[Advances in Immunology] Advances in Immunology Volume 48 Volume 48 || Immune Privilege and Immune Regulation in the Eye
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ADVANCES IN IMMUNOLOGY, VOL. 48
Immune Privilege and Immune Regulation in the Eye
JERRY Y. NIEDERKORN
Department of Ophthalmology, University of Texas Southwestern Medical Center,
Dallas, Texas 75235
The eye has been likened to an imunological microcosm in which virtually all forms of immunological events can take place and, in doing so, often produce unique results (1). Indeed, when the topic of immuno- logical privilege is raised, the anterior chamber of the eye (Fig. 1) is offered as the classic example of a privileged site in which histoincompa- tible allografts escape immunological recognition and enjoy prolonged, and sometimes permanent, residence (2). Likewise, discussions of other immunological phenomena, such as tolerance, often turn to the eye for examples to illustrate basic immunological principles. For example, one pathway for maintaining immunological tolerance of host tissue epitopes is the sequestration of self-antigens behind anatomical barriers. In this regard, the crystallin antigens of the lens are offered as examples of tissue-specific immunogens that are sequestered early in ontogeny but, although potentially capable of arousing an autoimmune response, do not dq so since they are incarcerated within an impervious capsule. Thus, tolerance of host antigens (e.g., lens crystallins) can be maintained, at least in part, by anatomical sequestration.
The eye possesses other unique features that warrant the immunolo- gists attention. Perhaps the oldest and most successful form of organ transplantation takes place in the eye. Corneal transplantation has been performed on animal subjects for over 150 years and on human patients for over 80 years (3). In the United States alone, 30,000 corneal trans- plants are performed each year, with a success rate well over 90%. The extraordinary success of corneal transplantation is often attributed to the mysterious and undefined immunological privilege of the cornea and the eye. What is the basis for this immunological privilege, and is it restricted to the eye? In this review I examine those features of the cornea and the eye which conspire to promote successful corneal transplan- tation.
The putative immunological privilege of the cornea, corneal graft bed, and anterior chamber would seemingly create an environment free of
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192 JERRY Y . NIEDERKORN
Area Of I Choroid ., , activitv Retina L
Area of immunologic
FIG. 1. Schematic representation of ocular anatomy demonstrating regions of immunological privilege and areas especially vulnerable to autoimmune activity.
immune-mediated diseases. However, this is not the case, as the eye is vulnerable to an interesting array of autoimmune diseases uniquely suited to precise immunological analysis.
Thus, the eye offers interesting opportunities for analyzing a panorama of immunological phenomena, including organ transplan- tation, immunological privilege, tolerance, and autoimmunity. More- over, each of these immunological processes can have a profound bear- ing on the normal functioning of one of the most precious of the five senses: our vision.
II. Corneal Allografts: lmmunogenically Privileged Grafts on Immunologically Privileged Graft Beds
The prospect of replacing an opaque diseased cornea with a healthy one was suggested as early as 1796 by Erasmus Darwin, who suggested that I. . . a slight and not painful operation might be facilitated by cutting the cornea with a kind of trephine, about the size of a thick bristle or a small crow-quill, an experiment I wish strongly to recommend to some ingenious surgeon or oculist (4). In 1837 an Irish surgeon, Samuel Bigger, described how he had successfully placed a corneal allograft into a pet gazelles eye while he was a prisoner of the Egyptians (5). The first recorded attempt at therapeutic corneal transplantation on a human
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subject was performed by Richard Kissam, who sutured the cornea of a 6-month-old pig onto a blind Irishman (6). This xenograft ultimately failed, as did subsequent attempts at corneal grafting. However, suc- ceeding decades brought the introduction of general anesthesia, antisep- sis in surgery, improvements in ophthalmic surgical instruments, and, at the turn of this century, the first successful human corneal transplant (7). In the 75 years following the first successful human corneal allograft, literally thousands of corneal transplants have been performed each year to correct blndness produced by corneal edema, trauma, inflammation, or congenital abnormalities.
The success of corneal transplantation is unrivaled by all other forms of organ transplantation. Corneas transplanted onto diseased, but other- wise avascular, graft beds (e.g., keratoconus), remain clear and healthy in greater than 90% of the recipients (8,9). This extraordinary success rate is even more impressive when one considers the conditions surrounding corneal transplantation. Human leukocyte antigen matching of donor and recipient is normally not performed, except in high-risk patients. Immunosuppressive drugs are restricted to topical corticosteroid eye- drops, which are gradually tapered to maintenance levels following su- ture removal. Such conditions would certainly lead to graft failure with any other form of organ transplantation, yet corneal allografts thrive in spite of such immunological handicaps.
A. ESCAPING IMMUNOLOGICAL RECOGNITION The apparent ease with which corneal grafts avoid immunological
recognition suggests that the cornea is endowed with unique immunolog- ical characteristics and thus possesses an immunological privilege not shared by other organ grafts (10). Historically, three basic hypotheses have been offered to account for the privileged existence of corneal allografts (10). The simplest explanation suggests that the cornea, like certain neuronal tissues, is devoid of conventional major histocompatibil- ity complex (MHC) antigens. Accordingly, potentially alloreactive T cells would be blind to alien corneal cells. The putative absence of MHC antigens would not only prevent the arousal of an alloimmune response, but even if a response were initiated, the grafts would be immunologically invisible. This hypothesis, although appealing in its simplicity, has been unequivocally disproved. Several findings indicate not only that the cor- nea is vulnerable to immunological attack, but that it is also capable of eliciting an alloimmune response that results in graft rejection. Studies in rats, rabbits, and mice indicate that heterotopic transplantation of cor- neas to subdermal graft beds leads to rapid sensitization and swift rejec- tion of the allografts (1 1-14).
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Moreover, MHC antigens have been detected on all three cell layers of the cornea (15-18) (Fig. 2). In fact, the grafting procedure itself has been shown to elicit increased expression of MHC class I1 antigens on the corneal epithelium (19, 20). Although MHC antigens can be found on cells of the corneal epithelium, stroma, and endothelium, the density of antigen expression differs markedly among these three cell layers. In the rat, corneal endothelial cells express meager detectable amounts of MHC class I antigens and no detectable MHC class I1 antigens (2 1).
Despite the paucity of MHC antigens, corneal endothelial grafts stimu- late robust cytotoxic T lymphocyte (CTL) responses following hetero- topic grafting in the rat (22). Although MHC class I antigen expression is greater in the epithelium than in the endothelium, heterotopic trans- plantation of isolated allogeneic corneal epithelium fails to induce detect- able anti-MHC class I CTL responses in the rat (22). Thus, there are significant antigenic and immunogenic gradients within the corneal allo- graft. The mere expression of histocompatibility antigens on corneal cells does not ensure the induction of an alloimmune response. Nonetheless, the corneal allograft has the full potential to be both immunogenic and antigenic.
FIG. 2. The cornea is composed of three distinct layers: epithelium, stroma, and endothelium. MHC class I antigens are expressed on cells of each layer. [Reprinted from Niederkorn and Peeler (153) by permission of S. Karger Pub- lishing, Inc.]
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The second hypothesis offered to explain the survival of corneal allo- grafts suggests that the donor cells were rapidly replaced by host cellular components in the graft bed. According to this hypothesis, the cellular elements of the graft were replaced before the host's immune machinery could be aroused. Like the previous hypothesis, this explanation has been refuted. Animal studies using sex chromatin markers to distinguish do- nor cells from recipient cells have demonstrated the long-term survival of donor cells in corneal grafts (23,24). Other investigators came to similar conclusions by radiolabeling donor corneas with [3H] thymidine (25,26). Clinical findings also support this conclusion, since immunological rejec- tion can occur over a decade after corneal transplantation (10).
The third and most widely accepted hypothesis to account for the high acceptance of corneal allografts relates to the nature of the avascular corneal graft bed. It is a well-recognized clinical observation that vascu- larization of the corneal graft bed is a harbinger of graft failure (27,28). The absence of blood and lymph vessels at the interface of the graft and the graft bed is thought to prevent the escape of alloantigens to the regional lymphoid tissues, thereby resulting in an afferent blockade of the immunological reflex arc.
Although the corneal allograft is potentially immunogenic and anti- genic, the anatomical sequestration promotes graft survival due to the privileged location of the graft. Thus, the avascular graft and the graft bed conspire to produce a state of immunological ignorance that permits allograft survival. Heterotopic and orthotopic corneal allografts in rats and mice have been used to examine this hypothesis. Corneal allografts can be transplanted heterotopically (i.e., to an abnormal anatomical site) onto subdermal graft beds richly endowed with blood vessels and lym- phatics, which would favor the induction and execution of alloimmunity.
