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

T-cell Receptor

Much interest has focused on the TCR. T cells need to produce the TCR on their cell surface to recognize the target immunogenic peptide on the MHCs of APCs. This complex interaction involves either CD4 or CD8 and their TCRs. The TCR is similar in structure to an immunoglobulin, having both α and β chains. 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 target 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. 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 small number of cells have a TCR made up not of α and β chains but, rather, of γ and δ chains (detailed later). In addition to these physiologic mechanisms, T cells may also be activated by “superantigens,” which are bacterial products, such as enterotoxins, or plant products, such as phytohemagglutinin. In addition, T cells may be activated by antibodies to certain surface antigens, mostly CD3 and CD28.

Major Populations of T Cells

The functions that have been briefly described are carried out by several subsets of CD4 T cells, identified by their products and functions. It was observed early on that T cells (and 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 1.2 ). The antibodies to the CD3 antigen in humans (e.g., OKT3) are directed against an antigen found on all mature human T cells in the circulation; approximately 70% to 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 (Th cells; about 60%–80% of the total T cells). These CD4+ cells respond to antigens complexed to MHCs of the class II type. The CD4 cells are particularly susceptible to human immunodeficiency virus (HIV) that causes acquired immunodeficiency syndrome (AIDS), with the percentage of this subset decreasing dramatically as this disease progresses. Furthermore, these helper cells are necessary components of the autoimmune response seen in the experimental models of ocular inflammatory disease induced by retinal antigens (see discussion of autoimmunity later in this chapter). Antibodies to the CD8 antigen (i.e., OKT8) distinguish a population that includes cytotoxic T cells, making up about 20% to 30% of the total number of T cells. Antibodies directed against the CD8 antigen block class I histocompatibility-associated reactions.

The two major populations of T cells (CD4 and CD8) are further divided into subpopulations that are detailed later. These subpopulations are generated by combinations of cytokines, which are products of other cells, and affect the immune system by the specific cytokines they produce.

T-cell Subsets

The population of Th cells has been further subdivided on the basis of their functional characteristics into several subsets. The major subsets are named Th1, Th2, and Th17. In normal conditions, Th1 cells defend against intracellular pathogens, Th2 cells defend against extracellular parasites and mediate antibody production, and Th17 cells defend against extracellular pathogens. Pathologically, Th1 and Th17 cells are responsible for initiation of “cell-mediated” immune responses, such as foreign tissue rejection and pathogenic autoimmune processes, whereas Th2 cells are involved in allergic responses and in immunoregulation. Th1 cells ( Fig. 1.3 ) produce mostly IFN-γ, IL-2, and tumor necrosis factor-α (TNF-α). The cytokine profile of Th2 cells consists of IL-4, IL-5, IL-10, IL-13, and perhaps TGF-β , and the major cytokines produced by Th17 cells are IL-17, IL-21, and IL-22. In many animal models of human diseases, Th1 and Th17 cells are associated with initiation of disease, whereas Th2 cells are associated with disease downregulation and allergy initiation.

Importantly, under experimental conditions, Th17 cells may switch to a Th1 phenotype, but Th1 cells maintain their phenotype and do not change.

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 its 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 disease and ulcerative colitis ^, and in both the serum and intraocular fluids of patients with uveitis. ^,

Gamma Delta (γδ) T Cells

γδ Τ cells, which constitute a small fraction of peripheral T cells, play important roles in inflammatory processes, such as EAU. Of particular importance is the involvement of γδ Τ cells in mucosal tissue inflammation in the conjunctiva, as shown by St Leger et al., where an inflammatory response to a commensal bacterium is mediated to a large extent by IL-17 produced by γδ cells.

T-regulatory Cells

It is now clear that just as the immune system needs cells to initiate a response, it needs cells to suppress or modify immune responses. One of the ways this need is met is with Treg cells. These cells derive from a naive T-cell population under the influence of cytokines that are different from those involved in Th1, Th17, or Th2 cell development (see Fig. 1.3 ). Treg cells can be found in the thymus or in the peripheral circulation, where a large portion is “induced” (iTregs). An interesting report by Kemper et al. described stimulating CD4+ cells with CD3 and CD46 (a complement regulator) and inducing Treg 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 Treg cells, and these cells have been reported in several organs, such as the gut, where peripheral immune tolerance needs to be induced. Certain Treg cells are characterized by their ability to produce IL-10 and TGF-β. They are capable of downregulating both CD4- and CD8-mediated inflammatory responses and apparently require cell-to-cell contact. The majority of Treg cells bear CD25 (the IL-2 receptor) on their cell surface and express the transcription factor forkhead/winged helix (FoxP3), which is a reliable marker for the development and function of naturally occurring Treg cells. When we evaluated the T cells of patients with ocular inflammatory disease, we found that the FoxP3 marker varied considerably among patients and was not a very good indicator of poor Treg cell function. An interesting observation is the noted increase in a subset of NK cells (so-called CD56 “bright”) after daclizumab therapy; this subset makes large amounts of IL-10, indicating the regulatory nature of these cells. The increase is seen when the patient’s disease is well controlled, and it has also been seen in patients with multiple sclerosis receiving daclizumab therapy.

