Severing corneal nerves in one eye induces sympathetic loss of immune privilege and promotes rejection of future corneal allografts placed in either eye.

Less than 10% of corneal allografts undergo rejection even though HLA matching is not performed. However, second corneal transplants experience a threefold increase in rejection, which is not due to prior sensitization to histocompatibility antigens shared by the first and second transplants since corneal grafts are selected at random without histocompatibility matching. Using a mouse model of penetrating keratoplasty, we found that 50% of the initial corneal transplants survived, yet 100% of the subsequent corneal allografts (unrelated to the first graft) placed in the opposite eye underwent rejection. The severing of corneal nerves that occurs during surgery induced substance P (SP) secretion in both eyes, which disabled T regulatory cells that are required for allograft survival. Administration of an SP antagonist restored immune privilege and promoted graft survival. Thus, corneal surgery produces a sympathetic response that permanently abolishes immune privilege of subsequent corneal allografts, even those placed in the opposite eye and expressing a completely different array of foreign histocompatibility antigens from the first corneal graft.

IFN-γ blocks CD4+CD25+ Tregs and abolishes immune privilege of minor histocompatibility mismatched corneal allografts.

Th1 CD4+ cells are believed to be the primary mediators of corneal allograft rejection. However, rejection of fully allogeneic C57BL/6 corneal allografts soared from 50% to 90% in both interferon-gamma (IFN-γ)(-/-) and anti-IFN-γ-treated BALB/c mice. In contrast, similar deficits in IFN-γ in BALB/c hosts enhanced immune privilege of BALB.B (minor histocompatibility [minor H] antigen-matched, major histocompatibility complex [MHC]-mismatched) and NZB (MHC-matched, minor H antigen-mismatched) corneal allografts-decreasing rejection from 80% to ~20%. This effect of IFN-γ was independent of CD4+ T cell lineage commitment as both anti-IFN-γ-treated acceptor and rejector mice displayed a Th2 cytokine profile. The presence of IFN-γ prevented the generation of alloantigen-specific CD4+CD25+ T regulatory cells (Tregs) in hosts receiving either MHC only mismatched BALB.B or minor only histocompatibility (minor H)-mismatched NZB corneal allografts. Tregs in these hosts promoted corneal allograft survival by suppressing Th2 effector cells. By contrast, IFN-γ was necessary for the generation of CD4+CD25+ Tregs that prevented rejection of fully allogeneic C57BL/6 corneal allografts in BALB/c hosts. These findings suggest that MHC-matching in combination with blockade of IFN-γ holds promise as a means of enhancing corneal allograft survival.

Allergic conjunctivitis renders CD4(+) T cells resistant to t regulatory cells and exacerbates corneal allograft rejection.

Allergic diseases rob corneal allografts of immune privilege and increase immune rejection. Corneal allograft rejection in BALB/c allergic hosts was analyzed using a short ragweed (SWR) pollen model of allergic conjunctivitis. Allergic conjunctivitis did not induce exaggerated T-cell responses to donor C57BL/6 (B6) alloantigens or stimulate cytotoxic T lymphocyte (CTL) responses. Allergic conjunctivitis did affect T regulatory cells (Tregs) that support graft survival. Exogenous IL-4, but not IL-5 or IL-13, prevented Treg suppression of CD4(+) effector T cells isolated from naïve mice. However, mice with allergic conjunctivitis developed Tregs that suppressed CD4(+) effector T-cell proliferation. In addition, IL-4 did not inhibit Treg suppression of IL-4Rα(-/-) CD4(+) T-cell responses, suggesting that IL-4 rendered effector T cells resistant to Tregs. SRW-sensitized IL-4Rα(-/-) mice displayed the same 50% graft survival as nonallergic WT mice, that was significantly less than the 100% rejection that occurred in allergic WT hosts, supporting the role of IL-4 in the abrogation of immune privilege. Moreover, exacerbation of corneal allograft rejection in allergic mice was reversed by administering anti-IL-4 antibody. Thus, allergy-induced exacerbation of corneal graft rejection is due to the production of IL-4, which renders effector T cells resistant to Treg suppression of alloimmune responses.