Such grafts can be compared to similar grafts placed orthotopically (i.e., to the normal anatomical site) onto avascular graft beds in the eye. If the avascular graft bed contributes to the survival of the allograft, one would predict that heterotopic corneal grafts would suffer significantly higher rejection rates than their orthotopic counterparts. This is indeed the case, as 100% of the fully allogeneic heterotopic corneal allografts are rejected in a mouse model of corneal transplantation (13, 14, 29), while only 55-57% of fully allogeneic grafts fail when grafted orthotopically (30, 31).
The pioneering studies of Maumenee (32) provided strong support for the afferent blockade theory. In these studies rabbits bearing long-term orthotopic corneal allografts rejected skin grafts from the same donors which provided their corneal grafts. Skin graft rejection occurred at a tempo indicative of a first-set rejection, thereby supporting the notion
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that the initial corneal allograft failed to stimulate alloimmunity and that the graft bed produced an afferent blockade of the immune response. However, skin graft rejection led to the rejection of 90% of the previously clear corneal grafts. Thus, the corneal grafts initially displayed immuno- genic privilege, but were antigenically vulnerable to an ongoing systemic immune response: Afferent blockade was present, but efferent blockade was not. Callanan and co-workers (30) came to similar conclusions, using a rat orthotopic corneal allograft model.
In these studies the appearance of antigen-specific CTL activity coin- cided with graft rejection, while the absence of CTL responses was a consistent feature of hosts bearing long-term corneal allografts. It is interesting that in both of these studies a small but significant number of corneal grafts were initially clear and avascular, yet subsequently under- went immunological rejection. Thus, the presence of an avascular graft bed does not necessarily ensure permanent graft survival or the mainte- nance of an afferent blockade.
Recent studies from our laboratory lend further support to the affer- ent blockade theory of corneal graft survival. Ross et al. (33) have shown that orthotopic corneal grafts differing from their hosts only at MHC class I1 loci do not undergo rejection unless the host is systemically immunized with skin grafts from the same donor strain.
The importance of an efferent blockade in promoting corneal graft survival is not clear. The previously mentioned studies by Maumenee (32) and by Ross et al. (33) argue against an effective efferent blockade. However, Khodadoust and Silverstein (34) confirmed earlier findings by Billingham and Boswell(1 l), which indicated that the corneal graft bed served as an effective barrier that shielded the graft from sensitized effector elements of the host. Results from the Khodadoust and Silver- stein study indicated that 95% of the lamellar corneal grafts and 75% of the penetrating grafts remained healthy, even though the hosts had rejected large skin grafts from the same donors of the corneal grafts.
B. ROLE OF DONOR LANCERHANS CELLS IN INITIATING CORNEAL GRAFT REJECTION
Numerous studies have demonstrated the presence of MHC class I antigens on cells in all three layers of the cornea (15-18); however, MHC class I1 antigens are not normally expressed in detectable amounts on corneal cells. MHC Class I1 antigen-bearing Langerhans cells are abun- dant in the peripheral outermost regions of the cornea that interface with the conjunctiva (i.e., limbus), but are conspicuously absent from the central corneal epithelium in adult humans, mice, rabbits, and guinea pigs (35-38).
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The absence of Ia Langerhans cells in the central cornea is of more than casual interest, since these cells represent an important immuno- genic component of an allograft. Indeed, it has been suggested that Ia+ passenger cells are the major barrier to successful organ transplan- tation (39). Over three decades ago Snell (40) suggested that donor leukocytes present in transplanted tissues were a major source of tissue immunogenicity. Interest in passenger cells, however, remained dor- mant until Lafferty et al. (39) reconsidered the role of Ia cells in thyroid allografts and pancreatic islet grafts. Subsequently, numerous studies have confirmed that Ia passenger cells are indeed major obstacles to successful organ transplantation and that their prior removal has a favor- able effect on graft survival.
The conspicuous absence of Langerhans cells in the central corneal epithelium offers a unique opportunity to determine whether grafts typically devoid of passenger cells are capable of inducing the normal array of alloimmune responses and whether the presence or absence of such cells affects graft survival. Results from our laboratory indicate that the absence of resident Langerhans cells has a profound effect on the grafts ability to induce allospecific delayed-type hypersensitivity (DTH) responses following heterotopic transplantation (4 1) . Surprisingly, grafts differing from the host at MHC classes I and I1 loci as well as multiple minor H loci were consistently rejected, but failed to induce detectable DTH responses before, during, or after immunological rejection (20,29, 4 1). Although DTH responses were not induced, Langerhans cell-free grafts elicited potent CTL responses that coincided with the onset of graft rejection (20, 29,41).
It is generally agreed that solid-organ transplants, such as skin or cornea, are rejected by either CTL- or DTH-mediated processes (42). Central corneal allografts fail to induce DTH, yet undergo immunologi- cal rejection at both orthotopic and heterotopic sites. Therefore, it is reasonable to conclude that DTH plays little, if any, role in the rejection of corneal allografts. Moreover, adoptive transfer studies in adult thy- mectomized bone marrow reconstituted mice (ATXBM) demonstrate that lymphoid cell suspensions depleted of CD4 (i.e., T helper/DTH), but containing CD8 (CTL/suppressor), cell populations promoted cor- neal graft rejection in T cell-depleted ATXBM recipients (29).
Categorizing effector T cells into either CTL or DTH populations, based on the expression of cell surface markers, is an artificial and potentially misleading approach for understanding the mechanism of corneal graft rejection. CD4 T cell clones can demonstrate cytolytic activity against MHC class I1 alloantigens (43), whereas some CD8 T cell populations secrete lymphokines and demonstrate T helper cell activity
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(44). Whether corneal allograft rejection is solely due to CTL or DTH is more of a semantic question than an immunological one. It is more productive to consider the relevance of the T cell phenotype in terms of the class of MHC antigen that it recognizes rather than with a presumed effector function.
Recent studies of skin allograft rejection in mice have shown that the elimination of CD8' T cells (MHC class I-restricted T cells) enhances the survival of skin allografts differing from the host only at MHC class I loci (45). In similar studies Rosenberg et al. (46) found that the elimination of CD4' cells does not alter rejection of MHC class I-disparate skin grafts, whereas adoptive transfer of CD4+/CD8- T cell populations produced rapid rejection of MHC class I1 and minor H-disparate skin grafts. The rejection of a skin allograft, whether the MHC disparity is at class I only or class I1 only, most likely occurs by the same immunological process, even though different T cell populations are probably involved. Like- wise, the basic mechanism of corneal allograft rejection, although funda- mentally different from skin graft rejection, is most likely a CTL- dominated process, regardless of the nature of the allodisparity.
Although the absence of Langerhans cells does not affect the recogni- tion and rejection of heterotopic corneal grafts involving MHC class I or I1 disparity (20, 29, 41), the situation with orthotopic grafts is markedly different. Using a rat model, Callanan et al. (30, 47) recently demon- strated that orthotopic central corneal allografts differing from the recip- ient at MHC classes I and I1 loci were rejected in 55% of the naive recipients. However, if similar corneal grafts were pretreated with sterile latex beads to induce the infiltration of donor-derived Langerhans cells (before transplantation), 98% (and more recently, 100%) of the grafts were rejected (30; unpublished observations). Moreover, rejection was invariably accompanied by the development of antigen-specific CTL responses. Thus, the presence of graft-borne Langerhans cells almost doubled the risk of rejection of orthotopic corneal allografts involving combined MHC classes I and I1 disparities.
Recent findings from our laboratory indicate that graft-borne Langer- hans cells are the major immunogenic stimulus for the rejection of male corneal grafts by female recipients (19, 48). Langerhans cell-free male- heterotopic corneal grafts were incapable of inducing either a CTL or DTH response against the male-specific H-Y antigen (19,48). Moreover, male grafts were not rejected, even though they were residing on vascu- larized graft beds. The male grafts did, however, express H-Y antigen, since female mice preimmunized with male skin grafts rapidly rejected central corneal grafts from male donors. The immunogenic privilege of male grafts could, however, be breached by the presence of male donor
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Langerhans cells. Corneas pretreated with latex beads, as a means of inducing the infiltration of donor Langerhans cells, induced strong CTL and DTH responses and underwent prompt rejection following hetero- topic transplantation onto naive female recipients ( 19).
