Ординатура / Офтальмология / Английские материалы / Ocular Periphery and Disorders_Dartt, Bex, Amore_2011

Niederkorn, J. Y. (2006). Anterior chamber-associated immune deviation and its impact on corneal allograft survival. Current Opinion in Organ Transplantation 11: 360–365.

Niederkorn, J. Y. (2006). See no evil, hear no evil, do no evil: The lessons of immune privilege. Nature Immunology 7: 354–359.

Niederkorn, J. Y. and Wang, S. (2005). Immune privilege of the eye and fetus: Parallel universes? Transplantation 80: 1139–1144.

Skelsey, M. E., Mellon, J., and Niederkorn, J. Y. (2001). Gamma delta T cells are needed for ocular immune privilege and corneal graft survival. Journal of Immunology 166: 4327–4333.

Stein-Streilein, J. (2003). Invariant NKT cells as initiators, licensors, and facilitators of the adaptive immune response. Journal of Experimental Medicine 198: 1779–1783.

Glossary

Adaptive Immunity – The part of the immune system utilizing T cells and B cells that adapt to specifically target, eliminate, and prevent pathogenic infections. Through adaptive immunity, immunological memory is established to further adapt immunity to mount stronger responses each time the pathogen is encountered.

Immune homeostasis – The state of immunity in a stable, unperturbed tissue environment.

Immune privilege – The immune status of the ocular microenvironment that has evolutionally adapted itself to prevent the induction of excess inflammation, thereby protecting its delicate structures from the damages of inflammation. It is also defined as any tissue site, such as the brain and eye, which affords survival of incompatible grafts without immunosuppressive therapy.

Innate immunity – A more primitive defensive mechanism against infecting pathogens. The activation of innate immunity is through pathogenassociated molecules that bind specific recognition receptors on innate immune cells. Innate immunity is associated with phagocytosis, complement activation, and infiltration of neutrophils and macrophages. Neuropeptides – The low-molecular-weight proteins that are found in, and released from, centrally derived neurons; however, their production is not limited to neurons. They are produced by cells of endocrine glands, immune cells, and cells that make up immune-privileged tissue microenvironments.

Immunosuppression and

Anti-Inflammatory Activity in Aqueous

Humor

The immune-privileged microenvironment of the eye suppresses the induction of inflammation. One of the most dominant mechanisms of this suppression, by which the immune-privileged eye prevents induction of inflammatory immunity, is the manipulation of the functionality of immune cells that enter the ocular microenvironment.

A well-characterized set of neuropeptides that targets specific immune cells and their activities mediates this manipulation. The result of this immunosuppression and immunoregulation is the induction of immune cells that are not only prevented from expressing proinflammatory functionalities, but also regulate other immune cells. The regulatory activity itself is not unique; it occurs at the resolution of immune responses and is part of the mechanisms that prevent the induction of autoimmunity. These are the mechanisms of immune homeostasis that tailor the immune response, and prevent uncontrolled immunity. What is unique is that within the ocular microenvironment these mechanisms are constantly active, and are mediated by constitutively present soluble factors that provide the eye with its unique form of immune homeostasis.

Over the past two decades, we have come to understand that the ocular microenvironment is rich with immunosuppressive molecules that influence the activity of immune cells. Many of these molecules are found in the aqueous humor. The first indication that soluble mediators in the eye manipulate the function of immune cells was the finding that aqueous-humor-treated macrophages process and present antigen in a manner that promotes activation of suppressor T cells. This activity is associated with a phenomenon of systemic antigen-specific immunosuppression induced by placing an antigen into the anterior chamber of the eye. This phenomenon is called anterior- chamber-associated immune deviation (ACAID), which may be responsible for the highly successful acceptance of corneal grafts, and may be important in the survival of ocular stem cell and retinal transplants.

Macrophages and dendritic cells stimulated with bacterial products that activate innate immune-mediated inflammation fail to mediate inflammation when they are treated with aqueous humor. The expected promotion of macrophages and dendritic-cell-antigen-presenting cell functionality to promote proinflammatory T-cell activation are also suppressed by aqueous humor treatment. Moreover, aqueous-humor-treated macrophages and dendritic cells produce anti-inflammatory cytokines, and present antigens in a manner that promotes immune regulation. Therefore, resident ocular macrophages and dendritic cells, while still able to respond to infectious agents and unhealthy cells, are inhibited from mediating an inflammatory response. It is not completely understood how aqueous humor manipulates the functionality of macrophages. It is possible that ocular resident macrophages function perfectly well in

clearing and defending the ocular microenvironment from a pathogen, but cannot recruit other immune cells to help control the infection through inflammation, or to mediate an effective wound response.