Induction of anterior chamber-associated immune deviation requires an intact, functional spleen.

Anterior chamber-associated immune deviation (ACAID) expresses itself in BALB/c mice inoculated intracamerally with P815 cells in three ways: progressive growth of the tumor within the eye, transient growth of P815 cells injected subcutaneously, and prolonged acceptance of DBA/2 skin allografts. The spleen was found to play a crucial role in the development of ACAID. Splenectomized animals bearing intracameral P815 tumors reject DBA/2 skin grafts in an accelerated manner. A functioning spleen was required during the first 10 d after intracameral inoculation of P815 cells, but not thereafter. Reconstitution experiments revealed that the spleen's ability to support the induction of ACAID depends partly upon its constituent lymphoid cells, but also upon either a stromal component or a unique architectural arrangement that can only be restored with splenic fragments. The data hold promise that therapeutic protocols using appropriately timed splenectomy and specific immunization can be devised to induce hosts bearing intraocular tumors to mount an immune response sufficiently vigorous to destroy the tumor within the eye, and sufficiently precise to preserve the functional and anatomic integrity of the eye.

Systemic immune unresponsiveness induced in adult mice by anterior chamber presentation of minor histocompatibility antigens.

The ability to introduce carefully controlled numbers of viable cells into the anterior chamber of mouse eyes made it possible to examine the interrelationship between presentation of antigens into the anteior chamber and into conventional body sites and their synergistic/antagonistic effects on the immune system. P815 mastocytoma (DBA/2; H-2d) cells are syngeneic with BALB/c hosts at the major histocompatibility locus, but differ at multiple minor histocompatibility loci. When P815 cells were injected subcutaneously, they were rejected by BALB/c recipients who became specifically immune. By contrast, when P815 cells were injected intracamerally, they grew progressively into massive intraocular tumors; moreover, these BALB/c hosts proved subsequently unable to reject subcutaneously injected P815 cells, and, more impressively, failed to reject DBA/2 skin allografts placed orthotopically. Minor histocompatibility antigens, presented first through the anterior chamber of mouse eyes, elicit a suppressive rather than an aggressive host immune response that protects cells that bear these antigens from a destructive alloimmune reaction at both intracameral and systemic sites.

Presentation of the H-Y antigen on Langerhans' cell-negative corneal grafts downregulates the cytotoxic T cell response and converts responder strain mice into phenotypic nonresponders.

We have used the murine cornea is an allograft model to investigate the relative roles of graft-derived IA+ APC (Langerhans' cells) and host-derived APC during the induction of CTL responses to H-Y. The natural exclusion of LC from the immunizing corneal graft led to a specific state of unresponsiveness to H-Y in responder strain mice, while inclusion of LC resulted in responsiveness. Failure to respond to H-Y could not be attributed to the absence of H-Y or IA antigen expression on the surface of LC-deficient grafts but instead, appeared to be due to active suppression of the T helper cell response during in vivo priming. Reprocessing of the H-Y antigen by host APC did not occur after immunization with H-Y presented on H-2-incompatible grafts unless presented initially by graft-derived LC. H-2 as well as some non-H-2 alloantigens were presented to the host without a requirement for donor-derived LC. Thus there appear to be differential requirements for the processing and presentation of alloantigens.

Allergic airway hyperreactivity increases the risk for corneal allograft rejection.