Ray-Keil and Chandler (49) came to the same conclusions, using a similar heterotopic corneal graft model. However, in their study a signifi- cant number (i.e., 36%) of the male grafts were rejected by naive female hosts. The discordance between their results and ours might be ex- plained by the presence of donor Langerhans cells in their grafts, since our grafts were approximately 2.5 mm in diameter, while theirs were slightly larger (3.0 mm in diameter) and therefore more likely to con- tain the peripheral regions that are known to contain large concentra- tions of Langerhans cells. Nonetheless, it is intriguing that Ray-Keil and Chandler found that elimination of donor Langerhans cells (i.e., hyper- baric oxygen treatment) virtually eliminated graft rejection: Only one of 23 grafts underwent rejection. Thus, in the case of at least one minor histocompatibility antigen system (i.e., H-Y), the absence of Langerhans cells has a profound effect on the immunogenicity and fate of the corneal graft.
At this point the question arises as to whether it is the mere expression of alien MHC class I1 antigens on cells of the corneal graft or the expres- sion of these antigens on the surface of a cell with antigen-presenting potential (i.e., a Langerhans cell) that is crucial in provoking an immune response and graft rejection. In vitro treatment with y-interferon readily induces the expression of MHC class I1 antigens on human (50) and rabbit (5 1) corneal endothelium. Male corneal grafts incubated in y-in- terferon (1) displayed extensive expression of Ia antigen, (2) induced potent H-Y-specific DTH and CTL responses, and (3) were promptly rejected following heterotopic transplantation to female C57BL/6 recipi- ents (19).
I t is interesting, however, that the expression of donor Ia antigens on the corneal grafts occurs independently of the rejection process. Central corneal grafts from male donors become Ia' 2 days after grafting into syngeneic female recipients and remain Ia' through day 7 posttrans- plantation, yet the grafts fail to induce either a CTL or DTH response and survive indefinitely (19). By contrast, male grafts incubated in y-interferon, and therefore Ia' at the time of heterotopic grafting, are promptly rejected (19). Thus, the time at which Ia antigens are expressed determines whether the corneal graft is destined to survive or undergo rejection.
A similar situation occurs with corneal grafts differing from the host only at MHC class I1 loci. Streilein et al. (52) found that MHC class
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11-disparate heterotopic corneal grafts were not rejected and failed to induce alloimmunity. Such grafts failed to sensitize the host against the donors MHC class I1 antigens, because subsequent challenged ortho- topic skin grafts from the corneal donor strain were rejected in a first-set tempo, indicating that the MHC class I1 antigens were being perceived by the hosts immune system for the first time. In subsequent studies we used the same donor-host combinations (i.e., A.TL and A.TH) and eval- uated the ability of such grafts to induce allospecific DTH and CTL re- sponses (20). Surprisingly, MHC class 11-disparate grafts containing the Langerhans cell-rich limbus region or grafts pretreated with latex beads, so that they contained donor Langerhans cells at the time of grafting, induced vigorous DTH and CTL responses, yet were not rejected (20).
Using an orthotopic rat model, Katami et al. (53) reported a 95% rejection rate for grafts differing from the host at MHC classes I and I1 loci, but only a 25% rejection rate for MHC class 11-incompatible grafts. Ross et al. (33) observed similar results for orthotopic corneal allografts involving MHC class 11-disparate grafts. Although fully allogeneic grafts (i.e., MHC classes I and I1 disparate) were rejected in 55% of the hosts, MHC class 11-disparate grafts did not undergo rejection (i.e., experi- enced 100% survival), even though such grafts were found to express donor MHC class I1 antigens 4-7 days following transplantation. The transient expression of MHC class I1 antigens, however, is of sufficient duration to render the graft vulnerable to the efferent arm of the im- mune apparatus, since recipients preimmunized with MHC class II- disparate skin grafts reject subsequent orthotopic corneal grafts (33).
It is interesting, however, that grafts pretreated with latex beads prior to transplantation did not undergo immunological rejection unless the host was subsequently immunized with skin grafts from the same MHC class 11-disparate donor strain (33). Under these circumstances both the orthotopic corneal graft and the skin graft underwent rejection. Thus, the MHC class 11-disparate corneal graft is not immunogenic, but can be induced to express alien MHC class I1 antigens that serve as targets for alloimmune effector elements.
Collectively, the results indicate that in the case of corneal grafts repre- senting only minor histoincompatibilities or MHC class I1 disparities, the timing of MHC class I1 antigen expression is critical and determines whether the graft is destined to survive or perish.
C. DYNAMIC DISTRIBUTION OF CORNEAL LANGERHANS CELLS The well-defined geometric arrangement of Langerhans cells in the
peripheral limbus and the abrupt exclusion of these cells from the central regions of the corneal epithelium suggest that the distribution of these
IMMUNOLOGY OF THE EYE 20 1
cells is under stringent regulation. Recent studies suggest that the juxta- positioning of corneal Langerhans cells is a dynamic physiological process. Various stimuli can induce corneal Langerhans cell migration from the periphery to the central regions. For example, electrocautery (54), bacterial and viral infections (54-58), and phagocytic stimuli (59) can induce Langerhans cell migration (Fig. 3). Since Langerhans cells rulson dftre is antigen presentation, one might suspect that the aforementioned stimuli somehow mimic signals that beckon Langerhans cells to sites for antigen processing. With this in mind, investigations were conducted to determine whether corneal cells could phagocytose anti- gens and, as a result, elaborate chemoattractants that induced the cen- tripetal migration of peripheral Langerhans cells into the central regions of the cornea.
It has been previously demonstrated that rabbit corneal cells elaborate an interleukin (1L)- 1 -like molecule, corneal epithelial cell-derived T cell-
Latex beads, //
FIG. 3. Distribution of epithelial Langerhans cells. IA Langerhans cells are concentrated at the periphery of the cornea and at the limbus (i.e., corneal/ conjunctival border). The central corneal epithelium is normally devoid of Langerhans cells. However, sterile latex beads deposited into shallow incisions in the corneal epithelium will induce the rapid migration of peripheral Langerhans cells into the central regions of the cornea. [Reprinted from Niederkorn and Peeler (153) by permission of S. Karger Publishing, Inc.]
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activating factor (60). The quantity of this factor elaborated by rabbit corneal cells is increased 50-70% following phagocytosis of either Staph- ylococcus or latex beads (59). Moreover, Shams et al. (61) recently con- firmed these findings, using human corneal epithelial cells, and demon- strated that either y-interferon or Staphylococcus aureus, would stimulate secretion of biologically active IL-1p. The hypothesis that IL- 1 and/or CETAF functioned as a chemoattractant to promote the migration of peripheral Langerhans cells into the central corneal epithelium was sup- ported by findings in which intracorneal injection of purified IL-1 in- duced rapid centripetal migration of peripheral Langerhans cells to the site of cytokine inoculation (59).
The specificity of the chemoattractant was supported by the observa- tion that this effect could be blocked by anti-IL-1 antibodies (59). Che- motaxis could not be mimicked by intracorneal injection of other cy- tokines, such as IL-2, or by irrelevant proteins (e.g., hen egg lysozyme). Thus, corneal epithelial cells have the capacity to regulate the distribu- tion and migration of potential antigen-presenting cells within this organ.
The orderly distribution of Langerhans cells and the corneas ability to regulate their migration and distribution suggest that a dynamic regula- tory process is at work. At this point one can only speculate on the mechanisms that maintain the normal circumferential arrangement of limbal Langerhans cells. Perhaps local chemotactic factors-either cell membrane bound or soluble-prevent Langerhans cell migration from the limbus, unless a stronger stimulus (e.g., IL-1) is released from the central corneal epithelium.
An attractive, yet unproven, hypothesis is that the corneal epithelial cells might serve as accessory antigen-processing cells. Since the central cornea is devoid of conventional antigen-presenting cells, such as dendri- tic cells, some immunological provision must be made to accommodate pathogens and antigens that may insult the corneal epithelium. Potential antigens or pathogens (e.g., Staphylococcus or herpes simplex virus) are phagocytosed by corneal epithelial cells. This, in turn, would stimulate the elaboration of IL-I, which then induces the migration of peripheral Langerhans cells to the area of antigen accumulation. Partially processed antigens could then be displayed on the cell membranes of corneal epi- thelial cells and then be presented to the itinerant Langerhans cells for further processing and presentation to peripheral lymphoid elements. As the levels of IL-I dissipate, the Langerhans would be stimulated to return to the periphery and eventually to the regional lymphoid appara- tus, where they would interact with antigen-specific T cells and thus initiate a conventional immune response.