T cells treated with aqueous humor are inhibited in proliferation, cytotoxic activity, and production of proinflammatory cytokines. The CD4+ T cells treated with aqueous humor are not only suppressed in their production of the proinflammatory cytokine interferon-gamma (IFN-g), but they also produce the regulatory cytokine, transforming growth factor-beta (TGF-b). This change in cytokine production is associated with a change in T-cell functionality from inflammatory to regulatory. The resistance of the ocular microenvironment to the activation of inflammation mediated by T cells is seen when T cells that mediate delayed-type hypersensitivity are placed into the anterior chamber of the eye along with antigen and antigen-presenting cells. These T cells normally mediate inflammation if injected into the skin, a conventional immune site, with their antigen and antigenpresenting cells; however, in the anterior chamber, the T cells do not mediate inflammation. In addition, treating the T cells in vitro with aqueous humor and then transferring them into the skin fails to produce inflammation. Moreover, aqueous-humor-treated Tcells function as regulatory T cells that can suppress the activation of other hypersensitivity-mediating T cells. Therefore, the constitutive immunoregulation and immunosuppressive soluble factors of aqueous humor prevent the activation of inflammatory immunity while turning the immune response onto itself to further regulate the immune response within the immune-privileged ocular microenvironment.

There is an ever-growing list of identified factors in aqueous humor that have the potential to suppresses and regulate immunity (Table 1). Neuropeptides form a major group of these factors and most of these factors are found throughout the eye, suggesting that they regulate immunity in all tissue sites of the ocular microenvironment. The factors important for the regulation of T-cell activation and innate immunity in aqueous humor are TGF-b, alpha- melanocyte-stimulating hormone (a-MSH), calcitonin- gene-related peptide (CGRP), somatostatin (SOM), and vasoactive intestinal peptide (VIP) (Figure 1). Other factors, such as Fas ligand (FasL), maybe important in eliminating activated T cells within the ocular microenvironment, and factors such as macrophage migration inhibitory factor (MIF), and complement inhibitors are important in the regulation of innate immunity. In addition to neuropeptides, the retina produces thrombospondin-1 (TSP-1), and pigment epithelium-derived factor (PEDF), which influence the activation and regulation of immunity. It is the collective activity of these molecules within an intact blood–ocular barrier that maintain the unique immune homeostasis of the ocular microenvironment, a process called immune privilege.

Table 1 Immunoregulating and immunosuppressive factors of ocular immune privilege

aProduction of proinflammatory cytokines and antimicrobial molecules induced by bacterial products or by interleukin-1 and tumor necrosis factor.

bAssayed for APC activation of hypersensitivity-mediating T cells.

cAssayed for antigen-stimulated T-cell production of proinflammatory cytokines, proliferation, cytotoxic activity, and survival. dAssayed in an adoptive transfer of treated APC for induction of immune deviation.

eComplement regulatory proteins.

The Immunoregulatory and

Immunosuppressive Factors of The

Immune-Privileged Eye

Transforming Growth Factor-Beta

TGF-b was the first identified immunosuppressive factor in aqueous humor. While it is common to discuss TGF-b in a generic manner, the most interesting aspect of the TGF-b produced in the eye is that a single isoform, TGF-b2, is the dominant form found in aqueous humor, with very little of the other forms of TGF-b being present in the aqueous humor. The dominant expression of TGF- b2 over the other isoforms of TGF-b is a common feature of neurological tissues. The significance of expressing TGF-b2 over the other isoforms of TGF-b is not known. In addition, TGF-b is normally expressed in a latent form, requiring activation by other enzymes or binding proteins. It is through TGF-b2 that aqueous humor induces ACAID-mediating antigen-presenting cells (APCs). Treating macrophages in vitro with TGF-b2 induces the characteristics of the aqueous-humor-induced and the in-vivo-induced ACAID APCs. When exposed to TGF-b2, the macrophages increase their expression of the surface marker F4/80, and have reduced expression of coreceptors needed in T-cell activation. The macrophages express anti-inflammatory cytokines of interleukin-10 (IL-10) and activated TGF-b. The activated TGF-b production by the macrophages is probably associated with an

Cornea

Aqueous

humor

Figure 1 Distribution of soluble immunomodulating proteins in the eye. TGF-b, transforming growth factor-beta; a-MSH, alpha- melanocyte-stimulating hormone; VIP, vasoactive intestinal peptide; CGRP, calcitonin-gene-related peptide; SOM, somatostatin;

