New thoughts on the immunology of corneal transplantation.

Significant advances derived from rodent models of penetrating keratoplasty have transformed our understanding of the pathogenesis of rejection of orthotopic corneal transplants. The high rate of success of corneal allografts placed in low-risk eyes without cover of immunosuppression arises from immune privilege of the cornea graft itself, and of the anterior chamber where it forms the anterior wall. Immune privilege owes its existence in penetrating keratoplasty to an absence of blood and lymph vessels in the graft and its bed, the absence of MHC class II(+) antigen presenting cells in the graft, reduced expression of MHC-encoded alloantigens on graft cells, constitutive expression of T cell-deleting CD95 ligand on corneal graft endothelium, the existence of an immunosuppressive local microenvironment (aqueous humor), and the capacity of the graft to induce anterior chamber associated immune deviation (ACAID). The results of recent experiments provide answers to pertinent questions regarding cornea graft failure: How does the cornea as a graft suppress inflammation and angiogenesis locally? How does the graft promote ACAID to the alloantigens it expresses? and How do corneal cells reduce their vulnerability as targets of effector T cells? The answers offer the possibilities of novel strategies for preventing immune-based corneal allograft failure.

[Angiogenesis and lymphangiogenesis in the cornea. Pathogenesis, clinical implications and treatment options]. / Angiogenese und Lymphangiogenese in der Hornhaut. Pathogenese, klinische Bedeutung und Therapieoptionen.

BACKGROUND: Whereas the normal cornea is devoid of blood and lymphatic vessels,both can invade the cornea secondary to a variety of corneal diseases and after surgery. This not only reduces visual acuity, but also renders such a cornea high-risk, if subsequent corneal transplantation is performed. METHODS: A PUBMED-based literature search was carried out. RESULTS: Current knowledge on pathogenesis, clinical implications and treatment modalities for corneal neovascularization is discussed. CONCLUSIONS: Novel anti-angiogenic and antilymphangiogenic therapeutic strategies should reduce blindness associated with corneal neovascularization and subsequent graft rejection.

Light-induced migration of retinal microglia into the subretinal space.

PURPOSE: To explore the effects of light exposure and deprivation on the distribution and function of microglia in the subretinal space of mice. METHODS: Using a monoclonal antibody, 5D4, that identifies resting, ramified microglia, the distribution and density of microglia in the retina, and the subretinal space were determined by confocal microscopy and by immunohistochemistry of cryopreserved sections of eyes of albino and pigmented mice exposed to diverse levels of light, ranging from complete darkness to intense brightness. Axotomized retinal ganglion cells were retrograde labeled by fluorescent tracer to determine whether the marker colocalizes to 5D4+ cells. Electron microscopy was used to evaluate microglia for evidence of phagocytosis. RESULTS: 5D4+ microglia in pigmented eyes were limited to the inner retinal layers, but in albino eyes 5D4+ cells were found in the outer retinal layers and subretinal space as well. The subretinal space of eyes of albino mice raised from birth in complete darkness contained few 5D4+ cells, but exposure to light caused the rapid accumulation of 5D4+ cells at this site. 5D4+ cell density in the subretinal space correlated directly with intensity of ambient light. Retrograde labeling of axotomized ganglion cells resulted in 5D4+ cells in the subretinal space that contained the retrograde label. Subretinal microglia contained phagocytized rod outer segment discs. On intense light exposure, 5D4+ cells adopted an active morphology, but failed to express class II major histocompatibility complex (MHC) molecules. CONCLUSIONS: Light exposure induced retinal microglia migration into the subretinal space in albino mice. Subretinal microglia appeared to augment through phagocytosis the capacity of pigment epithelium to take up the photoreceptor debris of light toxicity. The unexpected presence of these cells in the subretinal space raises questions concerning their potential contribution to immune privilege in this space and to the fate of retinal transplants.

Evidence that ultraviolet B radiation transiently inhibits emigration of Langerhans cells from exposed epidermis, thwarting contact hypersensitivity induction.

