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Purpose: To investigate the effects of anterior chamber pigment dispersion on ocular immune privilege and the possible mechanisms involved in a DBA/2J mouse model of pigmentary glaucoma., Methods: DBA/2J mice were utilized as a pigment dispersion model, and age-matched C57BL/6J mice were used as the control group in this study. Proteins in the aqueous humor (AH) and serum were quantified using the bicinchoninic acid assay. Immune cells in the AH were detected using hematoxylin and eosin staining and immunocytochemistry. The expression of TGF-β2 in the AH and cytokine levels (IL-10, IFN-γ) in serum were measured using ELISA. Anterior chamber-associated immune deviation (ACAID) was induced in DBA/2J mice by injecting antigens into the anterior chamber. Delayed-type hypersensitivity (DTH) assays were used to assess the induction of ACAID. In DBA/2J mice, before and after pigment dispersion, following anterior chamber injection of pigment particles, and after ACAID modeling, the expression of regulatory T cells (Tregs) was detected using flow cytometry., Results: Compared to C57BL/6J mice, the protein concentration, immune cell count, and TGF-β2 levels in the AH were elevated in DBA/2J mice. Protein concentration and IL-10 levels in serum were increased, while IFN-γ levels were decreased in DBA/2J. Additionally, the expression of Treg cells in the spleen of DBA/2J mice was significantly increased after pigment dispersion and anterior chamber injection of pigment particles. At 3 and 6 months, DTH responses in DBA/2J mice were not inhibited, thus preventing ACAID induction. However, the opposite was observed at 9 months in DBA/2J mice. Furthermore, the ACAID group exhibited an augmented expression of Treg cells., Conclusions: Dispersion of pigment particles in the anterior chamber of the eye enhances the state of ocular immune privilege by influencing the immunosuppressive microenvironment and inducing more Treg cells to reestablish ACAID.

Purpose: Severing corneal nerves during orthotopic corneal transplantation elicits the elaboration of the neuropeptide substance P (SP), which induces the generation of CD11c+ contrasuppressor (CS) cells. CS cells disable T regulatory cells (Tregs) that are induced when antigens enter the anterior chamber (AC), either by direct injection or by orthotopic corneal transplantation. This study examined the crucial cell surface molecules on Tregs that are adversely affected by CS cells that are generated by severing corneal nerves., Methods: CS cells were induced by producing shallow 2.0-mm circular incisions in the corneal epithelium in BALB/c mice. CD8+ Tregs were generated by injecting ovalbumin into the AC. The effects of CS cells and SP on the expression and function of two cell surface molecules (CD103 and the receptor of interferon-γ) that are crucial for the induction and function of CD8+ Tregs were analyzed., Results: SP converted CD11c+, but not CD11c- , dendritic cells (DCs) to CS cells. Severing corneal nerves resulted in a 66% reduction in the expression of CD103 on CD8+ AC-associated immune deviation (ACAID) Tregs, and a 50% reduction in the interferon-γ receptor (IFN-γR). These effects could be mimicked in vitro by coculturing CS cells with CD8+ ACAID Tregs., Conclusions: The elaboration of SP in response to corneal nerve ablation converts CD11c+ DCs to CS cells. CS cells disable CD8+ ACAID Tregs by downregulating two crucial cell surface molecules, CD103 and IFN-γR, by an SP-dependent pathway. Blocking this pathway may provide a means of restoring ocular immune privilege in corneas subjected to corneal nerve injury.

Purpose: Severing of corneal nerves in preparation of corneal transplantation abolishes immune privilege of subsequent corneal transplants placed into either eye: a phenomenon termed sympathetic loss of immune privilege (SLIP). SLIP is due to the disabling of T regulatory cells (Tregs) by CD11c+ contrasuppressor (CS) cells. This study characterized the induction, function, and manipulation of CS cell activity and the effect of these cells on Tregs induced by anterior chamber-associated immune deviation (ACAID)., Methods: CS cells were induced using a 2.0-mm trephine to score the corneal epithelium. CD11c+ CS cells were evaluated by adoptive transfer and by their capacity to disable CD8+ ACAID Tregs in local adoptive transfer (LAT) of suppression assays. CD11c+ cells were deleted from the ocular surface by subconjunctival injection of clodronate-containing liposomes., Results: CD11c+ CS cell were radiosenstive and long lived. As few as 1000 CS cells blocked the suppressive activity of previously generated CD8+ ACAID Tregs, indicating that CS cells act at the efferent arm of the immune response. Depletion of resident CD11c+ cells at the ocular surface prevented the generation of CS cells., Conclusions: Corneal nerve injury that occurs during keratoplasty converts ocular surface CD11c+ cells into CS cells that block CD8+ Tregs, which are induced by introducing antigens into the anterior chamber (i.e., ACAID Tregs). Depletion of CD11c+ cells at the ocular surface prevents the generation of CS cells and may be a useful strategy for preventing SLIP and enhancing the survival of second corneal transplants.

Purpose: Severing corneal nerves during corneal transplantation does not affect first corneal transplants, but abolishes immune privilege of subsequent corneal allografts. This abrogation of immune privilege is attributable to the disabling of T regulatory cells (T regs) induced by corneal transplantation. The goal of this study was to determine if severing corneal nerves induces the development of contrasuppressor (CS) cells, which disable T regs that impair other forms of immune tolerance., Methods: Effect of corneal nerve ablation on immune tolerance was assessed in four forms of immune tolerance: anterior chamber-associated immune deviation (ACAID); oral tolerance; corneal transplantation, and intravenously (IV) induced immune tolerance. T regulatory cell activity was assessed by adoptive transfer and by local adoptive transfer (LAT) of suppression assays., Results: Corneal nerve ablation prevented ACAID and oral tolerance, but did not affect IV-induced immune tolerance. Contrasuppressor cells blocked the action of T regs that were generated by anterior chamber injection, oral tolerance, or orthotopic corneal transplantation. The neuropeptide substance P (SP) was crucial for contrasuppressor activity as CS cells could not be induced in SP-/- mice and the SP receptor inhibitor, Spantide II, prevented the expression of CS cell activity in vivo. Contrasuppressor cells expressed CD11c surface marker that identifies dendritic cells (DC)., Conclusions: The loss of immune privilege produced by corneal nerve ablation following corneal transplantation extends beyond the eye and also affects immune tolerance induced through mucosal surfaces and appears to be mediated by a novel cell population of CD11c+ CS cells that disables T regs.

Purpose: We evaluated the allogeneic response after corneal endothelial cell transplantation in the anterior chamber (AC) in a new mouse model by examining the acquisition of a delayed-type hypersensitivity (DTH) response, induction of allogeneic AC-associated immune deviation (ACAID), and acquisition of delayed transplantation tolerance., Method: The corneal eyecups from C57BL/6 mice were prepared. The epithelial layer was detached with EDTA solution and treated with trypsin to release mouse-derived primary corneal endothelial cells (mpCECs). The mpCECs (1 × 104 cells) were transplanted into the AC of the eye or subcutaneously (SC) into the neck of BALB/c mice. In the mouse model of endothelial cell transplantation, the endothelial cells in a 2-mm central area of the cornea were eliminated by cryoinjury. The mpCEC transplant model was evaluated by measuring allogeneic cell survival and corneal thickness. The allospecific DTH response and ACAID induction were evaluated 1 week after transplantation. The long-term transplantation tolerance was evaluated by observing a secondary penetrating keratoplasty (PKP) performed on the same donor C57BL/6 mice., Results: The SC injection of mpCECs induced a DTH response, whereas the AC injection induced ACAID. However, eyes inflamed by cryoinjury showed neither the DTH response nor ACAID following AC injection. The mpCECs survived for at least 1 week after injection. Penetrating keratoplasty allografts at 8 weeks after mpCEC transplantation survived indefinitely (100%)., Conclusions: The mpCECs display low allogenicity in the AC and are capable of inducing allogeneic tolerance. Corneal endothelial cell transplantation into the AC may represent a safe technique for allogeneic transplantation.

Purpose: Immune privilege of the eye protects the nonregenerative ocular tissues from innate and adaptive immune-mediated inflammation. In the case of intraocular tumors, immune privilege can be arrested to allow for immune-mediated rejection. Activation of innate immune cells can contribute to necrosis of the intraocular tumor and bystander ocular tissue. Identifying the cellular components of the innate immune system that contribute to ocular destruction, but are not needed for tumor rejection, provides insights into the immunopathological sequelae in intraocular tumor rejection., Methods: Wild-type (WT), Jα18 knockout (KO) mice lacking type I natural killer T (NKT) cells, and CD1d KO mice lacking all NKT cells, were used to identify the role of type II NKT cells in intraocular tumor rejection immunopathology., Results: CD1d KO mice had significantly lowered rates of necrotic eye destruction during tumor rejection compared to WT or Jα18 KO mice. Transcriptome and protein analyses revealed that CD1d KO mice had significantly lower expression of CXCL3 compared to WT or Jα18 KO mice, and this was associated with decreased neutrophil recruitment. The presence of type II NKT cells in WT or Jα18 KO mice led to increased CXCL3, which attracted neutrophils to the intraocular tumor and culminated in destruction of the eye., Conclusions: We found that type II NKT cells are critical in initiating a damaging inflammatory antitumor response involving the recruitment of neutrophils that compromises the integrity of the eye. Loss of type II NKT cells or depleting neutrophils allows for a productive intraocular tumor response that converts the rejection phenotype to preserve the eye.