NPY, neuropeptide Y. Adapted from Taylor, A. W. (2009). Neuropeptides, aqueous humor, and ocular immune privilege. In: Troger, J., Kieselbach, G., and Bechrakis, N. (eds.) Neuropeptides in the Eye, pp. 79–91. Research Signpost, Kerala, India.

autocrine pathway involving TSP-1. This induction of ACAID-inducing APCs is not limited to the anterior chamber and aqueous humor. Injecting antigen into the retina induces a similar immune deviation involving TGF-b and TSP-1 as well. While the phenomenon of ACAID is an antigen-specific systemic immunosuppression initiated by ACAIDogenic APCs that migrate to the spleen, it is not clear how these APCs may function within the ocular microenvironment. Macrophages or dendritic cells that take up residence with the ocular microenvironment will likely be exposed to some level of activated TGF-b2, and as a result, they express low levels of molecules that promote T-cell action, and simultaneously produce anti-inflammatory cytokines. There is evidence to support this hypothesis. Expression of antigen-presenting molecules within the ocular microenvironment is rarely detected. However, the expression of anti-inflammatory cytokines, such as IL-10, has not been found in abundance in normal aqueous humor. This suggests that while the presentation of antigen by resident APCs in the eye is impaired by TGF-b, other functionalities induced by TGF-b in vitro may not occur within the ocular microenvironment. Some of the suppression of T-cell activation by aqueous humor may involve TGF-b2; however, neutralization of TGF-b2 in aqueous humor does not eliminate all of the T-cell suppression by aqueous humor. Aqueous- humor-treated T cells function as regulatory T cells, which produce TGF-b and suppress the action of other T cells. Therefore, it appears that one of the effects of TGF-b2 in the ocular microenvironment is the

suppression of antigen presentation that promotes inflammatory T-cell activation. If there is activation, it results in the generation of immune cells that produce additional TGF-b that further promotes immune privilege.

Alpha-Melanocyte-Stimulating Hormone

The neuropeptide a-MSH is a 13-amino-acid-long neuropeptide that is derived from sequential endoproteolytic cleavage and post-translational modifications of the protein proopiomelanocortin (POMC) hormone. Originally described for its melanin-inducing activity in frogs, a-MSH has a fundamental role in modulating inflammatory responses. Injections of a-MSH suppress systemic inflammatory responses to endotoxin, and proinflammatory cytokines such as IL-1 and tumor necrosis factor (TNF). Its anti-inflammatory activity is greatest on macrophages, dendritic cells, and neutrophils where it can suppress the induction of reactive oxygen intermediates, nitric oxide, proinflammatory cytokine production, and immune cell migration. It also enhances its own receptor expression and production in macrophages, which in turn, promotes an anti-inflammatory autocrine loop. a-MSH also induces IL-10 production by macrophages and dendritic cells. The anti-inflammatory activity of aqueous humor resembles the anti-inflammatory activity of a-MSH. a-MSH is constitutively present in the aqueous humor in pg ml–1 amounts that are highly antiinflammatory. In aqueous humor, a-MSH has two roles: it suppresses proinflammatory cytokine production by endotoxin-stimulated macrophages and it induces regulatory activity in T cells. While a-MSH suppresses proinflammatory cytokine production, it does not affect antigen presentation other than causing the APCs to present antigen in a manner that does not promote inflammatory T-cell activation. a-MSH-treated macrophages that are stimulated with endotoxin have their intracellular signaling pathway from endotoxin-bound receptors blocked by a-MSH treatment. Thus, instead of a classically activated macrophage producing proinflammatory cytokines and anti-microbial molecules, it produces anti-inflammatory cytokines. This suggests that the ocular microenvironment has a pathway to clear microbial molecules or pathogens without inducing an inflammatory response. Whether this is an effective process is yet to be confirmed.