Langerhans cells (LC) play a critical role in the induction of contact hypersensitivity (CH), and ultraviolet B radiation (UVR) impairs CH induction in UVB-susceptible (UVB-S) mice via a TNF-alpha-dependent mechanism. A possible explanation of this effect is that UVR impairs CH in UVB-S mice by immobilizing LC transiently in the epidermis and upper dermis, thereby preventing their timely migration to draining lymph nodes. To test this hypothesis we examined in vitro and in vivo the effects of in vivo UVR on migration of Ia(+) cells from skin of UVB-S and UVB-resistant (UVB-R) mice. Dorsal surfaces of ears of mice were irradiated with 400 J/cm(2) UVB and either explanted in vitro or transplanted orthotopically to the thoracic wall of syngeneic recipients. After 24, 48, and 72 h the epidermis was recovered from these explants/grafts and the number of Ia(+) cells determined by immunohistochemistry. Culture medium obtained after explants were removed was also evaluated for content of Ia(+) cells. The density of Ia(+)-bearing cells in the epidermis of cultured untreated skin explants and of grafted skin fell progressively for both UVB-S and UVB-R skin during the observation period. The rate of decline in Ia(+) cells density during this interval was greatly impaired if the skin was exposed to UVR prior to excision; this effect was seen equally in UVB-S and UVB-R skin. Recovery of Ia(+) cells in the medium after removal of cultured untreated skin explants was maximum after 24 h and comparable in UVB-S and UVB-R skin. However, the number of Ia(+) cells recovered in the medium from UVB-exposed skin was significantly reduced only if the skin donor was UVB-S. We conclude that the ability of UVR to impede LC migration from epidermis is significantly greater for UVB-S mice, accounting in part for the failure of these mice after UVR to acquire CH.

Immune privilege persists in eyes with extreme inflammation induced by intravitreal LPS.

Since immune privilege is believed to exist in the eye in order to suppress sight-destroying inflammation, we wondered whether eyes with intraocular inflammation retain the immune privileged state. Intraocular inflammation was induced by injection of lipopolysaccharide (LPS) into the vitreous cavity of BALB/c mouse eyes, which showed a peak in intensity at approximately 9 h. At this time point, inflamed eyes were examined for their capacity to afford immune privilege to injected allogeneic tumor cells, and to promote anterior chamber-associated immune deviation (ACAID) to antigens injected locally. In addition, aqueous humor (AqH) harvested from inflamed eyes was tested for its ability to suppress T cell activation. Surprisingly, eyes with acute, intense intraocular inflammation allowed allogeneic tumor cells to form progressively growing tumors, and these same eyes promoted ACAID. Moreover, AqH harvested from inflamed eyes strongly inhibited T cell activation. We conclude that the type of extreme, intraocular inflammation evoked by intravitreally injected LPS fails to abolish immune privilege in the eye. These findings are discussed in light of the effects of other types of inflammation on the integrity of ocular immune privilege, and with respect to the capacity of the eye to maintain immune privilege by more than one mechanism.

Acute rejection of orthotopic corneal xenografts in mice depends on CD4(+) T cells and self-antigen-presenting cells.

PURPOSE: Guinea pig corneal xenografts have been reported to be rejected acutely in eyes of normal adult mice. Rejection of this type is independent of xenoreactive antibodies, and mice deficient in CD8(+) and NK T cells are unable to reject guinea pig corneal grafts acutely. Therefore, a study was conducted to determine the extent and manner by which CD4(+) T cells are responsible for rejection of orthotopic corneal xenografts. METHODS: Xenogeneic corneas were prepared from eyes of normal guinea pigs and grafted orthotopically into normal eyes of C57BL/6 mice, class II major histocompatibility complex (MHC) knockout (KO) mice, and class II MHC KO mice reconstituted with syngeneic (C57BL/6) CD4(+) T cells and/or bone marrow cells. Graft survival was assessed clinically, and success of cellular reconstitution was assayed using flow cytometric analysis of peripheral blood leukocytes. T cells from rejector mice were analyzed for proliferative responses to guinea pig xenoantigens in vitro. RESULTS: Median survival times (MST) of corneal xenografts in MHC class II KO mice was significantly delayed (31 days) compared with grafts in wild-type C57BL/6 eyes (9 days). Acute rejection was restored almost completely when MHC class II KO mice were reconstituted simultaneously with C57BL/6 bone marrow and CD4(+) T cells, but not when the KO mice were reconstituted with either CD4(+) T cells or bone marrow cells alone. Mice that rejected guinea pig corneas possessed only CD4(+) T cells capable of responding to guinea pig xenoantigens in vitro. CONCLUSIONS: Acute rejection of orthotopic corneal xenografts in mice is mediated by CD4(+) T cells that detect guinea pig xenoantigens that are presented on MHC class II(+) syngeneic antigen-presenting cells. These results strongly suggest that rejection occurs exclusively through the indirect pathway of T-cell activation.