Corneal allografts transplanted into hosts with allergic conjunctivitis experience an increased incidence and swifter tempo of immune rejection compared to corneal allografts transplanted to nonallergic hosts. Previous findings suggested that increased risk for rejection was not a local effect produced by an inflamed eye, but was due to perturbation of the systemic immune responses to alloantigens on the corneal allograft. We tested the hypothesis that another allergic disease, airway hyperreactivity (AHR), would also increase the risk for corneal allograft rejection. Induction of AHR with either ovalbumin (OVA) or short ragweed (SRW) extract prior to keratoplasty resulted in a steep increase in the speed and incidence of corneal allograft rejection. Delayed-type hypersensitivity (DTH) responses to corneal alloantigens were closely associated with corneal allograft rejection. However, the deleterious effect of AHR on corneal allograft survival was not reflected in a heightened magnitude of allospecific DTH, cytotoxic T lymphocyte and lymphoproliferative responses to the alloantigens on the corneal allograft. Unlike Th2-based immediate hypersensitivity, CD8+ T-cell-based contact hypersensitivity to oxazolone did not increase the risk for corneal allograft rejection. Thus, Th2-based allergic diseases significantly reduce the immune privilege of the corneal allograft and represent important risk factors for consideration in the atopic patient.

Promotion of syngeneic intraocular tumor growth in mice by anterior chamber-associated immune deviation.

Regressor tumors (P91 and UV-5C25), known to express potent tumor-specific transplantation antigens, were briskly rejected when transplanted either sc or ip into syngeneic mice (DBA/2 and BALB/c, respectively). These tumors demonstrated markedly different behavior following transplantation into the anterior chamber of syngeneic mice. Tumors grew to significant masses and survived for prolonged periods within the anterior chamber of the eye. The extended growth and survival of regressor tumors in the anterior chamber (i.e. immune privilege) were abrogated by splenectomy and thus resemble anterior chamber-associated immune deviation. However, this immune privilege proved to be temporary and was superseded by complete tumor resolution. Studies of mice immunosuppressed with UV light or X-ray irradiation demonstrated that spontaneous intraocular resolution of regressor tumors was due to specific systemic immunity that not only led to intraocular tumor resolution but also prevented the spread of the primary intraocular tumor to distant organs and rendered the hosts highly resistant to secondary challenge with sc tumor inocula. The present findings were relevant to understanding human retinoblastoma, an intraocular neoplasm demonstrating a high incidence of spontaneous resolution, and will hopefully form a foundation for designing, immunotherapeutic strategies for treating human intraocular neoplasms.

Feline uveal melanomas induced with feline sarcoma virus: potential model of the human counterpart.

Uveal melanomas were produced by injecting Gardner strain feline fibrosarcoma virus intraocularly into 10- to 15-day-old noninbred kittens. Tumors developed in about 90% of the cats' eyes receiving virus. Progressing tumors (62 eyes of 36 cats) began as small hyperpigmented lesions at the site of injection and grew to fill the anterior chamber by 3-5 months after infection. About 30% of cats with these tumors developed secondary tumors and died. Nonprogressive tumors characterized by flat, pigmented plaques on the iris at the site of injection developed in 25 eyes of 18 cats. These lesions did not enlarge except in proportion to the growth of the eye. Tumors were composed of pigmented spindle cells, pigmented epithelioid cells, and nonpigmented spindle cells. The cells could be grown for 5-8 passages in vitro. One culture assumed a transformed morphology and grew in noninbred athymic nu/nu mice. The lesions resembled human spindle cell melanomas.

Effect of donor Langerhans cells on corneal graft rejection.

Unlike other cutaneous surfaces, the central portion of the corneal epithelium is typically devoid of Langerhans cells. The absence of Ia+ Langerhans cells in the central cornea is of more than casual interest and may explain the immunologic privilege that is characteristic of corneal allografts. The present communication summarizes previous studies that examined the role of corneal Langerhans cells in eliciting alloimmune responses and corneal graft rejection in rodents. Under normal circumstances, corneal allografts are poorly immunogenic when residing in the avascular ocular graft bed even though the graft displays large quantities of alloantigens. The afferent blockade of the immune response can be circumvented by donor-derived Langerhans cells that serve as potent immunogens for all categories of corneal allografts except grafts involving allodisparity only at class I major histocompatibility complex loci. Thus, the presence of donor-derived Langerhans cells exerts profound effects on the fate of corneal allografts.

The pathogenesis of Acanthamoeba keratitis.

