaking. However, it has a practical aspect as well. Therapeutic interventions will be increasingly specific, tailored to the problem at hand. Therefore, in the not-too-distant future, an understanding of the mechanisms of ocular inflammatory disease will be invaluable in choosing the appropriate therapy for the patient.


Immune characteristics of the eye


It seems reasonable to begin a section on immune mechanisms that may be responsible for intraocular inflammatory disease by reviewing the characteristics of the eye that might influence these responses. For years the eye was considered to be a ‘privileged’ immune site. The implication of this was that the immune system somehow ignored or was tolerant of the antigens in the eye. We think it appropriate to consider the eye as being indeed immune privileged, but in a different way than implied by the original notion. Although the characteristics to be reviewed are not always unique to the eye, the combination of all these factors does elevate this organ to a special relationship with the immune system.


Absence of lymphatic drainage


Like the brain, placenta, and testes, the eye has no direct lymphatic drainage, although in mice submandibular nodes do collect antigen from the eye. The environment in which antigen presentation occurs plays an important role in the type of immune response the organism may mount. Experimentally, for example, antigen placed in an area with good lymphatic drainage will elicit an excellent immune response, with a measurable antibody response and cell-mediated immune response. However, the same antigen given intravenously may elicit a very different immune response, the ultimate response being immune tolerance (or anergy). Therefore this anatomic phenomenon may have a profound effect on the types of immune response elicited in the eye.


Intraocular microenvironment


It has been suggested that the eye has at least four ways to protect itself against unwanted or nuisance inflammatory processes. The first is having a barrier such as the blood–ocular barrier. The second is the presence of soluble or membrane-bound inhibitors that block the function of an organism. The third strategy is to kill an invading organism or cell that may be inducing an unwanted inflammation (by perhaps speeding up apoptosis or programmed cell death), and the fourth is to devise a method by which a state of tolerance is induced. All of these barriers appear to exist in the eye.


Anterior Chamber-Associated Immune Deviation (ACAID)


This could be seen as an example of the fourth strategy mentioned above. The immune response elicited by antigen placement into the anterior chamber has interested immunologists for some time and observations are constantly being added. Allogeneic tissue implants (i.e., tissue from the same species but not an identical twin) in the anterior chamber were noted to survive longer than those placed in other orthotopic sites. The placement of alloantigens into the anterior chamber of the eye has been noted to elicit a transient depression of cell-mediated immunity but an intact humoral response. This was initially called an F 1 -lymphocyte-induced immune deviation. A continued refinement and understanding of the phenomenon led to its being called ACAID. The model has been further extended to include hapten-specific suppressor T-cell responses to syngeneic splenocytes that are coupled with azobenzenearsonate (i.e., cell-bound antigens) and also has been obtained with soluble antigen alone, such as histocompatibility and tumor antigens. In addition, the induction of ACAID can be enhanced by placing a cell line or tumor that is syngeneic to the MHC of the host, and the capacity of the immune system to enhance or suppress tumor growth can be successfully manipulated by use of this phenomenon. Good antibody responses and cytotoxic T cells directed against the intraocularly placed tumor (or antigen) develop. However, although cells that mediate delayed hypersensitivity reactions do not form, antigen-specific suppressor cells do.


ACAID can be induced in primates, rats, and mice. , An antigen-specific ACAID will develop with the injection of IRBP into the anterior chamber of rats or mice. , Of interest as well is the fact that the mice susceptible to IRBP-induced experimental autoimmune uveoretinitis (EAU) will not develop the disease if IRBP is injected into the eye before systemic immunization.


Of prime import in ACAID is the presence of an intact ocular–splenic axis. The induction of suppressor T cells is enhanced when antigen processing bypasses the lymphatic drainage system normally present. There appears to be a unique processing of antigen in the dendritic cells of the eye. Cells then will carry the ACAID signal to the spleen for the activation of regulatory T cells. It has been reported that this signal in the blood was associated with F4/80+ macrophages, which populate the anterior uvea. It appears that this signal is water soluble. Of interest is the fact that in vitro exposure of APCs to aqueous humor – or TGF-β – will confer ACAID-like properties on these cells. Indeed, TGF-β appears to play one of the important roles in ACAID. Other investigators have noted a soluble factor that could be transferred by serum alone. This apparent contradiction might reflect the different experimental methods that were used. It could, however, also reflect the fact that several mechanisms may exist for the induction of ACAID. Indeed, during the disruption of the normal mechanisms, as happens with the addition of INF-γ into the eye, prostaglandins may replace TGF-β as the mediator of suppression. One might speculate on the following scenario: antigen enters into the anterior chamber and is taken up by APCs that live in the special environment of the eye. The APC brings the antigen to the spleen, secreting a chemokine (MIP-2) that will attract natural killer (NK) T cells. The NK T cells in turn will secrete IL-10 and TGF-β, both associated with a Th2 response. The T cells responding to this environment become regulatory cells that will suppress delayed hypersensitivity responses in the eye. In ACAID the afferent regulatory T cell is a CD4+ T cell, whereas the efferent regulator is a CD8+ T cell. The environment is such that lymphoid cells in the eye will not produce IL-12 or express CD40, important components of the immune response. This is different from the tolerance that is induced when an antigen is given intravenously.


The role of ACAID in clinical situations still needs to be evaluated; however, it is not difficult to speculate on its potential role in ocular tumors, as well as autoimmune and even infectious immune responses. This could be a mechanism by which nature attempts to limit unwanted inflammatory responses in the eye.