Evidence for antigen-specific immune deviation in patients with acute retinal necrosis.

BACKGROUND: Because experimental acute retinal necrosis (ARN) induced by herpes simplex virus in mice develops only if mice fail to acquire virus-specific delayed hypersensitivity (DH), although they produce antiviral antibodies (ie, anterior chamber-associated immune deviation), we sought to determine whether a similar inverse correlation exists for patients with varicella-zoster virus (VZV)-induced ARN. DESIGN: Patients with acute, VZV-induced ARN and age-matched control subjects were skin tested with VZV and purified protein derivative antigens to evaluate DH. Varicella-zoster virus-induced ARN was diagnosed using polymerase chain reaction and intraocular antibody quotient. Serum samples were collected and analyzed for anti-VZV and anti-herpes simplex virus antibody titers. Acute retinal necrosis activity was assessed clinically, and DH skin tests were repeated 3 months after onset when ocular recovery had taken place. RESULTS: Whereas controls displayed intense DH when tested with VZV and purified protein derivative antigens, a subset of patients with ARN displayed absent VZV-specific DH (although their purified protein derivative responses were normal). Patients with the most severe ARN had the lowest DH responses to VZV antigens. Serum anti-VZV antibody titers were higher in patients with ARN than in controls, and antiviral titer correlated inversely with the intensity of anti-VZV DH responses. Varicella-zoster virus-specific DH responses were restored in patients who recovered from ARN. CONCLUSION: Varicella-zoster virus-ARN develops in a setting where DH reactivity to viral antigens is absent, implying that virus-specific DH might ameliorate the severity of ARN. CLINICAL RELEVANCE: Linking virus-specific DH to vulnerability to ARN in individuals infected with VZV might reveal an underappreciated pathogenic mechanism.

Hapten-specific tolerance induced by acute, low-dose ultraviolet B radiation of skin requires mast cell degranulation.

The deleterious effects of ultraviolet B radiation (UVR) on cutaneous immunity are mediated in part by cytokines released from cutaneous cells following radiation exposure. On the one hand, TNF-alpha has been advocated as the primary mediator of failed contact hypersensitivity induction, and, on the other hand, IL-10 has been held responsible for tolerance. While keratinocytes exposed to UVR have been found to produce both TNF-alpha and IL-10, there is reason to question whether these major cellular constituents of the epidermis are the relevant source of immunomodulatory cytokines after UVR. Dermal mast cells also produce TNF-alpha and IL-10, and we have recently reported that mast cell-derived TNF-alpha is required for UVR-induced impairment of CH induction. In this study, we have examined whether mast cells are also a relevant source of IL-10 in UVR-dependent tolerance. We found that (a) UVR fails to induce tolerance in mast cell-deficient mice, and (b) that tolerance occurs if mast cells are triggered to degranulate after ligation of the IgE receptor. Both types of tolerance were neutralized with anti-IL-10 antibodies, are hapten specific, and are associated with regulatory lymphoid cells. We conclude that mast cells are required in UVR-induced tolerance and may be one of the major sources of IL-10 that mediates the tolerance induced by acute, low-dose UVR.

Dynamics of donor cell persistence and recipient cell replacement in orthotopic corneal allografts in mice.