Purpose: After uniocular anterior chamber (AC) injection of HSV-1, the anterior segment of BALB/c mice becomes inflamed and infected; however, virus does not spread from the anterior segment to cause retinitis in the injected eye. The purpose of these studies was to determine whether interferon (IFN-)-γ and Mac-1(+) cells play a role in preventing direct anterior-to-posterior spread of HSV-1 in the injected eye., Methods: One AC of adult female BALB/c mice was injected with HSV-1 (KOS). The location of IFN-α, IFN-β, and IFN-γ in the injected eye was determined by immunofluorescence, and mRNA expression was quantified by qPCR. Injected eyes of IFN-γ knockout or clodronate-treated macrophage-depleted mice were examined to determine whether the absence of IFN-γ or Mac-1(+) macrophages affected the sites or timing of virus spread., Results: IFN-α, IFN-β, and IFN-γ were observed in the anterior segment of injected eyes through 72 hours and mRNA levels of IFN-β and IFN-γ were increased in virus-infected eyes 48 to 120 hours after infection. However, the absence of IFN-γ or macrophages did not affect either the sites or the timing of HSV-1 infection in injected eyes., Conclusions: Protection of the retina of the injected eye does not depend on a single cell type or cytokine. In addition, in the eye, as in other sites of the body, there are redundancies in the innate response to virus infection.

Purpose: To compare and contrast the T regulatory cells (Tregs) induced by anterior chamber (AC) injection of antigen with those induced by orthotopic corneal allografts., Methods: Anterior chamber-associated immune deviation (ACAID) Tregs were induced by injecting C57BL/6 spleen cells into the AC of BALB/c mice. Delayed-type hypersensitivity responses to C57BL/6 alloantigens were evaluated by a conventional ear swelling assay. Corneal allograft Tregs were induced by applying orthotopic C57BL/6 corneal allografts onto BALB/c hosts. The effects of anti-CD25, anti-CD8, anti-interferon-γ (IFN-γ), anti-IL-17A, or cyclophosphamide treatments on corneal allograft survival and ACAID were evaluated., Results: Administration of either anti-CD25 or anti-IFN-γ antibodies prevented the expression of ACAID and abolished the immune privilege of corneal allografts. By contrast, in vivo treatment with anti-CD8 antibody abrogated ACAID but had no effect on corneal allograft survival. Further discordance between ACAID and corneal allograft survival emerged in experiments in which the induction of allergic conjunctivitis or the administration of anti-IL-17A abolished the immune privilege of corneal allografts but had no effect on the induction or expression of ACAID., Conclusions: Although orthotopic corneal allografts are strategically located for the induction of ACAID by the sloughing of corneal cells into the AC, the results reported here indicate that the Tregs induced by orthotopic corneal allografts are remarkably different from the Tregs that are induced by AC injection of alloantigen. Although both of these Treg populations promote corneal allograft survival, they display distinctly different phenotypes.

Purpose: The pathway between the glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR) and GITR ligand (GITRL) has been shown to control the function of regulatory T cells (Treg). The present study was conducted to investigate the role of this pathway and Treg in establishing immune privilege status for corneal allografts., Methods: Corneas of C57BL/6 mice were orthotopically transplanted into the eyes of BALB/c mice, and graft survival was assessed. A separate set of BALB/c mice received an anterior chamber injection of C57BL/6 splenocytes, and induction of allo-specific anterior chamber-associated immune deviation was assessed. Recipients were intraperitoneally administrated anti-GITRL, anti-CD25 monoclonal antibodies (mAb), or control immunoglobulin (IgG). Expressions of GITRL, GITR, and Foxp3 in the allografts were assessed. In vitro, cornea pretreated with anti-GITRL mAb or control IgG was incubated with T cells, and destruction of corneal endothelial cells and the population of Foxp3(+)CD25(+)CD4(+) T cells were assessed., Results: GITRL was expressed constitutively in the cornea and iris-ciliary body. GITRL-expressing allografts were infiltrated with Foxp3+GITR+CD25+CD4(+) T cells. Blockade of GITRL did not affect allo-specific ACAID but led to infiltration of Foxp3(-)CD4(+) T cells and allograft rejection. Depletion of CD25+CD4(+) Treg also accelerated allograft rejection. Destruction of corneal endothelial cells by T cells was significantly enhanced in GITRL-blocked cornea compared with control cornea. Foxp3+CD25+CD4(+) T cells were increased after incubation with GITRL-expressing cornea, but not with GITRL-blocked cornea., Conclusions: Presence of Foxp3+CD25+CD4(+) Treg in the allograft is necessary for allograft survival. GITRL-dependent expansion of Treg within the cornea is one mechanism underlying immune privilege in corneal allografts.

Purpose: Intracameral (anterior chamber) injection of antigen inhibits the development of delayed-type hypersensitivity, a phenomenon known as anterior chamber-associated immune deviation (ACAID). The authors investigated the effect of intracameral injection of interphotoreceptor retinoid-binding protein (IRPB) peptides on the development of IFN-gamma(+) and IL-17(+) pathogenic T cells., Methods: A uveitogenic (IRBP1-20) or nonuveitogenic (IRBP161-180) peptide was injected into the anterior chamber (AC) of B6 mice. Seven days later, the mice were primed with a pathogenic dose of IRBP1-20 in adjuvant. Thirteen days later, the pathogenic activity of the T cells isolated from the spleens of treated and untreated mice were compared, and the numbers of Th1 and Th17 T cells were assessed by intracellular staining. Regulatory T-cell activity was assessed by antibody staining and functional assays. The authors also compared the effect of inhibition on EAU of ocular injection to various sites, including the AC, the vitreous cavity, and the subretinal space., Results: Intraocular injection of the uveitogenic peptide (IRBP1-20), but not the nonuveitogenic peptide (IRBP161-180), inhibited the generation of IFN-gamma(+) and IL-17(+) uveitogenic T cells and the development of experimental autoimmune uveitis (EAU). AC administration of IRBP1-20, but not IRBP161-180, significantly decreased the number of circulating gammadelta T cells after subsequent systemic immunization with IRBP1-20. Absence of the gammadelta T-cell population prohibited the development of ACAID., Conclusions: Injection of a uveitogenic peptide into the AC inhibited the development of EAU by regulation of Th1 and Th17 IRBP-specific T cells. The circulating gammadelta T-cell population was reduced and was associated with decreased activation of IL-17(+) uveitogenic T cells.

Purpose: To study the expression and functional characteristics of programmed death-1 (PD-1) and its ligands in the spleens of mice undergoing anterior chamber-associated immune deviation (ACAID)., Methods: ACAID was induced in BALB/c mice by intracameral injection of ovalbumin (OVA). The expression of PD-1 and its ligands in the spleens of ACAID mice was determined by quantitative real-time PCR, Western blotting, and flow cytometry. In vitro proliferation assays, enzyme-linked immunosorbent assays, and adoptive transfer assays were used to investigate the functional characteristics of splenic CD4+PD-1+ T cells of ACAID mice., Results: Both mRNA and protein of PD-1, PD-L1, and PD-L2 were markedly upregulated in the spleens of ACAID mice compared with controls. CD4+PD-1+ T cells from ACAID mice produced large amounts of IL-10 and exhibited in vitro antigen-specific suppressive activity. CD4+PD-1+ T cells from ACAID mice were able to significantly inhibit the antigen-specific, delayed-type hypersensitivity response when adoptively transferred to naive mice., Conclusions: CD4+PD-1+ T cells from ACAID mice, as regulatory cells, are involved in the induction of antigen-specific suppression in association with enhanced expression of IL-10. CD4+PD-1+ T cells in the murine spleen may represent a substantial population of regulatory T cells possibly responsible for the induction of ACAID after intracameral injection of antigen.

Purpose: To determine the immunogenic characterization of amniotic epithelium (AE), by examining the fate of allogeneic AE grafts heterotopically transplanted in the eye., Methods: Intact AE from enhanced green fluorescence protein (EGFP) transgenic mice (C57BL/6 background) and wild-type C57BL/6 mice were transplanted onto cornea or conjunctiva, or inserted into the anterior chamber (AC) of normal BALB/c mice, C57BL/6 mice, or BALB/c mice presensitized to donor antigens. For repeated AE transplantation experiments, AE was grafted in the other eye 7 days after the first grafting. Graft fate was assessed clinically and histologically at selected intervals after grafting. Infiltrating inflammatory cells were examined immunohistochemically. Sensitization to alloantigens by AE was assessed by the delayed hypersensitivity (DH) response., Results: In normal recipients, GFP+ cells were absent in EGFP donor-derived AE grafts by day 21 on cornea and by day 7 on conjunctiva. AE grafts implanted in the AC survived for >8 weeks. In presensitized recipients and recipients that underwent repeated AE implantation, graft survival was markedly shorter than in normal recipients. DH was induced at 2 weeks, but failed to be induced at 4 weeks after grafting on cornea or at 8 weeks after grafting on conjunctiva and in the AC of normal recipients., Conclusions: Fresh allogeneic AE expressed immunogenicity when placed on the ocular surface, although no memory of allospecific DH was acquired. Allogeneic AE is clearly vulnerable to immune rejection in specifically sensitized recipients.