Fas-Fas Ligand Interactions and Programmed Cell Death (Apoptosis)


Fas ligand (FasL) is a type II membrane protein that belongs to the TNF superfamily. It is found in the eye and can induce apoptotic cell death in cells that express Fas. Fas is part of the TNF receptor family and is found on lymphocytes. It is believed that apoptosis is one method of immune privilege in the eye. It should be added that others may not feel it is the only way that cell death can occur among invading autoaggressive cells, but there is enough provocative evidence to suggest that it at least should be considered. Organs that appear to be able to limit immune responses, such as the eye, testes, and brain, express FasL. Other organs, such as the liver and the intestine, express this antigen only during severe inflammatory processes. Gene therapy experiments performed on other organs where FasL is transferred can confer immune privilege. It is clear that the Fas-FasL works in concert with several factors. One cofactor appears to be TNF. Activated lymphocytes producing TNF will be more at risk to become apoptotic. Other mechanisms induce apoptosis through IL-2 activation of lymphocytes. These highly activated cells will ultimately die a programmed death. This raises the interesting question whether blockage of part of either the TNF system or the IL-2 circuitry, despite being beneficial on the one hand, could prevent apoptosis of these cells, thereby leaving them at a site of inflammation longer or circulating longer.


Resident Ocular Cells and Immune System


Although communication between resident organ cells and the immune system is not unique to the eye, the number of cells potentially capable of fulfilling this role in the eye is indeed remarkable. The list begins at the cornea with Langerhans’ cells, and includes cells in the ciliary body that can express Ia antigens on their surfaces, the Müller cells, which are capable of profound effects on the immune response, and the RPE, with characteristics very similar to those of macrophages. Finally, the vascular endothelium of the eye, as in other organs, may be of great importance in regulating immune system activity.


Müller cells have been shown to have a profound affect on T cells. Isolated pure cultures of rat Müller cells will downregulate the proliferative capabilities of S-Ag-specific T cells capable of inducing experimental uveitis. Cell-to-cell contact is needed to see this phenomenon. It is interesting to note that when Müller cells are killed with a specific poison, the disease induced by S-Ag immunization in rats appears to be worse than in rats with ‘intact’ retinal Müller cells in the retina. Such experiments would suggest that Müller cells play a role similar to that of ACAID – that is, as part of the protective mechanisms that downregulate ‘nuisance’ inflammatory responses in the eye.


A very different story seems to emerge with both corneal endothelial cells and the RPE. Kawashima and Gregerson reported that corneal endothelial cells block T-cell proliferation, but T-cell activation signals from an APC were not blocked. This inhibition was not neutralized by the addition of neutralizing antibodies to TGF-β 1 or TGF-β 2 .


As mentioned, the RPE has many characteristics of macrophages. These cells have the capacity to migrate and engulf particles and have characteristics that strongly suggest a capacity to participate in the local immune response. The RPE has been shown to produce cytokines, the one of most note to date being perhaps IL-6, a lymphokine capable of inducing intraocular inflammatory disease when injected into the eye. RPE cells, which express MHC class I antigens constitutively on their surface, can express class II antigens when activated (see later discussion). Further, RPE cells in culture can act as APCs for S-Ag-specific T cells. Here, then, it would appear that we have an example of an ocular resident cell capable of augmenting (or initiating?) an immune response in the eye, but there is no clinical proof to support this concept. However, we do have further experimental evidence that it could indeed happen. We have shown that the glucocorticoid-induced TNF-related receptor ligand (GITRL) is expressed constitutively at low levels on the RPE (and other ocular cells). When GITRL expression is upregulated on RPE cells, the suppressive effects of the RPE on T-cell proliferation is abrogated and so is the production of TGF-β, an important contributor to the downregulatory environment. GITRL upregulation also induced proinflammatory cytokines in T cells. Interestingly, GITR serves as a negative regulator for NK cell activation. Indeed, one may argue that there are so many APCs, such as macrophages and dendritic cells, in the eye that it really does not seem reasonable to think that these ocular resident cells would initiate an immune response.


Cytokines and Chemokines and the Eye


A large number of cytokines, some produced locally by ocular resident cells and others by cells of the immune system, have been implicated in the ocular immune response. In addition to cytokines, numerous neuropeptides and other factors have been cited as being involved in the ocular immune response (see Fig. 1-9 , which shows the complex nature of this response). As a result of numerous experiments, cytokines can be termed ‘proinflammatory’ or ‘immunosuppressive’ in the intraocular milieu ( Box 1-1 ). Some cytokines have been noted to both stimulate and suppress the immune response, depending on the environment in which the cytokine is found. Instead of considering it contradictory, this phenomenon should be viewed as evidence of the complex immune response we are studying. IL-6 (produced locally), IL-2, and IFN-γ are perhaps the most important cytokines to be considered when an intraocular inflammatory response occurs. Foxman and co-workers evaluated the simultaneous expression of several cytokines, chemokines, and chemokine receptors in the eye during an inflammatory episode. Of interest were the relatively high levels of chemokine activity in noninflamed eyes. For experimental autoimmune uveitis, IL-1α, IL-1β, IL-1 receptor antagonist, IL-6, and TNF-α were highly expressed ( Fig. 1-10 ). Interferon-β is found in the serum of a large number of retinal vasculitis patients (including those with Behçet’s disease).


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Oct 21, 2019 | Posted by in OPHTHALMOLOGY | Comments Off on Elements of the Immune System and Concepts of Intraocular Inflammatory Disease Pathogenesis

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