PURPOSE: To determine the extent to which donor cells persist and recipient cells repopulate each of the three cell layers of orthotopic corneal grafts in mice. METHODS: BALB/c, C57BL/6, and enhanced green fluorescence protein (EGFP) transgenic mice (B6 background) were used as donors and recipients for orthotopic syngeneic and allogeneic corneal grafts. Graft-bearing eyes were harvested at 5, 10, 15, 28, and 56 days, stained with propidium iodide, and observed (layer by layer) by confocal microscopy. Bone marrow-derived cells in the grafts were assessed immunohistochemically. RESULTS: Donor epithelium was totally replaced by recipient epithelial cells within 15 days in both syngeneic and allogeneic grafts, whereas donor stromal keratocytes and endothelial cells were retained virtually intact in syngeneic grafts and in accepted allografts. In rejected allografts, neither donor-derived keratocytes nor endothelial cells were detected, and, instead, recipient-derived stromal fibroblasts, neovessels, and infiltrating leukocytes were heavily represented. The posterior surface of rejected grafts was devoid of corneal endothelium and was covered incompletely with bone marrow-derived cells of recipient origin. CONCLUSIONS: Whereas in mice graft-derived epithelium is largely irrelevant to corneal allograft outcome, persistence of donor-derived endothelium and keratocytes correlates perfectly with graft acceptance. Recipient endothelium is incapable of covering the posterior surface of accepted or rejected corneal grafts, whereas bone marrow-derived cells of recipient origin come to occupy this site in rejected grafts.

Role of recipient epithelium in promoting survival of orthotopic corneal allografts in mice.

PURPOSE: To determine whether epithelium-deprived corneal allografts covered with syngeneic epithelium display immune privilege in orthotopic transplantation and whether syngeneic epithelium containing antigen-presenting cells nullifies this effect. METHODS: Epithelium-deprived allogeneic corneas (C57BL/6) and epithelium-deprived allogeneic corneas reconstituted with syngeneic (BALB/c) epithelium (containing or deprived of Langerhans' cells) were transplanted orthotopically into normal eyes of BALB/c mice. Graft survival was assessed clinically and evaluated histologically. RESULTS: Epithelium-deprived corneal grafts survived in syngeneic recipients but were swiftly rejected in allogeneic recipients. These allografts incited intense stromal inflammation and neovascularization. Epithelium-deprived allografts that were resurfaced in vivo by syngeneic epithelium derived from immune-incompetent SCID mice also underwent intense acute rejection when placed in normal eyes of BALB/c mice. The epithelium of in vivo resurfaced grafts was replete with Langerhans' cells. By contrast, most of the epithelium-deprived allografts reconstituted in vitro with fresh, normal BALB/c corneal epithelium survived indefinitely when placed in eyes of BALB/c mice. Similar grafts reconstituted with BALB/c epithelium containing Langerhans' cells were swiftly rejected. CONCLUSIONS: Replacement of donor epithelium with Langerhans' cell-deficient syngeneic epithelium protects orthotopic allogeneic cornea grafts (stroma plus endothelium) from immune-mediated rejection. The presence of an intact, histocompatible layer of corneal epithelium has two important effects on orthotopic corneal allografts: It suppresses nonspecific inflammation and neovascularization within the graft, and it blunts the alloimmunogenicity of the histoincompatible stroma and endothelium.

Cytotoxic T cells play no essential role in acute rejection of orthotopic corneal allografts in mice.

PURPOSE: To determine whether cytotoxic T cells of the direct alloreactive type are activated and responsible for early, acute failure of orthotopic corneal allografts observed in eyes of C57BL/6 but not of BALB/c mice. METHODS: Corneas from BALB/c and BALB.B mice were placed orthotopically in eyes of C57BL/6 and beta-2 microglobulin knockout mice (deficient in CD8(+) cytotoxic T cells). Graft fates were assessed clinically, and the T lymphocytes of recipients were assayed for the capacity to lyse target cells bearing donor major (MHC) and/or minor histocompatibility (minor H) antigens (direct and indirect pathways, respectively). RESULTS. Similar to BALB/c recipients, C57BL/6 mice with rejected cornea allografts acquired donor minor H-specific T cells. Unlike BALB/c recipients, C57BL/6 mice-both rejectors and acceptors-acquired donor MHC-specific T cells. beta-2 Microglobulin knockout mice showed rejection of corneal allografts in a manner indistinguishable from C57BL/6 mice, including early, acute rejection, yet T cells from beta-2 microglobulin knockout recipients of corneal allografts displayed no cytotoxic T cells specific for either donor MHC or minor H alloantigens. CONCLUSIONS: Although C57BL/6 mice acquired donor MHC-specific cytotoxic T cells (direct alloreactive cells), neither these cells nor donor minor H-specific cytotoxic T cells (indirect alloreactive cells) play any essential role in corneal allograft rejection, including the early acute failure uniquely observed in C57BL/6 eyes.