Free-living amoebae of the genus Acanthamoeba produce a progressive, blinding infection of the corneal surface. The pathogenesis of Acanthamoeba keratitis involves parasite-mediated cytolysis and phagocytosis of corneal epithelial cells and induction of programmed cell death. Acanthamoeba spp. elaborate a variety of proteases which may facilitate cytolysis of the corneal epithelium, invasion of the extracellular matrix, and dissolution of the corneal stromal matrix.

Characterization of a plasminogen activator produced by Acanthamoeba castellanii.

Serine proteases play an important role in a diverse array of biological processes, including embryogenesis, metastasis, angiogenesis, thrombolysis and tissue invasion by certain parasites. The latter observation prompted us to explore the possibility that the tissue-invasive ocular parasite Acanthamoeba castellanii elaborates one or more serine proteases. Acanthamoeba sp. are pathogenic free-living amoebae that can produce an invasive, blinding inflammatory disease of the cornea, termed Acanthamoeba keratitis. The present study reports the preliminary purification and characterization of a novel plasminogen activator from an ocular isolate of A. castellanii. The parasite-derived enzyme has a molecular mass of approx. 40 kDa and produces a single band of lysis on fibrinogen-agarose zymographs. Activity of the enzyme is completely inhibited by treatment with diisopropylfluorophosphate, indicating that it is a serine protease. The parasite-derived serine protease is not inhibited by amiloride which is a strong inhibitor of urokinase-type plasminogen activator. Additionally, the enzyme is not inhibited by plasminogen activator inhibitor-1 which is the primary physiological inhibitor of both urokinase and tissue-type plasminogen activator. It does not cross-react with antibodies specific for human urokinase or tissue-type plasminogen activator. The parasite-derived enzyme activates plasminogen from several mammalian species, including human, cow and pig. Thus, it is possible that this parasite-derived serine protease contributes to the pathogenesis of Acanthamoeba keratitis.

Exogenous recombinant interleukin-2 abrogates anterior-chamber-associated immune deviation.

Alloantigens placed into the anterior chamber of the eye elicit antigen-specific suppression of systemic delayed-type hypersensitivity (DTH) responses and severe impairment of skin allograft rejection. This pattern of immunological impairment has been termed anterior-chamber-associated immune deviation (ACAID). We have previously proposed that ACAID is the underlying mechanism responsible for immunologic privilege within the anterior chamber of the eye. The present study examined the role of interleukin 2 (IL-2) in the induction of ACAID. As previously shown, intracameral inoculation of P815 cells into allogeneic BALB/c recipients resulted in antigen specific suppression of DTH responses. However, subcutaneous administration of recombinant IL-2 along with intracameral inoculation of P815 cells prevented ACAID and permitted the development of positive DTH responsiveness in BALB/c hosts. In order to abrogate ACAID, exogenous IL-2 was required only during the first 72 hr following anterior chamber presentation of alloantigens. Collectively, the results indicate that induction of ACAID and the antigen-specific suppression of systemic DTH responses are due to IL-2 deficiency during the early stages of antigen perception. This in turn implies that the immunological privilege within the anterior chamber of the eye may pivot on the deficiency of a single lymphokine, interleukin 2.

The immune privilege of corneal allografts.

BACKGROUND: Corneal transplantation is the oldest, most common, and arguably, the most successful form of tissue transplantation. In the United States alone, over 40,000 corneal transplantations are performed each year. Less than 10% of the uncomplicated, first-time corneal grafts will undergo immune rejection even though HLA matching is not routinely performed and the use of immunosuppressive drugs is limited to the topical application of corticosteroids. The success of corneal transplantations predates the use of corticosteroids and further emphasizes the remarkable privilege of corneal allografts. METHODS: Several laboratories have used rat and mouse models of orthotopic corneal transplantation (keratoplasty) in an attempt to understand the basis for the immune privilege of corneal allografts. RESULTS: The time-honored explanation for the immune privilege of corneal allografts was based on the conspicuous avascularity of the cornea, which was believed to sequester the graft from the immune apparatus. However, results from several laboratories indicate that at least three additional features of the corneal graft contribute to its immune privileged status: (a) absence of donor-derived, antigen-presenting passenger Langerhans cells in the corneal graft; (b) expression of Fas ligand on the epithelium and endothelium of the corneal allograft; and (c) capacity of the corneal allograft to induce immune deviation of the systemic immune response. CONCLUSIONS: The immune privilege of corneal allografts is a product of at least three unique qualities of the corneal allograft that conspire to interfere with the induction and expression of allodestructive immune responses.