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D. IMMUNOREGULATORY EFFECTS OF CORNEAL LANGERHANS CELLS: A DOUBLE-EDGED SWORD
There is little doubt that corneal Langerhans profoundly influence the immunogenicity and fate of corneal allografts. Ridding corneal grafts of these passenger cells greatly reduces the risk of rejection in experimental animals and would presumably have the same effect on human subjects. Although the absence of Langerhans cells in the central cornea is an important parameter in explaining the immunological privilege of cor- neal allografts, other considerations are also involved. A thorough un- derstanding of the corneal allografts exemption from immunological rejection may have a profound impact on other categories of organ transplantation.
It is clear that Langerhans cells serve a vital function as antigen- presenting cells and that their presence in the eye is crucial for maintain- ing protective immune surveillance against potential pathogens. The peculiar distribution of ocular Langerhans cells, however, leads one to suspect that the behavior and functioning of ocular Langerhans cells is much different than cutaneous Langerhans cells. Understanding the biology of corneal Langerhans cells remains an intriguing and formida- ble challenge for the immunologist.
111. Immunological Privilege of the Anterior Chamber
The immunological privilege of the anterior chamber was recognized over 100 years ago, when researchers found that xenogeneic tumor grafts survived in this ocular compartment significantly longer than they did at other sites (62). In the 1940s Greene and associates (63) used the anterior chamber of the rabbit eye as a tool for propagating and passag- ing human tumors. Moreover, Greene raised an interesting, although unproven, hypothesis that the growth and metastasis of such tumors from the anterior chambers of experimental hosts could be used to predict the original tumors malignancy. Nonetheless, in the 40 years following Greenes original observation, enormous insights have been gained regarding the nature of the immunological privilege in the ante- rior chamber of the eye.
The known absence of lymphatic drainage to regional lymph nodes led early investigators to propose that the privilege of the anterior chamber could be traced to the sequestration of alloantigens within this com- partment and, thus, afferent blockade of the immune apparatus (62). However, during the past decade it has become increasingly clear that the
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immunological privilege extended to allogeneic tissues transplanted into the anterior chamber is not an immunologically null event, but rather a dynamic immunoregulatory process of exceptional complexity.
A. ANTERIOR CHAMBER-ASSOCIATED IMMUNE PRIVILEGE: THE BASIS FOR IMMUNOLOGICAL PRIVILEGE
The notion that allografts placed into the anterior chamber are seques- tered from the systemic apparatus was soon disproved. Raju and Grogan (64) and Franklin and Prendergast (65) demonstrated that not only were alloantigens capable of emigrating from the anterior chamber to the peripheral immune apparatus, but that alloimmune effectors were gen- erated against the anterior chamber allografts which eventually under- went rejection. The first clue that antigens delivered directly into the anterior chamber could be processed in a manner that favored down- regulation of alloimmunity came from studies by Kaplan and Streilein (66-68). In their studies semi-allogeneic (i.e., F1 donor) cells served as alloantigens and were injected directly into the anterior chambers of parental-strain rats. Evidence that the alloantigens had been perceived by the immune system came in the form of serum antibodies against the donor alloantigens. More importantly, these hosts demonstrated a mod- est, albeit significant, delay in their ability to reject challenged skin grafts from the donors of the intracameral inoculum, but were able to execute normal first-set rejection of third-party skin allografts (66-68).
Thus, presentation of alloantigens via the anterior chamber produced an antigen-specific impairment of systemic cell-mediated immunity: Im- mune privilege was extended beyond the eye to extraocular sites. These results, along with those by Subba Rao and Grogan (69), indicated that the terms of immunological privilege were defined by the mag- nitude of the histocompatibility disparity between donor and host- immunological privilege not being an all-or-none proposition.
Subsequent studies in mice revealed that an impressive display of immune privilege could be demonstrated by transplanting tumor allo- grafts into the anterior chamber. DBA/2 mastocytoma cells (P815) un- dergo swift rejection following subcutaneous transplantation into al- logeneic BALB/c recipients, due to the hosts recognition of the minor histocompatibility antigens of the DBA/2 donor strain. However, equal numbers of DBAIP mastocytoma cells are not rejected following trans- plantation into the anterior chamber of BALB/c hosts. DBA/2 tumors not only grow progressively, but induce antigen-specific suppression of systemic cell-mediated immunity (70, 7 1). BALB/c hosts challenged or- thotopically with DBA/2 skin grafts fail to reject donor-strain skin grafts, but are fully capable of rejecting third-party skin allografts.
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The progressive growth of intraocular tumor allografts and the failure to reject relevant orthotopic skin grafts from the DBAI2 donor strain suggested that systemic allograft immunity was paralyzed. Surprisingly, this was not the case, as these hosts developed serum antibodies as well as CTLs specific for the DBA/2 alloantigens (72). The latter finding repre- sents a perplexing result, considering the long-term survival of the skin allografts and the ever-expanding intraocular tumor allografts. This unusual spectrum of immunological findings led to the generic term anterior chamber-associated immune deviation (ACAID) to convey the dynamic and diverse nature of this phenomenon (73).
Investigations with other antigen systems revealed that the ACAID phenomenon was not restricted to minor histocompatibility antigens. Wetzig et al. (74) and Waldrep and Kaplan (75) demonstrated that ante- rior chamber inoculation of hapten-derivatized lymphoid cells resulted in ACAID, as demonstrated by the down-regulation of antigen-specific DTH responses. Moreover, these studies also established that the sys- temic down-regulation of cell-mediated immunity was attributable to the development of a suppressor T cell system.
It is now apparent that a wide array of antigens elicit ACAID when delivered via the anterior chamber. Herpes simplex virus (76, 77), mela- noma antigens (78), bovine serum albumin (79), hapten-derivatized cells (74,75), and retinal S antigen (80,81) induce ACAID following anterior chamber inoculation. The facility with which ACAID can be induced has permitted further exploration into the mechanisms behind the induction and maintenance of this immunoregulatory phenomenon.
B. INDUCTION OF ACAID The absence of a patent lymphatic drainage route for grafts residing in
the anterior chamber indicates that antigens must leave this site via the blood vascular route. Accordingly, it has been suggested that anterior chamber inoculation of antigen is tantamount to an intravenous injection and is merely another form of immune deviation, similar to the one described by Asherson and Stone (82), in which small amounts of deag- gregated heterologous proteins injected intravenously led to the produc- tion of serum antibodies, but a conspicuous absence of DTH. However, several findings argue convincingly against the hypothesis that ACAID is simply a cumbersome method for producing intravenous immune devia- tion. Repeated attempts to mimic ACAID by intravenous injection of P815 cells have failed (83). More importantly, the nature of the sup- pressor mechanism of ACAID differs markedly from intravenously in- duced immune deviation. The suppressors of ACAID act at the efferent
206 JERRY Y . NIEDERKORN
level (84), while the suppressor system of immune deviation functions only at the afferent mode (85).
As is discussed later, ACAID requires the presence of an intact spleen (73); by contrast, immune deviation is not ablated by splenectomy (86). In the case of herpes simplex virus (HSV), the intravenous injection of virus induces suppressed DTH, but vigorous CTL, responses (87, 88). However, anterior chamber inoculation of HSV leads to the suppression of both CTL (77) and DTH responses (76,88).
The eye plays an active role in the inductive phase of ACAID. Removal of the eye 4-7 days after intracameral inoculation of antigen prevents the induction of ACAID (89). It is also within this time frame that allogeneic tumor cells (originating from the anterior chamber) can first be detected in the spleen of the host (90,91). Thus, significant events occur within the eye during the first 7 days; beyond this time the eye is superfluous.
There are two considerations that may provide insights for under- standing the induction of ACAID. One explanation is that the unique physicochemical environment of the anterior chamber alters antigens or antigen-perceiving cells. In this regard, Streilein et al. (92) and Granstein et al. (93) have reported the presence of transforming growth factor-p-a potent inhibitor of T cell proliferation-in the aqueous humor. More- over, these investigators, as well as others, have demonstrated that aqueous humor inhibits antigen-specific and -nonspecific lymphocyte- proliferative responses.
A second, not mutually exclusive, explanation relates to the possible presence of unique antigen-processing cells within the anterior chamber or lining the outflow channels that communicate with the venous drainage system of the anterior chamber. It is conceivable that the eye possesses a specialized population of antigen-presenting cells committed to delivering a down-regulatory signal to the hosts T cells in a manner analogous to the suppressor antigen-presenting cells of the skin (94).
Granstein et al. (94,95) have demonstrated the presence of ultraviolet radiation (UVR)-resistant antigen-presenting cells in the skin of mice. Under normal conditions, haptens painted onto the skin evoke positive contact sensitivity; however, hapten sensitization on UVR skin results in the suppression of a DTH response. Granstein (94) demonstrated that the suppression occurred via antigen presentation by UVR-resistant cells residing in the skin. It is feasible that similar suppressive antigen- presenting cells reside in the eye and are responsible for the obligatory ocular phase of ACAID.