Polymorphisms in the tumor necrosis factor (TNF) genes are associated with susceptibility to effects of ultraviolet-B radiation on induction of contact hypersensitivity.

We investigated the allelic distributions of single nucleotide polymorphisms (SNPs) of the TNFA, TNFB and IKBL genes, 3 microsatellites within the tumor necrosis factor (TNF) region of HLA locus, and the HLA phenotypes as well as the TLR4 gene in Chromosome 9 in 26 healthy Caucasian volunteers. These individuals were also assessed as ultraviolet B (UVB)-susceptible (S) or UVB-resistant (R). Our results identified 12 UVB-S and 14 UVB-R individuals. Attempts to correlate particular HLA-A, -B, -C, and -DR antigens with the UVB phenotypes failed. Similarly, attempts to correlate SNP at the NcoI-RFLP within intron 1 of the TNFB, IKBL and TLR4 gene with UVB phenotypes also failed. However, microsatellite analyses of TNFa, TNFc, and TNFd markers revealed a significant increase in the frequencies of TNFa2 in UVB-S individuals (P=0.00032) and of TNFd3 in UVB-R individuals (P=0.012). Moreover, DNA sequencing analyses of 5 SNPs of the TNFA promoter region revealed a significant increase in the frequency of the allele B of the TNFA gene (TNFApB) representing the nucleotide A at position -863 and C at position -1031 (P=0.015). Since it is known that TNFa2 and TNFApB is a high TNF-alpha responder, whereas TNFd3 is a TNF-alpha low responder, we propose that the TNF region of HLA contains polymorphic genes that confer susceptibility and resistance to the deleterious effects of UVB radiation on the induction of contact hypersensitivity. This proposal is consistent with previous reports that a unique microsatellite region of the Tnfa gene in mice contains alleles that dictate the UVB-dependent phenotypes in mice, and implicate TNF-alpha as the primary mediator of the immune-damaging effects of UVB radiation.

Hapten-specific tolerance promoted by calcitonin gene-related peptide.

Calcitonin gene-related peptide has been shown to modulate inflammatory and immune responses in various systems. Recent studies in our laboratory and colleagues have shown that intracutaneously injected calcitonin gene-related peptide impairs the induction of contact hypersensitivity in mice, and participates in the pathogenesis of failed contact hypersensitivity induction after acute, low-dose ultraviolet B radiation. In this study we investigated the ability of calcitonin gene-related peptide to induce tolerance in normal and mast cell deficient mice and we examined the extent to which calcitonin gene-related peptide contributes to the tolerance induced by acute, low-dose ultraviolet B radiation. Calcitonin gene-related peptide was injected intradermally followed by application of 2,4-dinitro-1-fluorobenzene to the injected skin surface. Tolerance was assessed by re-exposing the mice 2 wk later to a second, sensitizing dose of 2, 4-dinitro-1-fluorobenzene on uninjected skin. We found that calcitonin gene-related peptide induced tolerance to 2, 4-dinitro-1-fluorobenzene in both normal and mast cell deficient mice. Calcitonin gene-related peptide-induced tolerance was blocked by intradermal injection of a calcitonin gene-related peptide antagonist [CGRP-(8-37)] that selectively blocks the calcitonin gene-related peptide receptor. Tolerance was also abolished by intraperitoneally injected anti-interleukin-10, but not anti-tumor necrosis factor alpha, antibodies. When 2,4-dinitro-1-fluorobenzene was painted on skin into which splenic dendritic cells pretreated with calcitonin gene-related peptide had been injected, tolerance was observed. Calcitonin gene-related peptide- treated dendritic cells mixed with anti-interleukin-10 antibody prior to intradermal injection failed to promote tolerance. Finally, injection of CGRP-(8-37) into skin that was subsequently exposed to acute, low-dose ultraviolet B radiation partially prevented tolerance induced by local application of 2,4-dinitro-1-fluorobenzene. These results indicate that calcitonin gene-related peptide has the capacity to promote cutaneous tolerance through an interleukin-10-dependent mechanism. This mechanism, which does not require the participation of mast cells, contributes to the tolerance promoted by acute, low-dose ultraviolet B radiation. Thus, calcitonin gene-related peptide from cutaneous nerve endings plays a key role in the local immune aberrations caused by ultraviolet B radiation.