Purpose: Regulatory CD4+ T cells (T regs) arise in the spleens of mice with anterior chamber-associated immune deviation (ACAID), an eye-derived tolerance evoked by injection of antigen into the ocular anterior chamber (AC). The current study was conducted to investigate the possibility that these T regs express CD25 and are derived from natural CD4+CD25+ T cells., Methods: Naïve T cells from DO11.10 mice were activated in vitro by ovalbumin (OVA)-pulsed, TGFbeta-treated antigen-presenting cells (APCs), and the expression of CD25 assayed by flow cytometry. OVA-specific ACAID T regs were obtained from the spleens of DO11.10 mice with ACAID to OVA. Immunomagnetic enrichment was used to sort out CD4+CD25+, and CD4+CD25- ACAID T cells before they were injected into OVA-immunized mice or examined for mRNA expression of the regulatory T-cell transcription factor Foxp3. In addition, before AC injection of OVA, systemic depletion of CD25+ T cells was performed with injections of anti-IL-2 receptor antibody into the mice., Results: OVA-specific T cells from DO11.10 mice expressed CD25 when exposed to OVA-pulsed, TGFbeta-treated APCs, even when the DO11.10 T cells were depleted of CD25+ cells before their in vitro stimulation. In addition, DH was suppressed in naïve mice that were injected with CD4+CD25+ or CD4+CD25- ACAID T cells. The CD4+CD25+, but not the CD4+CD25-, ACAID T regs expressed Foxp3. Finally, OVA induced ACAID in mice depleted of CD25+ cells., Conclusions: Some of the CD4+ T regs of ACAID arise from CD25- precursors, and the induction of ACAID is not dependent on the presence of natural CD4+CD25+ T regs.

Purpose: To determine the role of tumor necrosis factor receptors (TNFRs) in corneal allograft rejection., Methods: Corneal epithelial and endothelial cells were examined by flow cytometry for the expression of TNFRI and TNFRII and their susceptibility to TNF-alpha-induced apoptosis. Corneal allografts from normal and TNFRI and TNFRII knockout (KO) C57BL/6 mice were transplanted to BALB/c hosts, and the fate of the allografts was monitored. C57BL/6 spleen cells were injected into the anterior chamber (AC) of BALB/c mice to induce anterior chamber-associated immune deviation (ACAID) and promote corneal allograft survival. The presence of ACAID suppressor cells in corneal allograft recipients was tested using a local adoptive transfer (LAT) assay., Results: Murine corneal epithelial and endothelial cells expressed TNFRI and TNFRII and were susceptible to TNF-alpha-induced apoptosis, yet corneal allografts from either TNFRI or TNFRII donors did not enjoy a lower incidence of rejection or a prolongation in survival time compared to corneal allografts from normal C57BL/6 donors. Moreover, all 31 of the TNFRII KO corneal grafts were rejected by naïve BALB/c hosts. Rejection of TNFRII KO corneal grafts occurred even though suppressor cells developed in the hosts and inhibited the expression of delayed-type hypersensitivity to donor alloantigens., Conclusions: Expression of TNFRII on corneal cells conveys a degree of protection against immune rejection of corneal allografts by a mechanism that is independent of ACAID. Moreover, induction of ACAID before the application of TNFRII KO corneal allografts fails to improve survival and does not replace the TNFRII-dependent protective mechanism.

Purpose: To characterize the role of B cells in the induction of anterior chamber-associated immune deviation (ACAID)., Methods: An in vitro model of the ACAID spleen was used to recapitulate the events that occur when antigen is introduced into the anterior chamber of the eye and culminates in the appearance of antigen-specific, CD8(+) suppressor cells., Results: In vitro-generated suppressor cells mimicked those produced by anterior chamber injection of antigen, as shown by their antigen specificity, surface expression of CD8, and capacity to suppress DTH, which is mediated by previously immunized T cells. B cells were found to be necessary for suppressor cell development. The B cell receptor (BCR) was necessary for the induction of ACAID and conveyed antigen specificity to the suppressor T cells. Lysosomal acidification of internalized antigen was necessary for B cells to induce ACAID; however, transporter of antigen processing (TAP) was not required for the generation of ACAID., Conclusions: The results suggest that B cells use the BCR to capture and internalize antigen from ACAID-inducing macrophages. Lysosomal acidification of the captured antigen is essential for the processing of the ACAID antigen before TAP-independent presentation to suppressor cells.

Purpose: Delivery of antigen to the anterior chamber (AC) of the eye induces a systemic form of tolerance, referred to as anterior chamber-associated immune deviation (ACAID). ACAID is characterized by decreases in delayed-type hypersensitivity responses and complement-fixing antibodies on subsequent challenge with an immunogenic form of the antigen. The current study was designed to test whether priming of antigen-specific CD8(+) cytotoxic T-lymphocytes (CTLs) are inhibited by injection of soluble antigen into the AC and whether gammadelta T cells play a role in the inhibition of such responses., Methods: Antigen was administered through the AC to normal gammadelta T-cell-deficient or reconstituted gammadelta T-cell-deficient mice. Seven days after the AC injection, the mice were primed with antigen in adjuvant and 10 days later, their spleen cells were cultured for 5 to 7 days and the CTL responses measured., Results: CTL responses were inhibited by antigen delivered through the AC in normal but not gammadelta T-cell-deficient mice. Tolerance was reconstituted in delta-chain knockout mice by the adoptive transfer of gammadelta T cells from normal mice. Moreover, spleen cells and splenic gammadelta(+) T cells, but not gammadelta(-) T cells, from mice injected with antigen through the AC inhibited development of CTL responses when cultured together with primed effector T cells., Conclusions: These data show, for the first time, that administration of soluble antigen in the AC inhibits development of CD8(+) cytotoxic T-cell responses and that gammadelta T cells play a critical role in inhibition of CTL responses.

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.

Purpose: To determine the immunosuppressive status of aqueous humor (AqH) from mouse eyes afflicted with endotoxin-induced uveitis (EIU) and to identify the relevant cytokines responsible for immunomodulatory activity within EIU AqH., Methods: Bacterial lipopolysaccharide (LPS) was injected into hind footpads of C3H/HeN mice; and AqH, collected at 6, 12, 24, and 48 hours, was evaluated for content of transforming growth factor (TGF)-beta, tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6, and interferon (IFN)-gamma and capacity to suppress anti-CD3-driven T-cell proliferation. Cytokine mRNA expression in iris-ciliary body (I/CB) was analyzed by RNase protection assays., Results: During 6 to 24 hours after LPS injection, total TGF-beta levels in AqH increased even though the fluid lost its capacity to suppress T-cell activation. At this time, AqH contained high levels of IL-6, and I/CB contained high levels of IL-6 mRNA. When IL-6 was neutralized with specific antibodies, inflamed AqH reacquired its capacity to suppress T-cell activation, which correlated with high levels of TGF-beta. Coinjection of IL-6 plus antigen into the anterior chamber of the eye of normal mice prevented antigen-specific anterior chamber-associated immune deviation (ACAID)., Conclusions: LPS-induced intraocular inflammation is associated with local production of IL-6, which robs AqH of its immunosuppressive activity, perhaps by antagonizing TGF-beta. The fact that IL-6 antagonized ACAID induction in normal eyes suggests that strategies to suppress the intraocular synthesis of IL-6 may reduce inflammation and restore ocular immune privilege.

Purpose: To explore immunologic issues involved in orthotopic corneal xenotransplantation in a discordant combination using guinea pigs as donors and mice as recipients., Methods: Two-millimeter-diameter guinea pig corneal buttons were transplanted into 1.5-mm-diameter graft beds on mouse corneas using 12 interrupted sutures. Eyelids were maintained occluded with tarsorrhaphy except at the times of clinical inspection. Grafts were considered to be rejected when the pupil margin was not visible clearly through the graft by slit-lamp microscopy., Results: Guinea pig corneas protected from desiccation by persistent tarsorrhaphy survived indefinitely in the eyes of C.B-17SCID mice but were rejected acutely (but not hyperacutely) in eyes of normal BALB/c and C57BL/6 mice (median survival times, MST, 16 and 10 days, respectively). Graft survival was not extended in mice deficient in micro heavy chain or beta-2 microglobulin genes, slightly extended in mice deficient in the C3 gene (MST of 21 versus 17 days) and greatly extended in mice deficient in the CD4 gene (MST of 26 versus 9 days). Reconstitution of CD4 knock-out (KO) mice with CD4+ T cells promoted acute rejection of corneal xenografts., Conclusions: Hyperacute rejection does not occur in guinea pig corneal xenografts in mouse eyes, indicating that corneal xenografts are less vulnerable to this type of rejection than other solid tissue xenografts. CD4+ T cells are the primary mediators of acute graft rejection, although complement may contribute in a minor way. Neither antibodies nor CD8+ T cells participate in acute graft rejection. Because guinea pig cornea grafts in eyes of CD4KO mice are rejected in a delayed fashion, other innate and/or adaptive immune effectors must also be able to cause rejection of orthotopic corneal xenografts.