The effect of aqueous humor a-MSH on T cells is profound. It is possible to change an antigen-specific proinflammatory T-cell response to an antigen-specific regulatory T-cell response by treating T cells with aqueous humor or with a-MSH. In this process, TGF-b2 helps in promoting a-MSH induction of regulatory activity in T cells. Flow cytometric analysis of the a-MSH-treated T cells has shown that they express the regulatory T-cell marker CD25 and that this activity is limited to CD4+

T cells. Unlike other types of regulatory T cells, TGF-b is the only cytokine that appears to be produced by these a-MSH-induced regulatory T cells. Such regulatory T cells can be generated in vitro and used in adoptive transfer experiments to show that they require antigen specificity to activate their suppressive functionality. When their antigen specificity is for retinal autoantigens, they can be used to suppress autoimmune uveitis, and promote retinal allograft survival. Recently, it has been reported that mice that naturally recovered from autoimmune uveitis produce regulatory CD4+ T cells that are specific for retinal autoantigens. Importantly, such CD4+ T-regulatory cells are not detected in mice that do not require the receptor for a-MSH on their T cells. There are four a-MSH receptors that are differentially expressed on immune cells. For T cells, it is the melanocortin 5 receptor (MC5r) that is required for a-MSH to induce regulatory T-cell activity. Interestingly, the retinas of postexperimental autoimmune uveitis (EAU) mice, which do not express MC5r, are severely damaged with losses in photoreceptors. This severe damage is not seen in normal post-EAU mouse retinas. The retina itself also expresses MC5r, and there is evidence that the melanocortin family of proteins is needed for normal ocular development. This suggests that a-MSH, which is produced by several layers of cells in the retina, is important for healthy retinal development, survival, and immune privilege. It has been proposed that immune privilege is an evolutionary adaptation to protect the eye from inflammation, and a-MSH may be one these adaptations of a molecule originally used for other purposes, but now has the added role of being immunosuppressive and immunoregulatory.

Other Neuropeptides

Three neuropeptides were examined for their presence, and a possible role in aqueous humor immunosuppression because they are found in the neurons that innervate the anterior chamber and some are produced by neural cells of the retina. SOM, CGRP, and VIPs are constitutively present in aqueous humor in ng ml–1 amounts, are expressed in the retina, and target different cells of the immune response.

While SOM is found in aqueous humor, its presence was not required for aqueous-humor-mediated immunosuppression. We discovered that SOM induced a-MSH production in T cells that in turn caused the T cells to become regulatory T cells. Therefore, SOM contributes to immune privilege by further promoting production of antiinflammatory and immunoregulating factors by immune cells. There are some contradictory findings regarding the role of SOM in the retina. A protein produced by a retinal pigment epithelial (RPE) cell called PEDF was found to suppress the induction of inflammatory activity in macrophages. However, this suppression was countered by SOM. Activating macrophages by treatment with RPE-derived

factors results in enhanced nitric oxide production, yet the macrophages continue to produce anti-inflammatory cytokines. The meaning for this contradictory finding is not clear. It is possible that macrophages are alternatively activated within the retina and possibly in other tissue sites of the ocular microenvironment.

The role of VIP in immune privilege still remains a bit of a mystery. This neuropeptide is in aqueous humor, and it suppresses the activation and proliferation of T cells; however, it does not induce regulatory T cells. The suppression of T-cell proliferation by VIP is only 50%, and there is some speculation that selected populations of T cells are responsive to VIP. Therefore, it is possible that whole aqueous humor selectively expands a subpopulation of T cells that is not responsive to VIP. Maybe these are the regulatory T cells. VIP receptors are present on macrophages, but there is nothing known about the role of aqueous humor VIP on antigen presentation, and on inflammatory activity of macrophages. One problem is that it is not clear whether VIP exists in the ocular microenvironment as a whole molecule or as immunoreactive functional peptide. It is possible that in aqueous humor, or in other regions of the ocular microenvironment, different types of VIP fragments are present and affect different target cells and have different effects on immunity. Whether it is a whole polypeptide or a fragmented peptide, VIP is a contributing factor to the immunosuppression seen within the immune-privileged eye.

Unlike the other neuropeptides in aqueous humor, GCRP does not target T cells, but instead, influences macrophage activity. Most mature T cells are unresponsive to CGRP. The CGRP in aqueous humor suppresses nitric oxide generation by macrophages that have been stimulated with endotoxin and IFN-g. Neutralization of CGRP activity in aqueous humor also eliminates aqueous-humor-mediated suppression of nitric oxide production by inflammatory macrophages. The concentration of CGRP in healthy aqueous humor is 20-fold less than its concentration in uveitic aqueous humor. At the higher concentration found in uveitic eyes, CGRP has no effect on inflammatory macrophage function. This suggests that in normal conditions, there is a specifically maintained concentration of CGRP for immunosuppression. Studies on CGRP in aqueous humor have brought to light several issues about the homeostatic environment of the immune-privileged ocular microenvironment. The immune-privileged ocular microenvironment needs to not only maintain a constitutive level of a specific set of factors, but also must maintain them at physiological and functional concentrations.