Analysis of primed donor-specific T cells in recipient mice bearing orthotopic corneal allografts.

BACKGROUND: Orthotopic corneal allografts placed in normal eyes of mice are often not rejected, whereas grafts placed in high-risk (neovascularized) eyes are routinely destroyed. Because rejection of solid tissue allografts is usually mediated by donor-specific T cells, we wished to determine the extent to which donor-specific T cells become primed in mice bearing orthotopic corneal allografts in normal and "high-risk" eyes. METHODS AND RESULTS: Our data indicate corneal allografts placed in neovascularized eyes were rejected within 2 weeks, and lymph nodes draining these grafts contained primed donor-specific T cells that proliferated in vitro and displayed cytotoxic activity. By contrast, only 50% of corneal allografts placed in normal eyes experienced rejection. Lymphoid cells from all of these mice displayed donor-specific proliferative activity, irrespective of whether the graft was accepted or rejected. At no time were donor-specific cytotoxic T cells detected. Failure to detect primed cytotoxic T cells was not the result of anergy or deletion of unprimed donor-specific precursors of CTL. CONCLUSIONS: We conclude that primed donor-specific proliferative and cytotoxic T cells directed at MHC alloantigens correlate well with rejection of orthotopic corneal allografts in neovascularized high-risk eyes. However, rejection of cornea allografts in normal eyes does not correlate well with proliferative T cells, nor are donor MHC-specific cytotoxic T cells detected. The possibility is discussed that graft rejection in normal eyes is not mediated by T cells that recognize MHC alloantigens via the direct pathway, but via T cells that recognize donor alloantigens presented by recipient MHC molecules (indirect pathway).

Interleukin-1 receptor antagonist therapy and induction of anterior chamber-associated immune deviation-type tolerance after corneal transplantation.

PURPOSE: Topical treatment with interleukin 1 receptor antagonist (IL-1ra) can promote corneal allograft survival by suppressing induction of allodestructive immunity. The purpose of these experiments was to determine whether IL-1ra could also promote induction of allo-protective tolerogenic pathways, including anterior chamber-associated immune deviation (ACAID), which has been shown to participate in long-term survival of corneal transplants. METHODS: Corneal buttons from BALB/c (syngeneic) or C57BL/6 (fully mismatched allogeneic) mice were orthotopically grafted onto BALB/c recipients. Topical IL-1ra or vehicle alone was applied to grafts three times daily. Donor-specific ACAID was measured in allogeneic grafted mice at 4 and 8 weeks after transplantation by ear-challenging grafted hosts with donor-derived splenocytes 1 week after SC immunization. In separate experiments, grafted mice were treated for 4 weeks before injecting ovalbumin (OVA) into their anterior chambers to determine their capacity to induce antigen-specific ACAID. RESULTS: Treatment with IL-1ra did not promote, or inhibit, induction of donor-specific ACAID compared with vehicle-treated controls at either the early or late time points studied. However, IL-1ra treatment after transplantation led to significantly earlier restoration of the grafted eyes' capacity for inducing ACAID to soluble antigen (OVA). CONCLUSIONS: Promotion of OVA-specific ACAID by IL-1ra suggests that suppression of IL-1-mediated mechanisms contributes to recovery of the anterior segment's immunosuppressive microenvironment at least 1 month earlier than would otherwise be seen after corneal transplantation. However, IL-1ra treatment does not alter induction of donor-specific ACAID after transplantation, suggesting that its anti-inflammatory activities do not lead to an ACAID-inducing signal per se. This suggests that IL-1ra promotes graft survival almost exclusively by virtue of suppressing inflammation and not by directly promoting tolerance or antigen-specific regulatory pathways.