The presence of donor-derived class II-positive cells abolishes immune privilege in the anterior chamber of the eye.

The anterior chamber is widely recognized as an example of an immune privileged site. It has become clear that the immunologic privilege of the anterior chamber is the result of active down-regulation of systemic cell-mediated immunity, a phenomenon termed anterior chamber-associated immune deviation (ACAID). In murine models ACAID has been demonstrated using tumor antigens, viral antigens, haptenated spleen cells, and minor histocompatibility antigens. In the present study, we examined the role of class II-positive cells of donor origin on the induction of ACAID. DBA/2 splenocytes were sorted into plastic-adherent, class II-positive, and nonadherent, class II-negative. populations and subsequently transplanted into the anterior chamber of allogeneic BALB/c hosts. Hosts primed intracamerally with class II-positive, adherent cells developed strong DTH responses (P less than 0.01) while hosts primed with nonadherent, class II-negative cells failed to mount detectable DTH responsiveness (P greater than 0.05). Similar results were found in a parallel study using the class II-negative CCL 46 and class II-positive AD.4 subclones of the P388D1 tumor line (DBA/2 origin). The class II-positive tumor grew transiently and stimulated a strong DTH response (P less than 0.01), while the class II negative tumor grew progressively and failed to stimulate DTH responsiveness (P greater than 0.05). The results indicate that donor-derived Ia+ antigen-presenting cells can deprive the anterior chamber of its immunologic privilege and lead to the induction of normal systemic alloimmunity.

Analysis of antibody production induced by allogeneic tumor cells inoculated into the anterior chamber of the eye.

Humoral immune responses to alloantigens on P815 mastocytoma cells placed into the anterior chamber of murine eyes were studied. The results show that: (1) the amount of specific serum antibody produced by recipients of intracamerally injected P815 cells is directly proportional to the degree of immunogenetic disparity between the recipient and the injected cells; (2) destruction of allogeneic intraocular tumors is mediated, at least in part, by specific antibodies; and (3) retention of allogeneic tissue within the anterior chamber for 3 to 4 days after inoculation is essential to the elicitation of specific antibody formation. We conclude that alloantigens introduced into the anterior chamber are presented to the systemic immune apparatus in a very unique manner resulting in the preferential stimulation of specific antibody production without awakening specific cell-mediated immunity.

Characteristics of rejection of orthotopic corneal allografts in the rat.

We have employed a rat model of orthotopic corneal transplantation to study the characteristics of rejection and development of systemic immunity in the host. Lewis (LEW) rats underwent a true penetrating keratoplasty using Wistar-Furth (WF) donor corneas. A rejection incidence of 55% with a mean survival time (MST) of 17.1 days was observed using these untreated allogeneic corneas. Animals undergoing rejection of these allografts developed cytotoxic T lymphocytes (CTL) capable of lysing WF lymphoblasts in a standard 51-chromium release assay. These same rats did not have delayed-type hypersensitivity (DTH) responses when compared to skin grafted controls. Rats with clear allografts had no demonstrable CTL or DTH activity. As expected, LEW rats that were preimmunized with WF skin grafts and subsequently received WF orthotopic corneal grafts rejected 100% of these corneas at an accelerated rate (MST = 9.7 days, P less than .02). We then employed a previously described technique of using latex beads to induce migration of Langerhans cells into the central cornea of the donor graft prior to transplantation. The presence of Langerhans cells in the donor cornea resulted in a higher incidence of rejection (96%) and an accelerated rate (MST = 11.8 days, P less than .02) when compared to untreated allografts. These rats also had a higher level of CTL activity and marked DTH responses. These data show that rejection of orthotopic allogeneic corneas is accompanied by the development of systemic alloimmunity as measured by CTL activity. However, these fully allogeneic corneas can be rejected in the absence of DTH responses. Langerhans cells have a dramatic effect on graft survival and are necessary for induction of DTH responsiveness in the host.