Other Molecules

There are several publications that individually describe the immunosuppressive activity of proteins that are not

always considered immunosuppressive. Two that have already been discussed above, PEDF acting as an antiinflammatory cytokine, and TSP-1 being important in TGF-b activation and in the process of ACAID, are usually considered anti-angiogenic factors. Their regulation of angiogenesis may be their main function within the eye, but may have evolved like a-MSH, also to function as an immunoregulatory factor. The inflammatory cytokine, macrophage MIF, was considered an important molecule produced by activated T cells to keep macrophages migrating from sites of inflammation. In the eye it has been found to be constitutively expressed in aqueous humor and has a role in preventing natural killer (NK) cells from killing cells not expressing major histocompatibility complex (MHC) antigens. Transformed and injured cells express altered or reduced MHC class I antigens on their surface, and NK cells see this as signal to kill the cell. Since MHC class I molecules are expressed at low levels within the ocular microenvironment, the presence of MIF protects these cells from NK cell attack. The regulation of NK cells and inflammatory macrophages is part of the ocular microenvironment that controls innate immunity. Another component of innate immunity is the complement cascade pathways that release protein fragments that induce migration and activation of immune cells, vascular leakage, and cellular lysis. There are constitutively expressed inhibitors of complement within the eye. In aqueous humor, there are two inhibitors that prevent activation of the alternative complement pathway. Although complement inhibitors are present in the eye, there is evidence that complement activation maybe occurring at a low level that might be necessary to maintain normal aqueous flow.

Finally, there is the expression of FasL, a membranebound immunosuppressive molecule that is expressed by cells throughout the ocular microenvironment. When FasL binds to Fas, a molecule on activated immune cells, it triggers programmed cell death. It has been suggested that it is a major contributor to immune privilege and is necessary for graft survival within the ocular microenvironment. However, its expression has not explained the finding that FasL expression can induce neutrophil activation and destruction of corneal grafts. Recently, there is some evidence that FasL exists as a soluble molecule in aqueous humor, and that soluble FasL may block neutrophil activation, and act as an immunosuppressive molecule. This could mean that the balance between soluble FasL and membrane-bound FasL is what is important for maintaining immune privilege in the eye. In addition, surviving allografts of other tissues when engineered to express FasL promote induction of graft-specific regulatory T cells. While expression of FasL may keep activated immune cells at bay, it could be a selective element within ocular microenvironment promoting activation of regulatory immune cells.

Application of the Lessons of Immune

Privilege

The rapidly expanding discoveries of the mechanisms of immune privilege demonstrate that within the ocular microenvironment are active processes for suppressing inflammatory immunity, promulgating alternative activation of immune cells, and mediating regulatory immunity are present. Many of these mechanisms are normally found at various phases of a conventional immune response, especially in the resolution phase, and it appears that the responsiveness of the immune cells to the ocular immunosuppressive and immunoregulating mechanisms is the same whether the cells are taken from the eye or from other tissues. Therefore, it is becoming clear that such mechanisms of ocular immune privilege can be imposed onto immunity to prevent, cure, and establish or reestablish immune tolerance in various hypersensitivity responses, autoimmune diseases, and prevent allograft rejection in tissues other than just the eye. The mechanisms of ACAID and a-MSH-mediated suppression of immunity have been used to demonstrate the potential of applying the lessons of ocular immune privilege as a therapy.

The therapeutic approaches for both mechanisms are direct applications that in some ways are personalized therapies tailored to the disease and the patient. The ACAID therapy involves the use of patient’s monocytes, which are treated ex vivo with TGF-b and antigen and reinfused into the same patient. The a-MSH therapy involves either injecting the neuropeptide into the tissue site or collecting the patients’ own immune cells and treating them ex vivo with a-MSH while they are restimulated with autoantigen. The feasibility of these therapeutic approaches has been demonstrated using rodent models of autoimmune diseases, allografts, and hypersensitivity. The therapeutic utilization of the mechanisms of ocular immune privilege is in its infancy, and has a strong potential in being a new direction in immunotherapy.

Conclusion

Starting with the first experimental description of ocular immune privilege by Medawar in the 1940s, the understanding of the mechanisms of immune privilege has grown. Along with this understanding is a change in the concept of immune privilege. At first, immune privilege was viewed as an interesting experimental phenomenon that was explained by the unique anatomical features of the ocular microenvironment, which include the anterior chamber’s lack of direct lymphatic drainage and the presence of an ocular–blood barrier. These passive mechanisms suggested that the immune system was ignorant of the presence of antigen within the eye. However, today we

know that the immune system perceives antigen placed into the eye, but it is the active engagement with the ocular microenvironment that regulates and controls immunity within the eye and systemically to the intraocular antigen. This active engagement is mediated by soluble immunoregulatory and immunosuppressive factors and neuropeptides. Our discovery and understanding of the mechanisms of ocular immune privilege will not only lead to potentially new immunotherapeutic approaches, but may also reveal the mechanisms of how the immune system regulates itself.

