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.

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.

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.

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.

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.)

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.

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.

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 .

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.

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.

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.

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

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.

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.