Prevention of the induction of allospecific cytotoxic T lymphocyte and delayed-type hypersensitivity responses by ultraviolet irradiation of corneal allografts.

The effect of ultraviolet radiation (UVR) on the immunogenicity of corneal allografts was examined in a mouse model. Corneal allografts differing from the host at the entire MHC and multiple minor H loci were subjected to 200 mJ/cm2 of UVB irradiation immediately prior to heterotropic transplantation. Analysis of cytotoxic T lymphocyte and delayed-type hypersensitivity responses revealed that UVR treated corneal grafts failed to induce either CTL or DTH responses in C57BL/6 recipients. UVB treatment abolished the immunogenicity of highly immunogenic corneal grafts containing either resident or infiltrating donor-specific Langerhans cells. Sequential grafting experiments demonstrated that UVB-treated grafts rendered the hosts anergic to subsequent immunization with highly immunogenic corneal limbus grafts that contained dense concentrations of Ia+ Langerhans cells of donor origin. The results indicate that UV treatment not only reduces the immunogenicity of the corneal allograft but may also render it tolerogenic.

Alloantigens introduced into the anterior chamber of the eye induce systemic suppression of delayed hypersensitivity to third-party alloantigens through "linked recognition".

It has been recognized for over a century that the anterior chamber of the eye is endowed with unique immunological properties that permit the long-term survival of histoincompatible grafts that would otherwise be rejected at extraocular sites. Immune privilege in the anterior chamber of the eye has been attributed, at least in part, to the active down regulation of systemic delayed-type hypersensitivity (DTH) that is induced when antigens are introduced into this ocular compartment. The antigen-specific suppression of systemic DTH that is induced by anterior chamber priming has been termed anterior chamber-associated immune deviation (ACAID) and has been demonstrated with a variety of antigens. The present report summarizes a systematic evaluation of various categories of histocompatibility antigens for their capacity to induce ACAID. The results indicate that ACAID can be induced against a wide variety of alloantigens ranging from a single minor histocompatibility antigen (H-Y) to mismatches involving the entire major histocompatibility complex plus multiple minor histocompatibility loci. Further investigation revealed that it was possible to induce suppression of DTH to third-party alloantigens providing the alloantigens introduced into the anterior chamber were coexpressed with the third-party alloantigens on cells used for extraocular immunizations.

The effect of oral immunization on corneal allograft survival.

The present study examined the potential of orally induced tolerance for preventing immunological rejection of corneal allografts. Orthotopic corneal allografts were transplanted from either C3H (MHC + multiple minor H-mismatched) or NZB (multiple minor H-mismatched only) donors to CB6F1 recipients on day 0. Tissue cultured corneal epithelial and endothelial cells from relevant donor strains were administered orally from day -14 to day -4 on a daily basis, The incidence of graft rejection, graft mean survival time (MST), and alloimmune responses, and the antigen specificity of induced tolerance were studied. Oral immunization induced a remarkable tolerance such that only 55% of the orally immunized hosts rejected their fully allogeneic corneal grafts (MST = 43 days) compared with 100% rejection (MST = 18 days) in normal controls. Likewise, rejection of MHC-matched, multiple minor H-mismatched corneal grafts fell from 80% in untreated controls to 36% in orally immunized hosts. Oral immunization was effective in desensitizing previously immunized hosts. Rejection of MHC-matched, multiple H minor-mismatched corneal allografts fell from 93% in preimmune, unfed hosts to 36% in preimmune, orally tolerized mice. Thus, oral immunization is a safe and effective method for desensitizing high-risk, preimmune hosts and promoting corneal allograft survival.