Purpose: When injected intravenously into naive mice, peritoneal exudate cells (PECs) incubated with ovalbumin (OVA) in the presence of transforming growth factor (TGF)-beta2 induce immune deviation similar to that evoked by injection of OVA into the anterior chamber of the eye. Intraocular antigen injection elicits two distinct populations of regulatory T cells that impair delayed hypersensitivity (DH) by two different mechanisms: a CD4+ T cell that suppresses the induction of DH (afferent) and a CD8+ T cell that inhibits DH expression. In an effort to understand the origin and mechanism of action of these regulatory cells, CD8+ T cells from OVA-specific T cell receptor (Tcr) transgenic mice (OT-1) were used., Methods: CD8+ T cells were harvested from Tcr transgenic OT-1 mice whose Tcr recognize an OVA peptide in the context of the class I major histocompatibility complex molecule Kb. These cells were stimulated in vitro with OVA-pulsed PECs exposed (or not) to TGF-beta2, then analyzed for their capacity to proliferate, to secrete various cytokines, to lyse OVA-expressing target cells, and to regulate bystander T cells in vitro and in vivo., Results: When OVA-pulsed PECs were used in vitro as stimulators, responding OT-1 T cells proliferated and preferentially secreted interferon (IFN)-gamma, interleukin (IL)-2, and tumor necrosis factor (TNF)-alpha, rather than IL-4 and IL-10. When the stimulator PECs were pretreated with TGF-beta2 and then pulsed with OVA, responding OT-1 T cells proliferated even more swiftly, but they secreted significantly less IFN-gamma, IL-2, and TNF-alpha, and no IL-4 or IL-10. OT-1 T cells, which constitutively display cytotoxicity toward OVA-expressing target cells, lost this activity when stimulated with OVA-pulsed, TGF-beta2-pretreated PECs. Moreover, OT-1 T cells stimulated in this manner displayed the capacity to inhibit proliferation of OVA-primed T cells exposed to OVA in vitro and to suppress in vivo the expression of OVA-triggered DH., Conclusions: OVA-pulsed PECs, pretreated with TGF-beta2, coerce naive OVA-specific CD8+ T cells to become efferent regulators of DH similar to the regulatory T cells evoked by intraocular injection of OVA.

Purpose: To determine whether the subretinal space can extend immune privilege to allogeneic tumor cell grafts that do not possess their own inherent immune privilege., Methods: P815 tumor cells were injected into the anterior chamber (AC), the subretinal (SR) space, or subconjunctivally in eyes of BALB/c (allogeneic), SCID (immune incompetent), normal DBA/2 (syngeneic), or DBA/2 mice presensitized with P815 cells transfected with interleukin-12 and B7.1. Tumor growth was observed clinically and histologically for up to 50 days. BALB/c recipients were tested for suppression of DBA/2-specific delayed hypersensitivity and concomitant immunity. The SR space of tumor-containing eyes was assessed for its capacity to support ovalbumin (OVA)-specific anterior chamber associated immune deviation (ACAID)., Results: P815 cells injected into the SR space of presensitized and normal DBA/2 and SCID mice grew progressively, resulting eventually in recipient death. Tumor cells injected into the SR space of eyes of BALB/c mice grew progressively until day 14, followed by tumor regression resulting in phthisis bulbi (14/35) or tumor elimination (19/35) with preserved ocular anatomy by day 35. Despite elimination of tumors from the SR space, BALB/c recipients exhibited DBA/2-specific ACAID and concomitant immunity. In addition, OVA injected into the SR space of eyes from which tumor has been eliminated induced ACAID., Conclusions: Various parameters of immune privilege, originally described for the AC, are characteristic of immune privilege within the SR space. However, because P815 cells placed in the AC prove lethal for BALB/c recipients, but P815 cells placed in the SR space resolve without jeopardizing the host's life, immune privilege in the SR space can be distinguished from immune privilege in the AC, and this may have implications for grafts of retinal tissue placed within the SR space.

Purpose: To determine whether the capacity to induce ACAID by antigen injection into the anterior chamber is altered in animals with genetically determined retinal degeneration and increased age., Methods: Anterior chamber-associated immune deviation (ACAID) induced by injection of ovalbumin into the anterior chamber of the eye was studied in three rodent strains with different forms of hereditary retinal degeneration (Royal College of Surgeon [RCS] rats, retinal degeneration [rd] mice, and Norrie-Disease [ND] mice) and in different age groups (age range, 1-23 months). The data were compared with those of age-matched controls. Aqueous humors of rd mice, RCS rats, and age-matched congenic controls were investigated for concentrations of transforming growth factor-beta2 (TGF-beta2) using enzyme-linked immunosorbent assay., Results: ACAID was readily induced in RCS rats and ND mice irrespective of amount of retinal degeneration or aging. In rd mice ACAID could be induced in young animals but not in animals more than 12 months of age. In old rd mice, loss of ACAID was accompanied by a marked reduction in total TGF-beta2 levels in aqueous humor., Conclusions: Rd mice more than 1 year of age lose the capacity of the anterior chamber to support the induction of ACAID by intracameral injection of soluble protein antigen. Because loss of ACAID correlated with a decrease in TGF-beta2 concentration in aqueous humor, it is proposed that eyes of rd mice are unable to maintain an immunosuppressive microenvironment necessary for ACAID.

Purpose: To determine, with the use of mice genetically deficient in expression of CD4 or CD8 molecules, which T cells are responsible for rejection of orthotopic corneal allografts in mice., Methods: Corneas were prepared from major histocompatibility complex (MHC)-only incompatible, minor histocompatibility (H)- only incompatible, and MHC-plus-minor H incompatible donors and grafted orthotopically to eyes of CD4 knockout (KO), CD8KO, and wild-type control mice. Graft survival patterns were assessed clinically and compared. Mice that retained healthy corneal allografts beyond 8 weeks were evaluated for evidence of donor-specific tolerance and anterior-chamber-associated immune deviation (ACAID) using local adoptive transfer reactions and challenge with orthotopic skin allografts., Results: Corneas grafted to CD8KO mice were rejected with an incidence and tempo indistinguishable from that in wild-type control animals. By contrast, MHC-only, and minor-H-only incompatible corneal grafts survived indefinitely in eyes of CD4KO mice. Approximately 50% of corneal grafts that confronted CD4KO recipients with both MHC and minor H alloantigens experienced delayed rejection, whereas similar grafts in wild-type recipients were rejected acutely. CD4KO mice with long-accepted grafts displayed neither donor-specific ACAID nor allograft tolerance., Conclusions: CD8+ T cells play little or no role in acute rejection of orthotopic corneal allografts. Instead, acute rejection is mediated almost exclusively by CD4+ T cells. Moreover, when corneal allografts survive for 8 weeks without acute rejection, CD4+ T cells promote donor-specific ACAID thereby insuring long-term graft acceptance.

Purpose: To evaluate the role of delayed-type hypersensitivity (DTH) and cytotoxic T-lymphocyte (CTL) responses on alloepithelial rejection in a murine keratoepithelioplasty model., Methods: C3H/He mouse corneal lenticules were grafted around the limbus in BALB/c mice, and alloepithelial rejection was assessed by microscopic evaluation. The relation between rejection scores and DTH or CTL responses to donor antigens was assessed by Spearman correlation analysis. Suppression of DTH responses by induction of anterior chamber-associated immune deviation (ACAID) was used to evaluate the contribution of DTH responses to allograft rejection. CTL responses were evaluated by in vitro and in vivo depletion of CD4+ or CD8+ cells., Results: DTH responses, which developed 2 weeks postoperatively, correlated significantly with rejection scores (correlation coefficient r = 0.55). ACAID induction by anterior chamber inoculation of C3H/He splenocytes significantly suppressed allospecific DTH responses and alloepithelial rejection. While allospecific CTL responses also developed 2 weeks postoperatively and increased by 4 weeks, CTL responses did not exhibit positive correlation with rejection scores (r = -0.36, P = 0.076). The CTL responses were mediated exclusively by CD8+ cells. Although in vivo depletion of CD8+ cells abolished the induction of CTL responses, it did not prevent allograft rejection. Immunohistochemistry showed infiltration of CD4+ and CD8+ cells into the alloepithelium. Positive staining for interferon gamma but not interleukin 4 further implicated the participation of a DTH response., Conclusions: Allograft rejection after keratoepithelioplasty appears to be mediated primarily by DTH responses, with CTL responses playing only a minor role perhaps in modifying the development of rejection.