Participation of pigment epithelium of iris and ciliary body in ocular immune privilege. 2. Generation of TGF-beta-producing regulatory T cells.

PURPOSE: To determine whether T cells exposed to cultured iris and ciliary body pigment epithelial (I/CB PE) cells acquire the capacity to modify the activation, differentiation, and effector functions of bystander T cells, and if so, to identify the mechanism. METHODS: T cells from naive BALB/c mice were cultured with I/CB PE cells, x-irradiated, and used as regulators (a) of T-cell activation in vitro and (b) of delayed hypersensitivity expression in vivo. Neutralizing anti-TGF-beta and -IL-10 antibodies were used to abolish regulatory function. T-cell activation was assessed for proliferation by [(3)H]thymidine incorporation and for IL-2, IFN-gamma, IL-4, and IL-10 production by semi-quantitative RT-PCR for mRNA and by supernatant analysis by ELISA. I/CB PE-exposed T cells were evaluated for mRNA content of IFN-gamma, IL-4, TNF-alpha, TGF-beta1, TGF-beta2, and IL-10, and their supernatants were analyzed for content of TGF-beta. RESULTS: T cells exposed to I/CB PE cells inhibited anti-CD3-driven activation of bystander naive T cells in vitro and suppressed the expression of delayed hypersensitivity in vivo. Bystander T cells cocultured with I/CB PE-exposed T cells failed to proliferate and secreted high levels of IL-4 and IL-10 but low amounts of IL-2 and IFN-gamma. Regulation of bystander T-cell activation was mediated via enhanced secretion of TGF-beta by I/CB PE-exposed T cells. CONCLUSIONS: T cells exposed to cultured I/CB PE cells were induced to secrete active and latent TGF-beta, which conferred on the T cells the capacity to inhibit the differentiation as well as the effector function of Th1-type cells.

Evidence that retinal pigment epithelium functions as an immune-privileged tissue.

PURPOSE: Tissues derived from immune-privileged sites sometimes possess special characteristics that promote their own survival when transplanted to a nonprivileged site. This study was undertaken to evaluate whether retinal pigment epithelium (RPE) behaves as an immune-privileged tissue when transplanted extraocularly. METHODS: RPE grafts were prepared from eyes of neonatal C57BL/6 or C57BL/6 gld/gld (deficient in CD95 ligand expression) mice. These grafts (or conjunctival grafts as positive controls) were transplanted into the anterior chamber, the subretinal space, the subconjunctival space, and underneath the kidney capsule of histoincompatible BALB/c mice. Transplant survival was evaluated by histology at selected time points after engraftment. Recipients were tested for acquisition of C57BL/6-specific delayed-type hypersensitivity (DH) and for the ability to suppress DH. RESULTS: Allogeneic neonatal RPE grafts from normal donors showed significantly enhanced survival at all graft sites compared with conjunctival grafts. However, allogeneic RPE cell grafts from gld/gld mice were rapidly rejected after transplantation beneath the kidney capsule. Allogeneic RPE grafts placed in extraocular sites induced systemic DH directed at donor alloantigens, whereas RPE allografts placed intraocularly induced suppression of systemic DH. CONCLUSIONS: Allogeneic neonatal RPE grafts, through constitutive expression of CD95 ligand, promote their own survival at heterotopic sites. Paradoxically, these grafts also display immunogenicity. Thus, neonatal RPE tissue owes its immune privilege to the capacity to prevent immune rejection rather than to inhibit sensitization.

Immune privilege and immunogenicity reside among different layers of the mouse cornea.