In searching for such cells, we have focused on the trabecular mesh- work of the eye-the aqueous outflow system which serves as the conduit for removing the contents of the anterior chamber. Sparse, but consis-
IMMUNOLOGY OF T I I E EYE 207
tent, numbers of Ia' cells can be detected in the trabecular meshwork of both mouse and human eyes (96,97). The immunoregulatory capacity of these cells, however, remains to be established. Preliminary results sug- gest that cells of the iris and the ciliary body may have immunoregulatory functions. Mixed cultures of iris and ciliary body inhibited antigen-driven lymphocyte-proliferative responses in an antigen-nonspecific manner (92). It bears noting that at least a portion of this inhibitory effect may be attributable to the aqueous humor, since cells of the ciliary body are the major producers of the aqueous humor in situ.
Recent findings lend support to the proposition that the induction of ACAID is due to aberrant antigen-processing cells, not unique humoral factors, in the eye. Williamson and Streilein (98) demonstrated that posi- tive allospecific DTH responses could be induced via the anterior cham- ber if the alloantigen-bearing cells were admixed with cutaneous Langer- hans cells of the recipient strain prior to anterior chamber inoculation. Presumably, the antigen-presenting capacity of the inoculated Langer- hans cells competed with down-regulatory resident ocular cells and processed antigen in a manner that promoted the development of sys- temic DTH. Conversely, it might be suggested that the host Langerhans cells neutralized a putative inhibitor molecule present in normal aqueous humor. The latter suggestion is difficult to reconcile when one considers that ACAID can also be ablated by splenectomy, a procedure that would not be expected to alter the makeup of the aqueous humor.
C. EXPRESSION OF ACAID-INSIDE AND OUTSIDE THE EYE
1. Effect on Intraocular Tumor Allografts
In its simplest form ACAID has been offered as the underlying mecha- nism to explain the phenomenon of immunological privilege in the ante- rior chamber (99, 100). According to this hypothesis, allografts placed into the anterior chamber escape or delay immunological rejection, due to an active down-regulation of systemic cell-mediated immunity. Im- mune privilege, therefore, is a dynamic ongoing process manifested not only in the eye, but systemically as well.
Using the DBA/2 mastocytoma (P815) model of ACAID, we have suggested that the antigen-specific down-regulation of DTH and the preservation of allospecific CTL and antibody responses suggested that immune privilege and ACAID translated into the active suppression of DTH (101). Moreover, in this model of ACAID, BALB/c hosts harboring anterior chamber P815 tumor allografts were incapable of rejecting or- thotopic DBA/2 skin allografts. The suppression of skin allograft rejec- tion was antigen specific, as the same hosts were able to reject third-party
208 JERRY Y. NIEDERKORN
skin allografts (102). However, as previously mentioned, ACAID can be ablated by splenectomy.
Three interesting results occur in splenectomized BALB/c mice. First, and perhaps most striking, is the obvious loss of immune privilege: Intracameral P8 15 tumors undergo a violent rejection in splenectomized BALB/c mice (72). A second, and equally important, event is the restora- tion of systemic cellular immunity: Orthotopic DBA/2 skin allografts are not only rejected, but the tempo is indicative of a second-set rejection (i.e., the anterior chamber inoculum sensitized the host). The third note- worthy finding is the restoration of DTH that occurs in splenectomized mice.
This mosaic of findings, summarized in Table I, has led to the follow- ing conclusions: (1) ACAID is principally an antigen-specific down- regulation of systemic DTH; (2) rejection of minor H-incompatible orthotopic skin allografts is DTH dependent; (3) the rejection of intra- ocular tumor allografts is DTH dependent; and (4) progressive growth of tumor allografts occurs in the face of antigen-specific CTL and antibody. Coincidentally, these results also strongly suggest that skin allografts involving only minor H histoincompatibilities are rejected by a DTH- dependent process if ACAID is ablated; however, if ACAID is estab- lished, the skin allografts survive in the face of allospecific CTLs and serum alloantibody.
The weight of evidence supports the conclusion that the intraocular tumor allografts are also rejected by DTH-dependent mechanisms (Ta- ble I). In splenectomized BALB/c mice the acquisition of positive DTH coincides with the onset of intraocular tumor rejection. Moreover, the histopathological features of intraocular tumor rejection are reminiscent of a vigorous DTH reaction, with evidence of infarction of the microva- sculature feeding the tumorous mass, ischemic necrosis e n masse, erosion of the vascular endothelium, and extensive innocent bystander dam- age. Although ablation of ACAID results in the prompt rejection of the intraocular tumor allograft, the consequences are severe, resulting in total destruction of the eye.
2. Effect on Ocular HSV Infections HSV- 1 infections of the cornea are one of the leading causes of corneal
blindness in the United States. The damaging effects of corneal HSV infections are believed to be primarily due to the cytopathic effect of the virus, although there is a growing body of evidence suggesting that stromal disease is an immune-mediated process. For example, Metcalf et al. (103) demonstrated that athymic nude mice failed to develop stromal
TABLE I EFFECT OF ACAID ON IMMUNOLOGICAL PRIVILEGE AND INTRAOCULAR TUMOR ALLOGRAFT BEHAVIOR^
Immunological profile Fate of allografts
Host ACAID DTH Ts CTL Antibody Orthotopic skin Intraocular tumor
Eusplenic + - + + + Permanent survival Progressive growth Splenectomized - + - + - Second-set rejection Rejection (DTH-like pathology)
a Data are summarized from references cited in text. DTH, Delayed-type hypersensitivity; Ts, T suppressor cells that down- regulate DTH, as shown in adoptive transfer assays; CTL, cytotoxic T lymphocyte activity.
210 JERKY Y. NIEDEKKORN
lesions following corneal infection with HSV- 1, while euthymic litter- mates displayed typical keratitis following ocular infection. However, nude mice could be rendered susceptible to stromal keratitis if provided T lymphocytes from sensitized euthymic donors (104).
With this in mind, Ksander and Hendricks (105) attempted to mitigate HSV-1 keratitis in mice by inducing ACAID prior to corneal infection. As expected, topical corneal infections with HSV- 1 produced potent DTH and CTL responses. However, anterior chamber inoculation of HSV- 1 prior to topical infection resulted in a profound suppression of both DTH and CTL responses and virtually complete protection from corneal stromal lesions produced by HSV- 1.
Further studies by Hendricks et al. (106) demonstrated that anterior chamber inoculation of a mutant strain of HSV- 1 having a deletion of the gene encoding the glycoprotein C resulted in the induction of normal DTH responses, but a conspicuous absence of CTL reactivity against HSV-1. Hosts primed in this manner had significantly reduced stromal disease following topical infection with the wild-type HSV- 1. Collectively, the results implicate HSV-specific CTLs, not DTH, in the pathogenesis of HSV stromal keratitis in this murine model. Thus, these studies not only offer a plausible hypothesis to account for the pathogenesis of HSV stromal keratitis, but they also offer a novel approach for using ACAID to reduce the severity of the putative immune-mediated ocular disease.
Anterior chamber inoculation of infectious HSV- 1 not only induces the suppression of systemic DTH, but also results in a curious pattern of ocular inflammatory diseases in which the retina of the contralateral uninoculated eye undergoes inflammation, necrosis, and complete de- struction (107, 108). Paradoxically, the retina of the eye initially inocu- lated with HSV-1 is preserved (107, 108). Atherton and Streilein (109) have shown that the development and severity of contralateral retinitis were intimately related to the spread of virus from the inoculated eye to the retina of the contralateral eye.
Moreover, it has been suggested that in the absence of ACAID (e.g., hosts immunized via nonocular routes), virus-specific DTH responses contribute to the clearance of virus and prevent viral replication in the central nervous system, which in turn prevents the spread of virus to the contralateral eye (109, 110). Thus, the development of normal DTH reactivity to viral antigens would be expected to protect the host from contralateral retinitis induced by anterior chamber inoculation of HSV. It remains unclear, however, whether the suppression of DTH that occurs following anterior chamber inoculation promotes the develop- ment of contralateral retinitis.