Purpose: To determine whether the subretinal space supports the induction of deviant immune responses to cell-associated and soluble antigens and to elucidate factors influencing the immunologic properties of the subretinal space., Methods: P815 mastocytoma cells were used as cell-associated antigens and were inoculated into the anterior chamber (AC), the vitreous cavity (VC), the subretinal space, and subconjunctivally in H2-compatible, but minor H-incompatible, BALB/c mice. Ovalbumin, as a soluble antigen, was similarly injected into eyes, after which recipient animals were immunized with ovalbumin and complete Freund's adjuvant. Delayed-type hypersensitivity (DTH) was assessed by ear challenge. To alter the conditions in the subretinal space, the outer blood-retinal barrier was disrupted by compromising retinal pigment epithelial (RPE) cells with a systemic injection of sodium iodate. Immune privilege in the AC was abolished by mild corneal cauterization., Results: Antigen-specific DTH did not develop in mice in which alloantigenic tumor cells or ovalbumin was injected into the AC, the VC, or the subretinal space, and the mice's spleens contained lymphocytes capable of suppressing DTH when adoptively transferred into naive mice. When RPE cells were compromised with sodium iodate, tumor cells or ovalbumin injected into the subretinal space or the VC did not induce immune deviation, although the AC of these eyes still promoted AC-associated immune deviation. By contrast, when immune privilege in the AC was abolished by corneal cauterization, neither tumor cells nor ovalbumin injected into the subretinal space or the VC of eyes elicited immune deviation., Conclusions: The subretinal space supports immune deviation for histoincompatible tumor cells and soluble protein antigens by actively suppressing antigen-specific DTH. Acute loss of immune privilege in the subretinal space and the VC does not cause loss of privilege in the AC, but abolition of immune privilege in the AC eliminates the capacity of the subretinal space and the VC to support immune deviation to antigens injected locally.

Purpose: To determine whether the capacity of Langerhans cells (LCs) to abrogate ocular immune privilege can be suppressed by the topical application of interleukin-1 receptor antagonist (IL-1ra)., Methods: Cautery was applied to corneas of BALB/c mice on day 0 to induce centripetal migration of LCs. Immune privilege was tested by the ability to induce anterior chamber-associated immune deviation (ACAID) to intracamerally injected soluble antigen 1 to 2 weeks after cautery application. The number of LCs was enumerated by immunofluorescent staining. In other experiments, freshly procured Thy-1-depleted epidermal cells, with or without LC depletion, were injected directly into virgin murine corneas before testing for ACAID. All test animals were randomized for treatment with either topical IL-1ra or placebo in a masked fashion for 1 to 2 weeks after induction of LC migration and before intracameral injection of antigen., Results: Intracorneal injection of freshly procured LC-depleted epidermal cells into normal eyes failed to abrogate ACAID, whereas LC-containing cell populations uniformly led to loss of immune privilege (P < 0.01). Topical treatment with IL-1ra led to retention of the cauterized eyes' capacity for ACAID induction (P < 0.01) and to a profound (>80%) suppression of LC migration compared with untreated controls (P < 0.01). Additionally, topical IL-1ra treatment of eyes with intracorneally injected LCs preserved immune privilege and ACAID induction (P < 0.001)., Conclusions: IL-1 mediates mechanisms of immunity in corneal inflammation that subvert the normal eye's immune privileged state. However, its antagonism with topical administration of IL-1ra preserves ocular immune privilege and ACAID through suppression of LC function.

Purpose: Although mice with long-accepted orthotopic corneal allografts display donor-specific anterior chamber-associated immune deviation (ACAID), this deviant response is not detected until well after the fate of the grafts is decided. To determine the efficiency with which corneal tissue itself can induce ACAID, allogeneic corneal segments were inserted into anterior chambers (AC) of normal mouse eyes., Methods: Central corneas from normal eyes of C57BL/6 (allogeneic) and BALB/c (syngeneic) donors were cut into wedge-shaped fragments measuring approximately 0.3 x 2.0 mm (in some experiments the corneal epithelium was removed) and inserted into the AC adjacent to recipient endothelium. Recipients of these fragments were evaluated for donor-specific delayed hypersensitivity (DH) and ACAID, and fragment-containing eyes were tested for their capacity to support ACAID to an irrelevant antigen., Results: Syngeneic and allogeneic corneas survived indefinitely in the AC without evidence of inflammation or rejection. Although fragments of cornea (with or without epithelial layers) placed at extraocular sites were potent inducers of DH, within the eye only epithelium-bearing grafts induced DH. Moreover, this DH response was short-lived. Recipients of allogeneic corneal fragments in the AC developed ACAID by 8 weeks, but not at 1 week. Moreover, fragment-containing eyes supported ACAID induction when bovine serum albumin was injected into the AC., Conclusions: Anterior chamber-associated immune deviation can be induced by allogeneic corneal tissue inserted into the AC, but its onset is delayed. The delay may be dictated by persistence of donor epithelium on the graft, which promotes DH. Once the epithelium is lost, DH disappears and ACAID emerges. Anterior chamber-associated immune deviation may contribute to the maintenance of corneal allograft viability.

Purpose: Anterior chamber-associated immune deviation (ACAID) is elicited by an antigen-specific signal that escapes the antigen-containing eye and travels through the blood to the spleen. Two types of ACAID-inducing signals have been described: those associated with blood-borne monocytes, and a soluble factor found in serum. The authors sought to understand the basis for the existence of two distinct types of ACAID-inducing signals., Methods: Different kinds of antigens (soluble, cell associated, particulate) were injected into the anterior chamber (AC) of normal, presensitized, and immunodeficient mice. In addition, peritoneal exudate cells were pulsed in vitro with different kinds of antigen in the presence of transforming growth factor beta and then evaluated for the ability to induce ACAID in naive (nonsensitized) as well as T- and B-cell-deficient recipients., Results: Among antigens injected into the AC, inert particulate antigens could not induce ACAID, but soluble and cell-associated (minor histocompatibility) antigens generated cell-associated ACAID-inducing signals. In contrast, antigens injected into the AC of presensitized mice generated ACAID-inducing signals that were soluble and located in the plasma fraction of blood. All ACAID-inducing signals created in vitro with soluble, particulate, or cell-associated antigens induced ACAID in vivo., Conclusions: Cell-associated ACAID-inducing signals are generated in naive mice regardless of the kind of antigen, and these signals arise from mobile intraocular antigen-presenting cells. However, when antigen is injected into the AC of presensitized mice, a soluble signal emerges, perhaps derived from T cells that enter the antigen-containing eye. Together, these signals dictate that subsequent exposures to ocular antigen will not evoke immunogenic inflammation.

Purpose: To determine whether immunosuppression using cyclosporine interferes with anterior chamber associated immune deviation (ACAID) and can promote survival of retinal allografts in the anterior chamber., Methods: Neonatal neural retinas of C57BL/6 mice or ovalbumin were injected into the anterior chamber of BALB/c adult mice. In the test group recipients were injected with cyclosporine (10 mg/kg per day) from day 0 to 11 or from day 11 to 34 after implantation. At 12 and 35 days after transplantation, lymphocytes from the test group were injected into naive BALB/c mice to assay for the presence of suppressor T cells (adoptive transfer). The fate of the retinal grafts was determined by histologic examination at day 12 and 35. To evaluate the potential neurotoxic effects of cyclosporine in the absence of immune rejection mechanisms, cyclosporine was given to SCID mice during days 11 to 34 after syngeneic neonatal neural retinal grafts were placed in the anterior chamber., Results: At 12 days after transplantation, spleens of both cyclosporine-treated and control mice contained suppressor cells against donor alloantigens. The retinal grafts in the anterior chamber of both groups of mice were fully developed and well differentiated. The same duration of administration of cyclosporine did not interfere with the production of efferent suppressor cells after inoculation of ovalbumin into the anterior chamber. At 35 days after transplantation, only spleen cells from the cyclosporine-treated group showed the capacity to suppress donor-specific delayed hypersensitivity. However, allografts in the cyclosporine group had deteriorated by 35 days in a fashion similar to the control group. Syngeneic grafts in SCID mice showed differentiated retinal layers 35 days after transplantation., Conclusions: Cyclosporine treatment does not interfere with the ability of allogeneic neonatal retinal grafts to induce anterior chamber associated immune deviation when placed in the anterior chamber, nor does prolonged treatment with this drug interfere with the persistence of allospecific suppressor cells for 35 days after the graft. Because 35-day grafts of cyclosporine-treated mice display histologic evidence of graft failure similar to grafts placed in the anterior chamber of untreated mice, graft destruction is either the result of immune effector mechanisms not inhibited by cyclosporine, or the consequence of nonimmunologic factors.

Purpose: To investigate the role of the eye and the spleen in maintaining suppression of delayed-type hypersensitivity (DTH) after anterior chamber (AC) inoculation of allogeneic splenocytes., Methods: Suppression of DTH response was tested in BALB/c mice after AC inoculation of allogeneic B10.D2 splenocytes. Seven days after AC injection, the antigen-inoculated eyes were enucleated or the spleens were removed. After enculeation or splenectomy at different time intervals, DTH responses in groups of the BALB/c mice were examined. Spleen components obtained from BALB/c mice that had been primed by B10.D2 splenocytes in the AC 7 days earlier were transferred intravenously to groups of naive syngeneic acceptors. At various intervals after adoptive transfer, variations of DTH responses were tested., Results: Inoculation of B10.D2 splenocytes to the AC of BALB/c mice induced antigen-specific suppression of DTH. Either enucleation of the antigen-inoculated eyes or splenectomy weakened the DTH-suppressive effect within 5 weeks and abolished it within 9 weeks, whereas the mice retaining both antigen-inoculated eyes and spleens maintained longstanding DTH suppression. Adoptive transfer of spleen components to syngeneic acceptors demonstrated DTH suppression for only 3 weeks., Conclusions: The antigen-inoculated eye and spleen are required for long-standing suppression of DTH after AC inoculation of allogeneic splenocytes.