See also: Adaptive Immune System and the Eye: T CellMediated Immunity; Antigen-Presenting Cells in the Eye and Ocular Surface; Innate Immune System and the Eye.

Further Reading

Apte, R. S., Sinha, D., Mayhew, E., Wistow, G. J., and Niederkorn, J. Y. (1998). Cutting edge: Role of macrophage migration inhibitory factor in inhibiting NK cell activity and preserving immune privilege. Journal of Immunology 160: 5693–5696.

Cousins, S. W., McCabe, M. M., Danielpour, D., and Streilein, J. W. (1991). Identification of transforming growth factor-beta as an immunosuppressive factor in aqueous humor. Investigative Ophthalmology and Visual Science 32: 33–43.

Granstein, R., Staszewski, R., Knisely, T., et al. (1990). Aqueous humor contains transforming growth factor-b and a small (<3500 daltons) inhibitor of thymocyte proliferation. Journal of Immunology 144:

3021–3027.

Griffith, T. S., Brunner, T., Fletcher, S. M., Green, D. R., and Ferguson, T. A. (1995). Fas ligand-induced apoptosis as a mechanism of immune privilege. Science 270: 1189–1192.

Han, D., Tian, Y., Zhang, M., Zhou, Z., and Lu, J. (2007). Prevention and treatment of experimental autoimmune encephalomyelitis

with recombinant adeno-associated virus-mediated alpha-melanocyte-stimulating hormone-transduced PLP139-151- specific T cells. Gene Therapy 14: 383–395.

Ng, T. F., Kitaichi, N., and Taylor, A. W. (2007). In vitro generated autoimmune regulatory T cells enhance intravitreous allogeneic retinal graft survival. Investigative Ophthalmology and Visual Science

48: 5112–5117.

Nishida, T., Miyata, S., Itoh, Y., et al. (2004). Anti-inflammatory effects of alpha-melanocyte-stimulating hormone against rat endotoxin-induced uveitis and the time course of inflammatory agents in aqueous humor. International Immunopharmacology 4: 1059–1066.

Taylor, A. W. (2003). Modulation of regulatory T cell immunity by the neuropeptide alpha-melanocyte stimulating hormone. Cellular and Molecular Biology (Noisy-le-grand) 49: 143–149.

Taylor, A. W. (2007). Ocular immunosuppressive microenvironment.

Chemical Immunology and Allergy 92: 71–85.

Taylor, A. W., Streilein, J. W., and Cousins, S. W. (1992). Identification of alpha-melanocyte stimulating hormone as a potential immunosuppressive factor in aqueous humor. Current Eye Research 11: 1199–1206.

Taylor, A. W., Streilein, J. W., and Cousins, S. W. (1994). Immunoreactive vasoactive intestinal peptide contributes to the immunosuppressive activity of normal aqueous humor. Journal of Immunology 153: 1080–1086.

Taylor, A. W. and Yee, D. G. (2003). Somatostatin is

an immunosuppressive factor in aqueous humor. Investigative Ophthalmology and Visual Science 44: 2644–2649.

Taylor, A. W., Yee, D. G., and Streilein, J. W. (1998). Suppression of nitric oxide generated by inflammatory macrophages by calcitonin gene-related peptide in aqueous humor. Investigative Ophthalmology and Visual Science 39: 1372–1378.

Wilbanks, G. A. and Streilein, J. W. (1992). Fluids from immune privileged sites endow macrophages with the capacity to induce antigen-specific immune deviation via a mechanism involving transforming growth factor-beta. European Journal of Immunology 22: 1031–1036.

Zhang-Hoover, J. and Stein-Streilein, J. (2007). Therapies based on principles of ocular immune privilege. Chemical Immunology and Allergy 92: 317–327.

Glossary

ACAID (anterior chamber-associated immune deviation) – Systemic inhibition of delayed-type hypersensitivity reactions to antigens which have previously been placed into the anterior chamber of the eye.

APC (antigen-presenting cell) – A cell that displays foreign antigen complex with major histocompatibility complex on its surface.

CX3CR1 – CX3CR1 (Fractalkine receptor) is important for homing of Langerhans-like dendritic cells to the corneal epithelium.