IMMUNOLOGY OF THE EYE 21 1
D. EFFECTOR MECHANISM FOR SUSTAINING ACAID
As previously described, a wide range of immunogens are capable of inducing ACAID. A common feature shared by all models of ACAID is the appearance of an antigen-specific suppression of DTH responses (99, 100). Wetzig et al. (74) were the first to demonstrate that the suppression induced via anterior chamber inoculation of hapten-derivatized splenic cells could be adoptively transferred with T cells that acted at the efferent mode, but did not bear detectable cross-reactive idiotype surface recep- tors. Subsequent studies by Waldrep and Kaplan (75) confirmed the presence of an efferent-level T suppressor system consisting of a primary pathway involving a cyclophosphamide-sensitive suppressor T cell and a secondary suppressor pathway that is antigen-nonspecific and mediated by a cyclophosphamide-resistant suppressor T cell population.
The suppression of DTH induced by anterior chamber presentation of alloantigens, like the hapten models, is mediated by a suppressor T cell population that acts primarily at the efferent mode (1 11). However, unlike the previously described hapten models, the suppressor T cells in the alloantigen model of ACAID are I-J sensitive and contain a Thy-1.2' L3T4- Lyt-2.2' subpopulation and a second subpopulation that is
In all studies to date, the suppressor cell population capable of sup- pressing systemic DTH following adoptive transfer to naive recipients resides in the spleen (99, 100). There is agreement among all current models of ACAID that, during the initial stages of ACAID, there is a strict requirement for both an intact eye and an intact spleen. Early removal of either the injected eye or the spleen prevents the induction of ACAID. Removal of the eye (i.e., within 7 days of anterior chamber inoculation) fails to induce either a positive or a negative signal for DTH.
By contrast, splenectomy has a striking effect on the development of DTH in anterior chamber-primed animals: Vibrant DTH responses are detected, intraocular tumor allografts undergo a necrotizing rejection process, and orthotopic skin allografts are swiftly rejected in a manner indicative of second-set immunization (73). However, the strict require- ment for the spleen is limited to the first 10 days following anterior chamber inoculation of alloantigen.
Therefore, ACAID can be sustained and DTH thoroughly suppressed in the absence of an intact spleen, provided the spleen is in place during the 10 days following anterior chamber inoculation of alloantigen (73). Removal of the spleen after this does not alter the panorama of immuno- logical findings that characterize ACAID. Therefore, the suppressor cells necessary to sustain the suppression of DTH have emigrated from the
Thy- 1 + L3T4' Lyt-2.2- (1 1 1).
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spleen and are disseminated to other anatomical regions by day 10 post- inoculation.
Recent findings by Ferguson et al. (1 12) have provided further insights into the role of the spleen in the induction and maintenance of ACAID. Using trinitrophenyl (TNP)-derivatized T lymphocytes, these investiga- tors proposed that hapten-derivatized T suppressor-inducer (Tsi) cells elaborated soluble suppressor-inducer factor that was antigen specific and immunoglobulin H restricted. The putative suppressor-inducer molecule was believed to leave the anterior chamber and enter the serum and was eventually filtered within the spleen, where it induced the devel- opment of a T suppressor-effector (Tse) population that maintained the persistent down-regulation of systemic DTH responses to TNP. The authors proposed that the serum-borne Tsi factor could not be detected in the eusplenic host, due to its rapid removal by high-affinity Tse cells in the spleen. However, sera from splenectomized hosts were capable of inducing the suppression of DTH when passively transferred to naive hosts.
Collectively, these results suggest that in the hapten model of immuno- logical privilege, derivatized T cells elaborate a molecule capable of in- ducing splenic effector cells that actively maintain down-regulation of DTH. However, it is not known whether a similar condition occurs with alloantigens or soluble antigens presented into the anterior chamber. Other antigens induce ACAID without being derivatized to histocom- patible T cells prior to anterior chamber inoculation. Therefore, to inte- grate this paradigm to fit other antigen systems would require that anti- gen processing and presentation to T cells occur within the anterior chamber in order for the host T cells to elaborate a similar Tsi molecule- a plausible, but unproven, proposition.
IV. Effect of Immune Regulation on lntraocular Tumor Rejection
The high incidence of spontaneous neoplasms that occurs in patients suffering from immunological disorders and in transplant recipients subjected to prolonged immunosuppression has been cited as evidence in support of the immune surveillance theory (1 13). It is reasonable to suspect that the same situation occurs with immunologically privileged sites, such as the anterior chamber of the eye, where immunological censorship creates an environment that would be expected to thwart the induction and expression of immunological surveillance. Contrary to the immune surveillance theory, this is not the case (2). The incidence of spontaneous neoplasms in immunologically privileged sites is not higher than that occurring at other anatomic regions. In the case of the eye,
IMMUNOLOGY OF T H E EYE 213
tumors of the cornea are virtually unknown and the most common intraocular tumors occur in the posterior compartments of the eye, not in the anterior chamber (1 14).
The topics of intraocular tumorigenesis and immune regulation raise interesting questions in the context of ACAID. For example, are highly immunogenic tumors that undergo spontaneous immunological rejec- tion at extraocular sites exempt from immunological rejection within the immunologically privileged confines of the anterior chamber? If so, are DTH-dependent mechanisms disqualified from participation? Are CTL- mediated tumor rejection mechanisms preferentially induced and exe- cuted in the anterior chamber?
We have addressed these and other questions relating to tumor-specific immunity in the anterior chamber, using immunogenic syngeneic re- gressor tumors. P91 mastocytoma, a highly immunogenic mutant of P8 15 mastocytoma, and UV5C25, an immunogenic ultraviolet light-induced fibrosarcoma, undergo immunological rejection following subcutaneous transplantation in syngeneic hosts (1 15, 116). Rejection of both tumors is T cell mediated and tumor specific (1 15, 116). Following anterior cham- ber transplantation, P9 1 mastocytoma (of DBAI2 origin) grows progres- sively for approximately 3 weeks. Between the 3rd and 4th weeks posttransplantation, an intense inflammatory response is elicited and culminates in ischemic tumor necrosis en muse (1 15). Tumor resolution is completed within another 5-7 days, leaving the eye irreparably damaged (i.e., phthisis bulbi). The histopathological features of the resolving tu- mors are characteristic of DTH lesions and include (1) microvascular infarction, (2) erosion of vascular endothelium, (3) perivascular cuffing, (4) ischemic necrosis en muse, and (5) extensive innocent bystander dam- age. Moreover, immunological findings supported the proposition that rejection is predominantly a DTH-mediated process (1 17).
Although the hosts develop tumor-specific CTLs and antibody, neither appears to be involved in the rejection of intraocular P91 tumors. Sur- prisingly, high levels of tumor-specific CTLs can be detected in regional lymph nodes as well as in the spleen, yet there is never any evidence of a lymphocytic infiltrate within the resolving intraocular tumors. Likewise, antibody is apparently not involved in intraocular P9 1 tumor rejection: Efforts to passively transfer tumor rejection have failed (1 17). Moreover, tumor rejection occurs in the absence of detectable serum antibody in splenectomized hosts and in the absence of complement in C3-deficient hosts (1 14). The results emphasize the importance of a tightly regulated immune response in the eye. Although the intense tumor-specific DTH responses eliminated the intraocular tumor and spared the hosts life, it did so at great expense: blindness.
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The pathophysiology of intraocular P9 1 tumor rejection is of more than casual interest, not merely an interesting immunological anomaly. The most common intraocular malignancy of childhood, retinoblastoma, has one of the highest frequencies of spontaneous resolution of any human tumor, including nonocular tumors (1 18). Spontaneous resolu- tion of retinoblastoma is known to produce extensive innocent bystander damage to ocular tissues and culminate in an atrophic eye-a pattern of tumor resolution not unlike the previously mentioned P9 1 rejection (1 18). Although these sequelae rid the eye of a life-threatening malig- nancy, the cost is high. Is intraocular tumor rejection always destined to result in immune-mediated blindness?
Studies involving another regressor tumor, UV5C25, indicated that intraocular tumor resolution could occur via a CTL-mediated mecha- nism and simultaneously exclude the participation of DTH responses in oculi (1 16). Immunological rejection of intraocular UV5C25 tumors oc- curs over a prolonged period of 10-14 days, while maintaining the normal anatomical integrity of the affected eye. The immunological and histopathological features of UV5C25 rejection are consistent with a CTL-mediated process: (1) Thy-l+ Lyt-2' L3T4- lymphocytes are bound to individual tumor cells in situ, (2) resolution occurs by piecemeal necrosis of individual tumor cells, (3) ischemia and innocent bystander damage are absent, and (4) intraocular tumor resolution can be adoptively transferred with CD8' tumor-infiltrating lymphocytes iso- lated from resolving intraocular tumors (1 16, 119). Not only is UV5C25 tumor rejection a CTL-dominated process, but it occurs in hosts pos- sessing tremendous DTH reactivity to UV5C25 tumor-specific antigens (116, 119). Therefore, it appears that the effector phase of DTH is actively excluded from the intraocular tumor nidus. The result of this exclusion is elimination of the malignancy without jeopardizing vision.