Purpose: The authors investigated the effect of anterior chamber corneal (AC) inoculation of genetically graft-identical antigen on T-cell immunity and the suppression of alloepithelial rejection in mice., Methods: Antigen-specific suppression of delayed-type hypersensitivity (DTH) and suppression transferability were tested in BALB/c mice injected with irradiated allogeneic B10.D2 splenocytes into AC. Other groups of BALB/c mice received irradiated B10.D2, BALB/c, or C3H/He splenocytes in the AC of the right eye. Seven days later, B10.D2 or C3H/He corneal lenticules were grafted at the limbus of the left eye (keratoepithelioplasty). Alloepithelial rejection of each grafted eye was evaluated according to clinical findings. The DTH response of the keratoepithelioplasty recipients against B10.D2 minor antigen was tested at the end of clinical observation (4 months after grafting). Also examined was spleen component transfer from BALB/c mice with AC inoculation of B10.D2 splenocytes to syngeneic acceptors and its effect on suppression of epithelial rejection against B10.D2 antigen., Results: Inoculation of B10.D2 splenocytes into BALB/c AC induced antigen-specific DTH suppression, which suppression was transferable. During the 4-month observation period, AC inoculation of B10.D2 minor antigen significantly enhanced the survival of B10.D2-derived epithelium, but not of C3H/He-derived epithelium, in BALB/c mice. However, AC inoculation of BALB/c or C3H/ He splenocytes did not enhance B10.D2 epithelial survival in BALB/c mice. Incapability of antigen-specific DTH response generation was observed in the BALB/c mice with B10.D2 splenocytes in the right AC and B10.D2-derived epithelium in the left eye. Single transfer of spleen components from BALB/c mice with AC inoculation of B10.D2 splenocytes to syngeneic acceptors only delayed B10.D2 minor antigen-stimulated epithelial rejection, whereas supplementary transfers of the identical spleen components at different time intervals showed more significant effect in rejection delay., Conclusions: The results showed that AC inoculation of B10.D2 splenocytes in BALB/c mice induced antigen-specific suppression of DTH response, in a phenomenon termed anterior chamber-associated immune deviation (ACAID). It also was shown definitely that ACAID can suppress alloepithelial rejection in a murine keratoepithelioplasty model. Adoptive transfer of splenocytes from ACAID-induced mice merely affords short-term suppression of epithelial rejection, suggesting that an additional mechanism may be involved in ACAID maintenance.

Purpose: To determine whether anterior chamber-associated immune deviation (ACAID) promotes corneal allograft survival., Methods: CB6F1 mice were grafted with orthotopic corneal transplants from C3H donors (mismatch at the entire major histocompatibility complex plus multiple minor histocompatibility loci) and from NZB donors (mismatch only at multiple minor histocompatibility loci). ACAID was induced by priming in the anterior chamber (AC) with either Ia- spleen cells, Ia+ spleen cells, corneal endothelial cells, or corneal epithelial cells from corneal allograft donors before orthotopic transplantation. The role of ACAID in promoting corneal allograft survival was examined by determining the fate of corneal allografts in splenectomized and eusplenic mice., Results: Anterior chamber priming produced a modest enhancement of the survival of fully allogeneic C3H corneal allografts. By contrast, AC priming with Ia- NZB spleen cells or NZB corneal endothelial cells results in the permanent acceptance of NZB corneal grafts in 60% and 90% of the CB6F1 hosts, respectively. Abolition of ACAID by splenectomy resulted in a sharp increase in the incidence of graft rejection in donor-host combinations involving multiple minor histocompatibility disparity., Conclusions: Anterior chamber priming with alloantigens promotes corneal allograft survival in nonimmune and preimmune hosts. Disruption of the camero-splenic axis prevents the induction of ACAID and greatly increases the risk for corneal allograft rejection.

Purpose: To delineate the time course for loss of immune privilege after induction of corneal neovascularization (NV), and to test whether treatment with angiostatic agents can restore the eye's capacity to induce anterior chamber-associated immune deviation (ACAID)., Methods: Corneal NV in murine eyes was induced by placement of intrastromal sutures. At different time points after NV induction, study eyes were initiated on a 10-day regimen of one of a variety of anti-inflammatory or angiostatic agents. After the completion of their treatment regimen, eyes were tested as to whether they could support ACAID. To test whether any observed effect on the delayed-type hypersensitivity response was because of a systemic absorption of the topically applied medication, certain animals had only their fellow eyes treated., Results: Inflammatory corneal NV leads to the loss of immune privilege during the first week of the NV induction. Left untreated, these eyes remain incapable of supporting ACAID, even weeks after the initial corneal insult. However, when treatment with an anti-inflammatory agent is initiated during the first 2 weeks after the NV induction, these eyes show a restored capacity for ACAID induction, and this appears to be unrelated to any systemic effect of the treatment regimen. Treatment started at later time points is not capable of restoring the eye's normal capacity for inducing deviant immunity., Conclusions: Corneal neovascularization leads to loss of immune privilege in the anterior segment manifested as the inability to sustain ACAID. Moreover, topical angiostatic strategies can lead to restoration of immune privilege when instituted sufficiently early in the course of the neovascular response.

Purpose: To use explants of cornea and iris and ciliary body (I/CB) of experimentally manipulated eyes to determine whether loss of the capacity to suppress T-cell activation in vitro correlates with other consequences of manipulation, i.e., the migration of Langerhans cells into the central corneal epithelium, the leakiness of stromal neovessels, and the presence of axons in the cornea., Methods: Mouse eyes were subjected to cautery of central corneal surface, suture-induced neovascularization, circumferential or criss-crossed corneal surface wounds, and orthotopic corneal grafting. Corneas and I/CB were excised, explanted in vitro, and assayed for capacity to suppress mixed lymphocyte reaction. Integrity of suture-induced corneal neovessels was assessed with high molecular weight dextran, Langerhans cells were enumerated in corneal epithelium, nerve axons were evaluated in corneal stroma, and capacity of manipulated eyes to support anterior chamber-associated immune deviation (ACAID) induction after anterior chamber injection of bovine serum albumin were assessed., Results: Experimental manipulations that abolished ACAID included cautery, neovascularization, and keratoplasty, whereas criss-crossed corneal excision wounds did not. Loss of ACAID correlated variably with loss of ability of corneal explants to secrete immunosuppressive factors in vitro, presence of Langerhans cells within the central corneal epithelium, leakiness of corneal neovessels, loss of axons in corneal stroma, and loss of ability of I/CB after keratoplasty to secrete immunosuppressive factors., Conclusions: The cornea plays an active role in ocular immune privilege and ACAID by creating a local immunosuppressive microenvironment, providing neural afferent stimuli that affect immunosuppressive properties of I/CB, and preventing neovascularization and infiltration with Langerhans cells.

Purpose: Corneal allografts placed in human eyes at high risk often fail, and immune rejection is thought to be a major pathogenic factor. To understand the immunologic factors responsible for rejection in this instance, the authors have created "high-risk" eyes in mice by inducing corneal neovascularization. The authors then examined the fate of orthotopic corneal grafts placed in these beds and assessed the development of donor-specific delayed hypersensitivity (DH) in recipient mice., Methods: Three interrupted sutures were placed in the central cornea of recipient BALB/c mice to induce corneal neovascularization. Two weeks later, when corneal vessels occupied more than two quadrants of the cornea, mice received orthotopic corneal grafts from donor mice expressing alloantigens encoded by major and minor histocompatibility loci. Corneal allografts were evaluated by slit-lamp examination after grafting, and recipient mice were examined at the time of the rejection to determine whether they had acquired DH to alloantigens expressed on the corneal grafts., Results: Compared to grafts placed in normal eyes, a much higher incidence of rejection was observed among corneal allografts placed in neovascularized eyes (96.7% versus 46.7%). Moreover, grafts in neovascularized beds were rejected much more swiftly (2 weeks versus > 3 to 4 weeks). Rejection of corneal allografts in high-risk eyes coincided temporally with development of intense donor-specific DH, and the specificity of this immune response was directed solely at minor H antigens (not major histocompatibility complex-encoded antigens) on the graft., Conclusions: These results indicate that eyes rendered high risk by virtue of corneal neovascularization fail to provide immune privilege for orthotopic corneal allografts. In this circumstance, the grafts rapidly induce intense donor-specific DH that is readily detectable within 2 weeks of engraftment, at which time the grafts are acutely and universally rejected. The recipient DH response is directed exclusively at minor H antigens on the graft, which is consistent with the view that neovascularization creates graft beds in which recipient antigen-presenting cells infiltrate the graft and carry antigenic information by lymphatics to draining lymph nodes. In this manner, anterior chamber-associated immune deviation is avoided, and potentially allodestructive DH is promoted.