Innate Lymphoid Cells

The recently discovered ILCs are lymphocytes that lack antigen specificity and are involved in the immune response by releasing cytokines or carry cytotoxic capacity (NK cells; see later). ILCs are mainly tissue resident and play major roles in keeping the homeostasis in these tissues. Unlike T or B cells, ILCs react promptly to stimulations, such as pathogen invasion. ILCs are separated into three major groups (ILC1, ILC2, and ILC3) that selectively collaborate with Th1, Th2, and Th17 lymphocytes in the defense against intracellular pathogens and tumors, large extracellular pathogens and allergens, and extracellular pathogens, respectively.

Natural Killer and Invariant Nature Killer T Cells

NK cells and invariant natural killer T (iNKT) cells are major components of the innate immune response, whose main function is to carry out the rejection by cytolytic activity of both tumors and virally infected cells. The main difference between NK cells and iNKT cells is their morphology, with NK cells being large granular lymphocytes, whereas iNKT cells express highly conserved TCRs. Both populations release large amounts of cytokines upon activation. NK cells seem to be involved in the pathogenesis of EAU because the disease was found to be diminished in mice with no NK cells. Grajewski et al. showed that similar to NK cells, iNKT cells ameliorate the EAU process. However, iNKT cells were also found to have a dual effect in the pathogenic process of EAU: The disease was enhanced in iNKT-deficient mice, but activation of these cells also exacerbated the pathologic process.

Cytokines

Intercellular communication, which is crucial for active immune response, is mediated by cytokines, chemokines, and adhesion molecules. Cytokines are produced by lymphocytes, macrophages, and other cells. They are hormone-like proteins capable of amplifying an immune response and of suppressing it. With activation of a T lymphocyte, the production and release of various lymphokines will occur. One of the most important cytokines is IL-2, with a molecular weight of 15 kDa in humans. The release of this lymphokine stimulates lymphocyte growth and augments specific immune responses, including stimulation of Treg cells. Of particular interest are cytokines involved in the inflammatory process. They include proinflammatory cytokines, IFN-γ, IL-1, IL-17, and TNF-α and the anti-inflammatory IL-4, IL-5, IL-10, and TGF-β. Of interest, IFN-γ and TGF-β are active in both activation and suppression of immune responses. The number of lymphokines that have been purified and for which effects have been described continues to grow rapidly. An incomplete list is shown in Table 1.3 and a more recent list has been provided by Akdis et al.

Chemokines

This family of chemoattractant cytokines is characterized by its ability to induce directional migration of movable cells. They 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, is found on chromosome 11); and CX3C (fractalkine is found on chromosome 16). The nomenclature is based on the 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 CX3C chemokines have three amino acids between their two N-terminal cysteines. Each chemokine family has special functions that affect different types of cells. An example of this fine specificity is seen with the CXC family. These 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 chemokine receptors. Organisms have adapted to these chemokines as well. HIV gp120 binds to CCR5 and CCR3, aiding its entry into the lymphocyte. This area of knowledge 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 (AMD), in which the immune components of the disease are just being explored but which may be important areas for therapeutic interventions.

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 (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. 1.5 ). In conventional responses of B2 cells, IgM is produced in minute amounts. Because of its size, IgM 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 confirm a newly acquired infection. IgM has a complement-binding site and can mediate phagocytosis by fixing C3b, a component of the complement system.

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 help 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 infections by pyogenic organisms, such as Streptococcus and Neisseria species. In addition, both 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% to 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, which then form a complex with the IgA dimer/J chain ( Fig. 1.6 ). 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. Furthermore, it can induce eosinophil degranulation.

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. 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 immunoglobulin). It is found simultaneously with IgM on B cells before specific stimulation. Little else is known about this antibody other than that 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 clinical and basic investigators 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 by using hybridoma technology has allowed for the production of these immune probes in 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 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 remain of mouse origin. The advantage of this is the reduced probability of an immune response against the foreign protein.

Mast Cells

This large (15–20 μm) cell is intimately involved in 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, chemotactic factors of anaphylaxis, and 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). Findings from human studies have supported 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, 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 factors of what we describe as a T-cell–mediated disorder.

The role of mast cells in the pathogenesis of EAU has been noted in early studies. Mochizuki et al. have noted that rat strain susceptibility to EAU induced with S-Ag was dramatically associated with the number of mast cells in the choroid, and de Kozak et al. have shown that mast cells in the choroid degranulate just before the influx of T cells into the eye, suggesting that these cells “open the door” into the eye for the T cells. This concept is especially provocative because Askenase et al. showed that mast-cell degranulation can be induced not only by IgE antibodies but also by T cells.

Eosinophils

These bilobed nucleated cells are about 10 to 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. The granules are almost entirely made up of major basic protein (molecular weight 9 kDa), 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; and cytokines, such as TGF-α and TGF-β, VEGF, TNF-α, IL-1α, IL-6, and IL-8.

The eosinophil arises in 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% of nucleated cells). These cells can be attracted to an area in the body through 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 eosinophil contains the anti-inflammatory agents histaminase and arylsulfatase, which are capable of neutralizing the effect of histamine release and slow-reacting substance, both products of mast cells. Furthermore, 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. Furthermore, 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 cells (WBCs), 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, IFN-γ, and C5a. One of their main functions is phagocytosis, in particular killing microbes by using reactive oxygen species (ROS) and hydrolytic enzymes. Although their role in innate immunity seemed clear, some thought-provoking findings have suggested a relationship with IL-17. IL-17 is made by not only by T cells and macrophages but also by neutrophils. Furthermore, IL-17 appears to mobilize lung neutrophils after a bacterial challenge. This would therefore suggest that neutrophils are responding to immune responses from both the innate and the acquired sides of the immune process.