Neither of these two categories of intraocular tumors is irrevocably committed to follow a specific pathway. For example, hosts bearing intra- ocular P9 1 tumors develop impressive tumor-specific CTL activity, yet there is no evidence of lymphocytes infiltrating resolving intraocular tumors. However, if the contralateral eye is challenged with P9 1 tumor cells, the intraocular tumor in the second eye undergoes a pattern of rejection characterized by a mononuclear infiltrate of predominantly Thy-1 + Lyt-2' lymphocytes. Rejection occurs by piecemeal necrosis, with negligible innocent bystander damage. Thus, destructive and nonde- structive patterns of intraocular tumor rejection can occur simultaneously in the same host. This further suggests that an active local process ex- cludes cytolytic T cells from entering the first eye, but not the second eye.
Recent studies indicate that this converse phenomenon occurs with
IMMUNOLOGY OF THE EYE 215
intraocular UV5C25 tumors. BALB/c mice reject intraocular UV5C25 tumors by a process dominated by cytolysis of individual tumor cells and mediated by infiltrating Thy- 1 + Lyt-2 lymphocytes, even though the hosts display intense systemic DTH responsiveness to the tumor antigens. However, the exclusion of DTH responses from the tumor-containing eye can be altered. BALB/c hosts treated systemically with anti-CD8 antibody develop rapidly growing intraocular tumors that eventually undergo a necrotizing pattern of tumor rejection, characterized by ex- tensive innocent bystander damage and the complete destruction of the eye (unpublished observations.)
Results from these studies, as well as investigations of ACAID, have led us to propose that two basic patterns of intraocular tumor rejection are available to the immunocompetent host (1 16, 119). The first pattern involves minimal damage to normal host tissues and occurs by piecemeal necrosis by cytolytic T lymphocytes. The second pattern is destructive not only to the intraocular tumor, but to the anatomical integrity of the entire eye. The latter form of immune rejection is characteristic of a DTH-like lesion in which tumor necrosis occurs en musse. Hemorrhagic necrosis of the tumor and the entire eye attest to the antigen-nonspecific characteris- tic of this form of intraocular tumor rejection. These findings demon- strate that intraocular immunological privilege is manifested to varying degrees and should not be viewed as an all-or-none proposition. More- over, understanding intraocular immune regulation has important im- plications not only for intraocular tumors, but for more common sight- threatening ocular autoimmune diseases.
V. Immune Regulation and Autoimmune Uveitir
Inflammatory diseases of the eye are important causes of blindness in the United States and throughout the world. The region of the eye composed of the choroid, ciliary body, and iris (i.e., the uveal tract) is particularly vulnerable to immune-mediated diseases, collectively termed uveitis. The notion that autoimmunity was the underlying cause of uveitic conditions was proposed by Elschnig over 80 years ago (120) and has been confirmed in numerous studies (1 2 1,122). In 1965 Wacker and Lipton ( 123) demonstrated that immunogens extracted from retinal tis- sue were effective in inducing an inflammatory disease in the uveal tracts of experimental guinea pigs.
Scores of subsequent studies have established that systemic immuniza- tion with retinal antigens consistently induces an ocular inflammatory disease designated experimental autoimmune uveitis (EAU). EAU can be induced either with the well-characterized S antigen, a 48-kDa protein involved in light signal transduction, or with interphotoreceptor
216 JERKY Y. NIEDERKORN
retinoid-binding protein (IRBP), a 140-kDa protein that transports vita- min A derivatives between the photoreceptor cells and the retinal pig- ment epithelium (124, 125). Both proteins are major components of the photoreceptor cell layer, which is the primary target of immune- mediated damage in EAU (1 2 1 , 122). EAU has been a valuable tool for studying the immunopathogenesis of a variety of inflammatory diseases affecting primarily the posterior segment of the human eye (Fig. l ) , including sympathetic ophthalmia, birdshot retinochoroidopathy , Vogt- Koyanagi-Harada syndrome, and Behcets syndrome (12 1 , 122).
Numerous studies have established that EAU is a T cell-mediated disease because (1) athymic nude rats do not develop the disease follow- ing immunization with S antigen (126); (2) EAU is inhibited completely by treatment with the immunosuppressive agent cyclosporine ( 1 27); (3) EAU can be adoptively transferred with helper T cell lines (128, 129) or T cells from donor rats immunized with S antigen (130); (4) the ability to transfer EAU with lymphoid cells can be eliminated by treatment with anti-CD4 antibody plus complement (130); (5) the development of EAU correlates with the appearance of systemic DTH to retinal S antigen (130); and (6) attempts to transfer EAU with hyperimmune serum have consistently failed (13 1). The spectrum of immunological findings and the histopathological features of EAU in rodents and subhuman pri- mates indicate that this disease is predominantly a DTH-mediated disor- der directed at a well-defined anatomical region of the eye: the posterior uvea and retina.
Although IRBP and S antigen are retinal proteins residing close to each other, the pathogenesis of EAU induced by these two antigens differs considerably. EAU induced by S antigen is an acute explosive disease with an active phase lasting 7-10 days, during which the photo- receptor cell layer is totally destroyed (121, 122). The destruction of the photoreceptor layer is believed to prevent the recurrence of intraocular inflammation in S antigen-induced EAU, since the destruction of this cell layer eliminates the source of offending antigen in situ. By contrast, EAU induced by IRBP occurs later, the duration is longer, and the course is much less acute than EAU induced by S antigen (121,122). In addition to eliciting EAU, both retinal antigens also induce inflammation of the pineal gland, an organ that resides in another immunologically privi- leged location-the brain.
VI. Uveitir: A Breakdown in Self-Tolerance and Immune Privilege
EAU has been an attractive model for studying the pathogenesis of a variety of human inflammatory ocular diseases, such as sympathetic oph- thalmia. Key, and still unanswered, questions are what is the etiology for
IMMUNOLOGY OF THE EYE 217
autoimmune diseases of the eye, and if retinal antigens are the inflicting immunogens, what triggers the autoimmune response to these proteins? In the simplest scenario one might suggest that physical trauma or infec- tion might result in the release of large immunogenic quantities of retinal antigens. Moreover, a perforating injury to the eye might introduce bacterial contaminants that provide an adjuvant effect for the released ocular autoantigens. It has been proposed that this scenario occurs in the development of sympathetic ophthalmia, a relatively rare sequela of penetrating ocular trauma (1 32- 134).
By clinical definition, sympathetic ophthalmia is a bilateral uveitis in which inflammation occurs first in the injured eye (inciting eye) and is followed by inflammation in the second, uninjured, eye (sympathizing eye). The appearance of inflammation in the second eye indicates that the pathological sequelae are the result of noninfectious organ-specific (i.e, autoimmune responses. Although immunohistological analyses have re- vealed a varied spectrum of inflammatory cells in sympathetic ophthal- mia lesions (134, 135), the consensus is that the immunopathogenesis is largely due to an intraocular DTH reaction (134).
The series of events that provoke immunopathogenic processes that culminate in EAU, sympathetic ophthalmia, and similar autoimmune diseases of the eye remains a mystery. The facility with which EAU can be induced in a wide range of laboratory animals and subhuman primates suggests that the eye is at considerable risk for the development of spontaneous autoimmune diseases. However, the low incidence of uveitis in the human population indicates that effective mechanisms guard against autoimmune responses within the eye.
Maintenance of self-tolerance in the eye, like any other organ, can be achieved by four basic strategies: (1) sequestration of autoantigens from the immune system, (2) clonal deletion of self-reactive immune cells, (3) active suppression of anti-self-immune cells, and (4) programming im- mature cells with a down-regulatory signal that renders them hyporeactive without killing them (i.e., clonal anergy). These strategies are not mutually exclusive, and there is evidence to suggest that at least two of the four strategies are involved in the self-tolerance of ocular autoantigens.
Sequestration of self-antigens behind impervious anatomical barriers has been offered as the simplest strategy for preventing autoimmunity, especially in the case of ocular autoantigens. The sequestration of lens proteins within an impervious collagenous capsule is often cited as an example of this pathway for self-tolerance (136). The importance of this strategy in protecting the eye from autoimmune responses to uveal and retinal antigens is debatable. Under normal conditions, potential retinal antigens are unavailable for immunological recognition at the level of the inductive stage (i.e., the afferent mode) of the immune response, due
218 JERRY Y . NIEDERKORN
to their sequestration within the photoreceptor layer of the eye (12 1, 122, 137).