Purpose: To determine whether the introduction of antigen into the anterior chamber induces the production of extracellular antigen-specific T cell proteins (T cell antigen-binding molecules [TABM] specific for the antigen., Methods: Balb/c mice received an intracameral or subconjunctival injection of trinitrophenylated spleen cells (TNP spleen cells) before skin sensitization and challenge with picrychloride. The production of TNP-specific TABM in serum was quantified by enzyme-linked immunosorbent assay-based antigen binding and immunoblotting using a rabbit antiserum raised against a monoclonal antigen-specific T cell protein that induces suppressor T cells., Results: Intracameral, but not subconjunctival, injection of TNP spleen cells before contact sensitization increased the level of TNP-specific TABM in serum. At 2 days, TABM levels began to rise and peaked at 5 days after contact sensitization. Anterior chamber injection of TNP spleen cells alone into nonimmunized mice did not induce a detectable increase of TNP-specific TABM in serum. Mice that did not receive an intracameral TNP spleen cell injection but were made contact sensitive to TNP showed a diminished TNP-specific TABM response. Specificity of the TABM induced by intracameral-injection and sensitization for the TNP hapten was documented by showing that the induced TABM binds TNP-bovine serum antigen (BSA) but does not bind either BSA alone or azobenzenearsonate-ovalbumin and that immune sera raised against ovalbumin did not contain an increase in TNP-specific TABM. TNP-specific TABM in the sera of mice receiving an intracameral injection of TNP spleen cells, followed by contact sensitization with picrylchloride (PCl), were purified by affinity for TNP and were resolved by polyacrylamide gel electrophoresis and immunoblotting as M(r) 110,000 polypeptides., Conclusions: Serum levels of TNP-specific TABM induced by contact sensitization alone are enhanced fourfold to fivefold when sensitization is preceded by the intracameral, but not the subconjunctival, injection of TNP spleen cells. The authors propose that besides suppression of systemic delayed type hypersensitivity, the intracameral injection of antigen into nonimmune mice may prime an increased production of TABM, which may indicate systemic activation of the immunoregulatory T cell circuit.

Purpose: Immunosuppressive factors in aqueous humor (AH) contribute to the immune-privileged status of the anterior chamber of the eye. One such factor is transforming growth factor-beta (TGF-beta); other relevant inhibitors have not been fully identified. The authors examined AH to search for other putative inhibitors and to determine their effect on TGF-beta inhibitory activity., Methods: Radioimmunoassays (RIA) were used to detect the presence of hydrocortisone, corticosterone, cortisol binding globulin (CBG), and alpha-melanocyte stimulating hormone (alpha-MSH) in AH. The ability of these factors to inhibit murine thymocyte proliferation stimulated by phytohemagglutinin-interleukin 1 (PHA/IL-1) and proliferation of a TGF-beta-sensitive cell line (CCL64) in vitro was examined. The ability of hydrocortisone to inhibit a one-way mixed lymphocyte reaction (MLR) and the ability of epidermal cells to present soluble tumor-associated antigens (TAA) for elicitation of immunity in mice in the concentration range present in AH was also examined., Results: Hydrocortisone was detected in mouse, rat, and human AH (10.8 +/- 1.1 ng/ml, 9.3 +/- 2.1 ng/ml, and 18.0 +/- 1.0 ng/ml, respectively; mean +/- SEM), as was corticosterone (2.7 +/- 0.9 ng/ml, 2.2 +/- 0.3 ng/ml, and 0.7 +/- 0.1 ng/ml, respectively). Whereas normal plasma contains a binding protein for corticosteroids (i.e., CBG), the concentration in mouse, rat, and human AH was less than the level detectable by an RIA. Hydrocortisone inhibited PHA/IL-1-stimulated murine thymocyte proliferation and CCL64 cell proliferation in the concentration range present in AH. When hydrocortisone was combined with TGF-beta 2 (125 pg/ml), the degree of inhibition observed was greater than with either alone. Corticosterone inhibited thymocyte costimulation only slightly at concentrations present in AH but was inhibitory for CCL64 cells. alpha-MSH was also detected in AH. The concentration present had only slight inhibitory effects for CCL64 cell proliferation and did not enhance TGF-beta 2-mediated (62 pg/ml to 250 pg/ml) inhibition of CCL64 or thymocyte proliferation. Hydrocortisone inhibited the one-way MLR in the concentration range present in AH and, at 10 ng/ml, inhibited the ability of epidermal cells to present TAA for elicitation of delayed-type hypersensitivity in tumor-immune mice., Conclusions: These results show that AH contains biologically relevant concentrations of glucocorticoids and that CBG is relatively absent so that glucocorticoids present are largely free, and they suggest that regional sites take advantage of the activities of multiple factors to maintain an immune-privileged status.

Purpose: To examine conditions that determine the nature of the blood-borne, ACAID-inducing signal produced after intracameral injection of antigen., Methods: Balb/c mice were splenectomized, rested, and injected in the anterior chamber with various antigens. Two days later the animals were bled, the plasma and white cells were isolated, and these fractions were transferred to naive mice (with spleens). Recipients were immunized subcutaneously within 2 to 7 days and delayed type hypersensitivity was assessed 10 to 14 days after immunization by challenge with the appropriate antigen., Results: The antigens HSV-1, TNP-coupled cells, and P815 tumors cells induced a soluble ACAID-inducing signal found in the plasma portion of blood. The soluble protein antigens bovine serum albumin (BSA) and conalbumin induced a cell-associated signal. When T-cells were included with protein antigens, a soluble (not cellular) ACAID-inducing signal was induced., Conclusions: Particulate antigens, such as HSV-1 and P815, that elicit intraocular T-cell responses or antigens that contain T-cells (e.g., TNP cells) induce a soluble, ACAID-inducing signal. Soluble antigens (e.g., BSA and conalbumin) induce a cell-associated ACAID signal. Additionally, T-cells are capable of modulating the type of ACAID signal produced. These results show that two methods of delivering the ACAID signal exist that are dependent on the nature of the antigen and the presence of T-cells. The authors conclude that the eye shows great versatility in regulating potentially damaging immune responses.

Purpose: Interleukin-1 and tumor necrosis factor (TNF) alpha are proinflammatory cytokines and crucial mediators in many aspects of immunity. In this study, their role in anterior chamber-associated immune deviation (ACAID) was investigated., Methods: The role of these cytokines was examined by the use of neutralizing antibodies to TNF and interleukin (IL)-1 alpha, IL-1 beta, and IL-1 receptor. These reagents were co-injected with antigen into the anterior chamber and the effect on ACAID assessed. In addition, reverse transcriptase polymerase chain reaction (RT-PCR) was performed on eyes injected with TNP-spleen or bovine serum albumin to determine the levels of TNF alpha mRNA induced., Results: Neutralizing antibody to TNF, when injected with TNP-spleen cells into the anterior chamber, blocked ACAID to the TNP hapten. Antibodies to IL-1 alpha, IL-1 beta, and IL-1 receptors either alone or in combination did not block the establishment of ACAID: Studies with reverse transcriptase polymerase chain reaction (Rt-PCR) confirmed that early (within 2 hours) after anterior chamber injection of TNP-cells, messenger RNA levels for TNF alpha were dramatically increased. The induction of ACAID to bovine serum albumin also required the production of TNF alpha. Further studies showed that the production of blood borne "ACAID-inducing" signals after anterior chamber injection of bovine serum albumin or TNP-spleen were dependent on TNF., Conclusions: TNF alpha plays a crucial role in ACAID: Induction of TNF alpha within the eye may be an important event in the complex series of events that induce ACAID and possibly maintain immunologic privilege.

Purpose: To investigate the contribution of CD4+ and CD8+ T lymphocytes to retinitis in the contralateral eye after anterior chamber inoculation of herpes simplex virus type 1 (KOS strain)., Methods: T-cell-depleted BALB/c mice were prepared by adult thymectomy and treatment with anti-L3T4 (anti-CD4) monoclonal antibody and/or anti-Lyt2.2 (anti-CD8) monoclonal antibody. On days 9 and 14 after inoculation of herpes simplex virus type 1 (KOS strain) via the anterior chamber, the titer of virus in the uninoculated eye was determined by plaque assay. The eyes were examined microscopically, and the severity of retinitis was evaluated using a histopathologic scoring system., Results: At day 14 postinoculation, significantly less severe destruction of the parenchyma and less inflammatory cell infiltration were observed in the retina of the uninoculated eye of CD4+ T-cell-depleted mice and of mice depleted of both T-cell subsets. The titer of virus in the uninjected eye of CD4+ T-cell-depleted mice and of mice depleted of both CD4+ and CD8+ T cells was also significantly higher at day 14 postinoculation., Conclusions: The results of the studies suggest three ways CD4+ T cells might contribute to the pathogenesis of herpes simplex virus type 1 retinitis in the uninoculated contralateral eye: (1) accumulation of massive inflammatory cell infiltrates in the retina, (2) induction of retinal destruction, and (3) clearance of herpes simplex virus type 1 from the contralateral eye. In infection of the contralateral retina of nondepleted mice after uniocular anterior chamber inoculation of KOS, CD4+ T cells are required to induce fulminant retinitis and to mediate clearance of virus from the uninoculated eye by day 14 postinoculation. The exact mechanism by which CD4+ T cells contribute to contralateral retinitis remains to be identified.