DC (dendritic cell) – Professional antigenpresenting cells.

DC-LAMP/CD208 – A member of the lysosomeassociated membrane glycoprotein (LAMP) family, specifically expressed by mature dendritic cells. DC-SIGN/CD209 – A type 2 transmembrane protein that also contains a mannose-binding (C-type lectin) domain, expressed on dendritic cells.

EAU (experimental autoimmune uveitis) –

A disease of the neural retina induced by immunization with retinal antigens.

F4/80 – Antibody that recognizes both dendritic cells and macrophages.

GFP (green fluorescent protein) – Originally isolated from the jellyfish Aequorea victoria that fluoresces green when exposed to blue light. The GFP gene is frequently used as a reporter of expression. Animals have been created that express GFP as a proof-of-concept that a gene can be expressed throughout a given organism.

LCs (Langerhans cells) – Dendritic cells that typically reside in the epithelium or epidermis.

MHC class II (major histocompatibility complex class II) – These are necessary to present antigen to T cells.

Introduction

Antigen-presenting cells (APCs) serve as the immune sentinels to the foreign world and can be subdivided into professional and nonprofessional APCs. In the eye, professional APCs, such as dendritic cells (DCs), epithelial

Langerhans cells (LCs), macrophages, and B cells, are derived from hematopoietic stem and progenitor cells in the bone marrow (BM), forming an integral part of the immune system. Nonprofessional APCs are found among nonlymphoid cells (e.g., vascular endothelial cells, corneal endothelial cells, and keratocytes) and have a low T-cell stimulatory capacity. However, they can gain requisite signals for T-cell priming under certain circumstances (e.g., inflammation).

DCs are specialized APCs that play a dual role in inducing adaptive immune responses to foreign antigens and in maintaining T-cell tolerance to self. DCs can also play an important role in innate immunity due to their capacity to respond acutely to inflammatory insults or danger signals in peripheral tissues. DCs consist of several distinct populations that can be differentiated by surface and intracellular phenotypic markers, immunological function, and anatomic location. In mice, DCs variously express the CD11c integrin and MHC class-II (MHC-II) molecules, and are further phenotypically distinguished by their differential expression of CD8a, CD4, and CD11b, as well as a growing list of other new markers. Irrespective of their phenotype and immunological role, DCs exert their activity in the eye remote from their place of origin, where they utilize their advanced migratory skills for navigation.

DC progenitors are not restricted to the BM and can be found in multiple locations. These progenitors can differentiate into DCs upon challenge in peripheral tissues. Fully differentiated DCs are found in healthy tissues as immunologically immature cells, being able to sample foreign antigens, but not able to prime naive T cells. Immature DCs express negligible amounts of MHC-II on their surface, and lack the requisite accessory (costimulatory) signals for T-cell activation, such as CD40, CD80 (B7-1), and CD86 (B7-2). In their immature state, they remain alert until signals in the extracellular milieu through inflammatory mediators (derived from microbes or distressed bystander cells) induce a rapid change in function, also known as activation or maturation. Maturation induces redistribution of MHC molecules from the intracellular endocytic compartments of DCs to the cell surface, allowing for T-cell stimulation.

Macrophages reside in virtually every tissue, are an integral part of the innate immune response, and synthesize and secrete a variety of powerful biological molecules. They develop from myeloid progenitor cells, enter the bloodstream as monocytes, and migrate into tissues as macrophages. Monocytes are circulating precursors for

tissue DCs and macrophages, being able to maintain or replenish populations in the peripheral tissues during homeostasis. Macrophages express low levels of MHC-II and costimulatory molecules that enable them to act as APCs, even though much less efficient than DCs. Macrophages are generally poorly responsive to activation signals, and also play a role in other processes, including immune regulation and suppression, tissue reorganization, angiogenesis, and lymphangiogenesis. APCs, including macrophages and DCs, are found in a variety of ocular tissues, including the cornea, conjunctiva, iris, ciliary body, sclera, retina, and choroid.

Antigen-Presenting Cells of the

Ocular Surface

The immune-mediated responses of the ocular surface are influenced by its unique anatomy and physiology. The ocular surface consists of three distinct anatomical regions: the cornea, the limbus, and the conjunctiva that function both independently and in concert as specific barriers against microbial, immunogenic, and traumatic insults. Although the conjunctiva and cornea are anatomically proximate and are bathed in the same tear film, their immune responses are distinctly different from each other. Two populations of BM-derived cells, (1) macrophages or

monocytes and (2) DCs/ LCs, form the main APC arm of the ocular surface immune response (Figure 1).