The possibility that the deletion of retinal antigen-specific T cell clones is involved in ocular self-tolerance is remote. Recently, Caspi et al. (138) demonstrated that EAU could be induced in selected strains of inbred mice and that susceptibility seemed to be correlated with the H-2' MHC haplotype. Although the induction of EAU was limited to but a few mouse strains, all mouse strains tested demonstrated lymphocyte- proliferative responses and antibody titers to both IRBP and S antigens. Thus, all hosts possessed potentially reactive T cell clones, but only a few host strains manifested autoimmunity. A similar situation may occur in humans. It has been reported that T cells capable of responding to retinal S antigen can be detected in the peripheral blood of normal human subjects with no history of uveitis (139).
Depending on how we define clonal anergy and clonal suppression, either or both mechanisms could be involved in self-tolerance to autoan- tigens of the retina and the lens. A feeble response to autoantigens might be construed as evidence of clonal anergy or an incomplete manifestation of a suppressor mechanism. Indirect evidence suggests that a partial suppressor system is expressed in autoimmune responses to retinal anti- gens. Induction of EAU in mice is not only restricted to certain inbred strains, but the experimental protocol requires the use of potent ad- juvants (i.e., Mycobacterium tuberculosis and Bordetella pertussis ) and treat- ment of the host with cyclophosphamide (138), a drug commonly used for eliminating suppressor cells in vivo (140).
Recent studies by Caspi et al. (141) demonstrated that organ-resident nonlymphoid cells of the retina (retinal glial cells = Miiller cells) exer- cised suppressive effects on T helper lymphocytes specific for retinal antigens. The prospect that Muller cells are instrumental in preventing autoimmune responses in the uvea and the retina is especially appealing for several reasons. Muller cells are a major component of the neural retina, where they are closely associated with the photoreceptor cells and ensheath the retinal blood vessels (142). As a result of their anatomically strategic location, they are in a position to serve as a second barrier (after the retinal vascular blood-brain barrier) through which an infiltrating lymphocyte (e.g., autoreactive T cells?) must pass in order to enter the posterior segment of the eye. Moreover, it has been shown that Miiller cells exercise their suppressive effect on retinal antigen-specific cells by direct cell-cell contact (14 1)-a situation uniquely suited for the retina and Miiller cells.
Recent studies have demonstrated that Muller cells become activated and undergo proliferation when exposed to lymphokines secreted by T
IMMUNOLOGY OF T l I E EYE 219
helper lymphocytes (14 1). This blend of characteristics leads to the fol- lowing scenario: Due to their strategic location, retinal Muller cells could serve as an anatomical barrier for the migration of T cells into the posterior segment of the eye. If antigen-specific T cells find their way to the retina, the close physical association between the photoreceptor cells and the Miiller cells places the latter close to lymphokines released by autoreactive T cells. As a result, resident Muller cells would be activated and would function to dampen further activity by the autoreactive T cells.
Finally, it has been reported that not only do Muller cells function as immunoregulatory cells, but that they are also active in healing and in scar formation in the terminal stages of EAU (143, 144). Although much needs to be learned and confirmed regarding the function of retinal Miiller cells, their potential in maintaining immunological homeostasis in the eye is a provocative topic for further analysis.
In addition to the organ-resident Muller cells, there may be additional mechanisms for actively suppressing autoimmune responses directed at retinal antigens. Mizuno et al. (80, 81) have reported that intracameral injection of retinal S antigen induced suppression of systemic DTH responses to S antigen and significantly reduced the severity of uveitis induced by extraocular immunization with S antigen. Other studies in rats support the conclusion that the induction of ACAID to S antigen mitigates EAU.
A suppressor T lymphocyte line was isolated from the spleens of rats primed via the anterior chamber with soluble retinal S antigen (145). Adoptive transfer of this suppressor T cell line was found to downgrade EAU in actively immunized syngeneic hosts. It is not known whether a similar suppressor T lymphocyte population is constitutively induced and maintained at a low level in normal individuals. It is interesting that in this experimental context an ocular antigen (i.e., S antigen) was intro- duced through the anterior chamber and the resulting suppressor T cell population served to mitigate an ocular autoimmune disease. One is tempted to entertain the hypothesis that ACAID may be an integral regulatory mechanism for maintaining the immunological homeostasis of the eye.
VII. Is Cataract Formation an Immune-Mediated Disease?
No discussion on the immunology of the eye and autoantigens would be complete without commenting on the autoimmune potential of lens crystallins. As stated earlier, one of the simplest mechanisms for ex- plaining self-tolerance is the anatomical sequestration of autoantigens.
220 JERRY Y . NIEDERKORN
By erecting an anatomical barrier between the hosts immune system and potential autoantigens, self-tolerance is assured. The lens serves as an excellent example of anatomical sequestration. Housed within a col- lagenous capsule, the lens is shielded from the systemic circulatory and lymphatic systems. Moreover, the lens capsule is suspended within an immunologically privileged compartment: the anterior chamber of the eye (Fig. 1). The early sequestration of the lens during ontogeny implies that its crystallins could act as autoantigens if released into the peripheral circulation.
The suspicion that lens components are potential autoantigens was suggested over 80 years ago by Uhlenhuth (146), who demonstrated that antibodies to bovine lens reacted with lens proteins from a wide range of vertebrate species. Verhoeff and Lemoine (147) subsequently sug- gested that such immune factors might be related to the persistent ocular inflammatory diseases that occasionally follow penetrating in- jury to the eye.
Recently, Angunawela ( 148) reconsidered the hypothesis that catarac- togenesis is an autoimmune phenomenon, based on findings indicating a significant increase in the incidence of antibody directed against lens antigens in the sera of patients with cataractous lenses and in the sera of diabetic patients. Moreover, immunoglobulin deposits were detected on the cells of the cataractous lenses removed from nondiabetic and diabetic patients. The close relationship between diabetes and senile cataracts is well known; however, interpreting the presence of lens-specific anti- bodies in the serum of cataractous patients should be viewed with cau- tion, since similar antibodies have been demonstrated in approximately 50% of the normal individuals tested (149, 150). It is possible that an in- creased incidence of antibody in these individuals was the result of cataractogenesis, rather than the cause. Nonetheless, these findings bring to our attention some interesting issues regarding immune regu- lation in the eye and the possible consequences of a malfunction of this regulation.
It has long been assumed that the lens capsule was impervious to leakage of lens proteins and prevented their escape into the aqueous fluid of the anterior chamber. However, a and y lens crystallins can be readily detected in the aqueous humor of normal individuals (151). Leakage of lens antigens into the aqueous humor and transport to the outflow system may occur with sufficient frequency to explain the presence of serum antibody against lens crystallins in approximately 50% of the normal population (149,150). Moreover, homologous lens antigens are known to induce antibody synthesis in a variety of mammals, without inducing demonstrable T cell-mediated immune responsiveness ( 152). It is tempt- ing to suggest that a constant leakage of lens antigens into the anterior
22 1 IMMUNOLOGY OF THE EYE
chamber results in the induction of ACAID. As in the case of the retina and the uvea, it appears that a vulnerable ocular tissue is protected from autoimmune attack by anatomical sequestration and possibly by a dy- namic immunoregulatory process. This hypothesis, although appealing, remains to be verified.
This chapter has addressed some of the unique immunological charac- teristics of the eye. The goal was not to provide an exhaustive review of ocular immunology, but to focus on the curious immunological ground rules peculiar to this organ. The weight of evidence suggests that the immune system is carefully regulated within the eye and, to a lesser degree, at the ocular surface (i.e., the cornea). Teleologically, it appears that this regulation is designed to restrict the expression of exuberant inflammatory responses-namely, DTH-that carry a heavy burden of innocent bystander damage to the ocular tissues possessing few regenera- tive capacities, if any. Although DTH is excluded, CTLs and antibody provide adequate coverage to protect the eye from pathogens without damaging juxtaposed normal ocular tissues. The rare incidence of immune-mediated inflammatory diseases of the eye is a testament to the effectiveness of the ocular irnmunoregulatory circuit. Gaining a better understanding of this circuitry will offer immunological implications that extend well beyond the boundaries of the eye.
ACKNOWLEDGEMENTS These studies were supported in part by National Institutes of Health grants
EY05631, CA30276, and EY07641 and by an unrestricted grant from Research to Prevent Blindness, Inc. The author is a Research to Prevent Blindness-Olga Keith Wiess Scholar.
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This article was accepted for publication on 4 October 1989.