PURPOSE: To determine the extent to which each layer of the mouse cornea displays alloimmunogenicity or immune privilege. METHODS: Intact corneas or individual or combined layers of corneas from normal or cauterized eyes of BALB/c, C57BL/6, and CD95L-deficient B6-gld mice were grafted beneath the kidney capsule of normal BALB/c, B10.D2, BALB.B mice or of BALB/c mice presensitized to donor antigens. Graft fate was assessed clinically and histologically and acquisition of donor-specific delayed hypersensitivity (DH) was assessed at selected intervals after grafting. RESULTS: Full-thickness allogeneic corneas induced vigorous DH and were rejected acutely. Similar results were obtained with allografts of corneal epithelium alone (if supported by syngeneic viable stroma), allografts of epithelium from cauterized corneas (containing Langerhans' cells), and stromal allografts deprived of endothelium. Grafts comprised of stroma plus endothelium (without epithelium) were not rejected, nor did they induce DH unless the graft had no CD95L expression. If stroma-endothelium grafts had no CD95L expression, DH directed against major histocompatibility complex (MHC), but not minor histocompatibility, alloantigens was induced. Moreover, CD95L expressed on stroma-endothelium grafts protected endothelial cells, but not stromal cells, from rejection in presensitized recipients. CONCLUSIONS: When grafted to a heterotopic site, the alloimmunogenicity of the normal cornea resides within its epithelial and stromal layers, whereas immune privilege arises from the endothelium. In normal mice, CD95L-expressing endothelium can inhibit the stroma from inducing immunity directed at MHC alloantigens, but in presensitized mice the endothelium can protect itself only from immune rejection.

Haptoglobin is a natural regulator of Langerhans cell function in the skin.

Langerhans cells (LC), the best-understood antigen presenting cells (APC) of the skin, are functionally plastic. Freshly obtained LC readily activate allogeneic T cells, but are incapable of activating autologous, naive T cells. When placed in culture in the presence of GM-CSF, LC up-regulate surface expression of class I and II MHC molecules along with co-stimulatory molecules, such as B7, CD40 and IL-12. This functional transformation enables the cells to activate naive, autologous T cells in vitro. It is paradoxical that intracutaneous administration of exogenous GM-CSF fails to induce intraepidermal LC to undergo functional transformation in situ. It has been reported that serum contains a factor that prevents fresh LC from undergoing functional transformation in culture, and the relevant serum factor has now been identified as haptoglobin (Hp), based on the following experimental results: (a) SDS-PAGE, amino acid sequencing, and mass spectrometric analyses of the inhibitory factor purified by high performance liquid chromatography (HPLC) from normal human serum revealed molecules completely homologous to Hp alpha-1 chain; (b) pure human Hp, but not serum depleted of Hp, inhibited fresh LC from acquiring the capacity to activate autologous T cells in vitro; (c) abundant Hp was detected in cytoplasmic compartments of fresh, but not cultured, LC. It was concluded that Hp, an acute phase protein, is a systemically-derived factor that prevents epidermal LC from spontaneously undergoing functional maturation in the skin. This novel property of Hp may be important in ameliorating or preventing certain T cell-dependent inflammatory skin diseases.

Suprathreshold doses of hapten are required to induce both contact hypersensitivity and tolerance.

Whereas both high (conventional) and low (optimal) doses of epicutaneously applied hapten induce contact hypersensitivity in normal mice, only conventional doses retain their capacity to induce contact hypersensitivity after acute, low dose ultraviolet B radiation in UVB-resistant mice. Recent evidence indicates that conventional doses of hapten as well as acute, low dose ultraviolet B radiation destroy virtually all epidermal Langerhans cells, which leads to the conclusions that (a) dermal antigen presenting cells have a prominent role to play in contact hypersensitivity induction, and that (b) Langerhans cell provide this function only in normal skin, and only if non-toxic amounts of hapten are present. We have now explored the ability of suprathreshold, threshold, and sub-threshold doses of hapten to induce tolerance when painted on or injected into normal skin or skin exposed to ultraviolet B radiation. Our results indicate that a single exposure of low dose, ultraviolet B radiation generated tolerance-promoting signals within the epidermis when a threshold dose of hapten was painted on the exposed site. By contrast, suprathreshold doses of hapten painted on skin after four consecutive daily doses of ultraviolet B radiation led to tolerance that arose exclusively from cells within the dermis. In absence of ultraviolet B radiation, epicutaneously applied hapten failed uniformly to induce tolerance, whether applied at suprathreshold, threshold or sub-threshold doses. We conclude that normal skin lacks cells with inherent tolerance-promoting capacity, but that cells of this type can emerge within either epidermis or dermis after exposure to acute, low dose ultraviolet B radiation.