Corneal APCs

Epithelial Langerhans cells

During homeostasis, peripheral resident LCs, a subset of DCs, are the only cells that constitutively express MHC-II in the corneal epithelium (Table 1). While a large number of LCs are MHC-IIþ in the periphery, a large population of MHC-II-negative immature LCs are present both in the periphery and the center of the epithelium, with the center being exclusively negative for MHC-II and costimulatory markers. These immature LCs are capable of expressing MHC-II and costimulatory markers during inflammation and migrate to draining lymph nodes (LNs) to present antigen. Phenotypically, both the peripheral and central murine LCs are CD11cþCD11b , with the density of these cells decreasing from the limbus toward the center. These LCs have a classic dendritic morphology with long processes interdigitating among the corneal epithelial cells. In mice lacking the chemokine receptor CX3CR1, homing of immature LCs to the epithelium is markedly impaired.

APCs can be observed in living healthy corneas by modern in vivo confocal microscopy. Similar to rodents, the density of APCs declines from the limbus to the

Sclera

Cell type Markers Location

CD45+, CD11c+ Limbal, equatorial,

peripapillary

CD45+, CD11b+ Limbal, equatorial,

peripapillary

Location

Nerve fiber layer ganglion cell layer, inner plexiform layer, outer plexiform layer

Juxtapapillary regions (strain variability)

Perivascular

center in the healthy human cornea. In the corneal limbal epithelium, DCs are present in almost every healthy subject, while in the central cornea only some 20–30% of healthy controls show APCs. LCs are located at a depth of 35–60 mm, mostly at the level of basal epithelial cells and the subbasal nerve plexus. Phenotypically, peripheral LCs in freshly cultured human corneas are Langerin (CD207)þ/CD1aþ/CD11cþ/HLA-DRþ, with no Langerin expression on central LCs. The expression of high levels of CD1a and Langerin on peripheral LCs suggests a unique role of these cells in initiating immune responses to microbial pathogens.

Stromal APCs

Resident DCs reside in the periphery and center of the anterior corneal stroma. Phenotypically, these DCs are CD11cþCD11bþCD8a demonstrating their monocytic lineage, although a small number of plasmocytoid DCs have been described. Peripheral stromal DCs are MHC-IIþ and positive for the costimulatory markers CD80, CD86, and CD40. The stromal center, however, contains exclusively MHC-II CD80 CD86 DCs, similar to those of the highly immature LCs in the epithelium. The density of murine stromal DCs decreases from the limbus toward the center of the cornea. A population of undifferentiated monocytic precursor cells distinct from DC and macrophage populations also resides in the corneal stroma. Thus, in contrast to other organs, where terminally differentiated populations of resident DCs and/or macrophages outnumber colonizing precursors, large numbers of DCs within the cornea remain in a relatively undifferentiated state. The absence of MHC-II

molecules in the normal cornea might actively maintain tolerance to foreign antigens, as antigen presentation to T cells by immature DCs can lead to anergy of T cells and subsequent tolerance, protecting the cornea from immune-mediated damage, when the insults are minor.

Resident CD11c CD11bþ corneal macrophages are present in the posterior stroma of the normal mouse and human cornea, and are distinct from the DCs described in the anterior stroma. They are located in the peripheral, paracentral, and central regions. These resident stromal macrophages likely provide a critical first line of defense against pathogens that breach the epithelial barrier of the cornea by producing antimicrobial substances, as well as other inflammatory cytokines and chemokines to attract and activate additional macrophages, neutrophils, and DCs.

In freshly cultured human corneas, DCs express DCSIGN and are detected mainly peripherally and in the anterior stroma, having only variable CD11c expression. Most of these cells are HLA-DR , with few mature DCs expressing DC-LAMP/HLA-DR or costimulatory markers. These DCs can be found in the cornea even after long-term culture. DC-LAMPþ mature DCs are only partially DC-SIGNþ, implying that the peripheral stroma harbors two sets of rare mature DCs, those that coexpress DC-SIGN (mostly CD11c ) and those that do not coexpress DC-SIGN (CD11cþ).

Studies in BM chimera mice have demonstrated a turnover rate for BM-derived cells in the stroma at around 24% at 2 weeks. Replenishment occurs initially in the peripheral cornea and the anterior stroma. By 8 weeks, turnover reaches 75%, reaching a plateau between 2 and 6 months. Close to one-third of migrating cells into the central and peripheral cornea are DCs.