Purpose: To determine whether injection of a soluble antigen, ovalbumin (OVA), into the anterior chamber of cynomolgus monkey eyes would impair the ability of these animals to subsequently develop delayed hypersensitivity when confronted by this antigen in immunogenic form., Methods: OVA or phosphate-buffered saline was injected into the anterior chamber of adult cynomolgus monkeys that were subsequently immunized with OVA in adjuvant and then skin challenged for delayed hypersensitivity with OVA., Results: Recipients of intracameral OVA proved unable to acquire antigen-specific delayed hypersensitivity when they received an immunogenic regimen of OVA in adjuvant. Since the flow of aqueous humor through the uveoscleral pathway of primate eyes can be promoted by topical treatment with PGF2 alpha isopropylester, a preliminary experiment is described in which induction of anterior chamber-associated immune deviation by OVA was prevented when the antigen was first introduced into monkey eyes treated with PGF2 alpha isopropylester., Conclusions: Monkeys resemble rodents in displaying anterior chamber associated immune deviation (impaired ability to acquire antigen-specific delayed hypersensitivity) when they first encounter an antigen via the anterior chamber. The findings suggest that the cellular and molecular mechanisms of immune privilege, recently described in rodents, may apply to immune responses to intraocular antigens and pathogens in primates, including humans. Primate eyes offer an opportunity to explore the mechanisms of anterior chamber-associated immune deviation using pharmacologic agents that modify the aqueous outflow tracts.

Injection of antigen into the anterior chamber of the eye results in the induction of suppressed systemic cell-mediated responses as measured by delayed-type hypersensitivity or contact hypersensitivity (CHS). Previous studies from the author's laboratories have determined that this response is governed by exposure of the eye to visible light during the initial intraocular encounter between T cells and antigen. To more fully understand the role of light, as well as to begin to understand the molecular mediators involved, the authors chose to explore the properties of light governing the effect. Neutral density filter were used to demonstrate that the minimum amount of light required to induce suppression of CHS following anterior chamber injection of antigen is 1-2 lux (lumens/meter2). With narrow band filters, the wavelengths responsible for suppression were shown to be 500-510 nm. The results show that the effect of light extends beyond the hapten-derivatized spleen cell system to other antigens placed in the anterior chamber of the eye. Studies also show that the retina and the pineal gland, two light absorbing structures, may not be involved. The results in this report show that light of very restricted wavelengths controls intraocular immune reactions.

The inflammatory response in endogenous uveitis or after anterior segment surgery was noted to be substantially greater in heavily pigmented eyes. Because varying amounts of melanin are released into the anterior chamber after intraocular inflammation, it was hypothesized that a proinflammatory effect of melanin might account for the enhanced inflammatory response in these eyes. To test this hypothesis, albino (BALB/c) or pigmented (C57BL/6) mice were challenged in the anterior chamber 2 weeks after a subcutaneous foot pad injection of horse serum or conalbumin dissolved in Freund's complete adjuvant. The degree of inflammation in the challenged eyes was determined by histologic examination 72 hr after the challenge. In all cases, the inflammatory infiltrate consisted mainly of polymorphonuclear leukocytes suggestive of an Arthus reaction. An anterior chamber challenge of horse serum-sensitized BALB/c or C57BL/6 mice with horse serum alone resulted in mild inflammation, which was augmented markedly by challenge with a combination of horse serum and melanin. The presence of melanin in the anterior chamber similarly increased the inflammatory response of conalbumin-sensitized mice to anterior chamber challenge with conalbumin. Melanin in the anterior chamber also significantly (P less than 0.05) augmented the inflammatory response of conalbumin-sensitized mice to a horse serum challenge, but it did not significantly augment the inflammatory response of horse serum-sensitized mice to a conalbumin challenge. The heterologous antigens induced minimal inflammation in the absence of melanin. Injection of melanin alone did not evoke an inflammatory response. Ocular challenge with melanin alone or in combination with antigen induced minimal inflammation in nonsensitized mice. However, preincubation of melanin with sera from horse serum-sensitized mice significantly increased its proinflammatory capacity when injected with horse serum into the anterior chamber of nonsensitized mice. In vitro binding studies using fluorescein isothiocyanate-conjugated mouse immunoglobulin G showed a high binding capacity of melanin for immunoglobulin G. It was concluded that the presence of free melanin in the anterior chamber can increase intraocular inflammation. Although the mechanism(s) by which melanin augments inflammation has not been defined, these data suggest that the binding of serum components (such as antibodies) to melanin may contribute to its proinflammatory effect.

The potential role of interleukin-6 (IL-6) was studied as an inflammatory mediator of endotoxin (or lipopolysaccharide [LPS])-induced uveitis (EIU) in the rat. In young Lewis rats, levels of intraocular IL-6, but not serum IL-6, correlated with the severity of uveitis and with aqueous humor protein levels in response to foot pad injections of LPS (P less than 0.001). Adult Lewis rats did not develop uveitis and had no intraocular IL-6, although IL-6 was released systemically. Resistance to EIU and absence of IL-6 levels in the aqueous humor, despite the ability to release serum IL-6, also were observed in brown Norway rats, irrespective of age and weight. Intravitreal injection of as little as 1 ng of human recombinant IL-6 induced uveitis in young Lewis rats. In adult Lewis rats, and in young animals made tolerant to LPS, intravitreal IL-6 still caused substantial leakage of plasma proteins into the anterior chamber but no influx of inflammatory cells. As early as 2 hr after intravitreal injection of IL-6, immunohistochemical analysis showed invasion of the iris, corneal stroma, and anterior chamber by polymorphonuclear leukocytes (PMN) and expression of major histocompatibility complex (MHC) class II antigen in the retina by large cells that were macrophage-marker ED2 negative. This was followed by massive PMN infiltration of the retinal layers and vitreous. The MHC class II antigen expression of ciliary and iris epithelium occurred at a later stage (greater than 8 hr).(ABSTRACT TRUNCATED AT 250 WORDS)

Onchocerciasis is a major cause of blindness worldwide, and much of the blindness is caused by onchocercal chorioretinitis. In an experimental animal model for ocular onchocerciasis, intravitreal injections of 10,000 live Onchocerca volvulus microfilariae isolated from infected humans into the eyes of cynomolgus monkeys (Macaca fascicularis) resulted in patchy, progressive loss of retinal pigment with pigment clumping. Areas of pigment loss were less extensive in animals that had been sensitized with microfilariae. Intravitreal injections of dead O. volvulus microfilariae resulted in mild vitritis with relatively less clinical change noted in the retina and choroid. Histopathologic examination revealed thinning and loss of outer retinal layers with pigment migration into the retina, and inflammation was more pronounced in eyes that received live microfilariae. Clinical changes appeared in eyes receiving live microfilariae before the development of significant antibody or cell-mediated immune responses. O. volvulus microfilariae appear to be more suitable than O. lienalis microfilariae in producing lesions which resemble human onchocerciasis in the primate model.

Failure of corneal grafts is thought to involve the development and activation of specifically sensitized T-cells. One method which might be used to circumvent the development of such cells is the phenomenon of anterior chamber-associated immune deviation (ACAID). Injection of antigen into the anterior chamber of the eye leads to an immune response characterized by normal antibody response coupled with depressed T-cell reactivity especially as measured by delayed-type hypersensitivity. To determine if this phenomenon could be used to alter the course of graft failure, potential recipients (Lewis rats) were injected intracamerally (IC) with allogeneic lymphoid cells (Wistar-Furth). Orthotopic, full-thickness, penetrating keratoplasty was done 0-30 days later, and the recipients were observed for at least 60 days. Approximately 75% of Wistar-Furth corneal grafts placed on uninjected Lewis rats failed as evidenced by continued opacity, edema, and infiltration of mononuclear cells into the grafts. The IC injection of Wistar-Furth lymphocytes decreased this failure rate to 25% and 50% when grafting was done 14 and 7 days after injection, respectively. Grafts of cornea from a third strain onto IC injected animals failed at an intermediate rate which demonstrated some immunologic protection. The results of these studies indicate that IC injection of allogeneic lymphocytes results in prolonged acceptance of corneal grafts syngeneic with the injected lymphocytes.

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 immunologic alteration has been termed anterior chamber-associated immune deviation (ACAID) and is the underlying mechanism responsible for immunologic privilege within the anterior chamber of the eye. Previous studies indicate that the immunologic privilege associated with the anterior chamber of the eye might be the result of a deficiency of interleukin-2 (IL-2) during antigen presentation. The present study examined the role of IL-1 in the induction of ACAID. It is well known that intracameral (IC) inoculation of DBA/2 mastocytoma cells (P815) into allogeneic BALB/c recipients results in antigen-specific suppression of DTH responses and progressive tumor growth. The authors found, however, that sublines of P388D1 (DBA/2 monocyte/macrophage tumor) that produce IL-1 not only do not grow progressively in the anterior chamber, but also they can prevent the suppression of DTH (P less than or equal to 0.01). The role of IL-1 in the abolition of ACAID was confirmed in studies with IC-inoculated P815 cells. Systemic administration of exogenous IL-1 (by subcutaneous miniosmotic pumps) prevented the induction of ACAID in hosts that received IC inocula of P815 cells (P less than or equal to 0.01). These results indicate that induction of ACAID and perhaps the immune-privileged character of the anterior chamber is dependent on an IL-1 deficiency during the processing of IC alloantigens.

Herpes simplex virus type 1 (HSV-1) inoculated directly into the anterior chamber of the mouse eye induced an acute inflammatory process in both the injected eye and its uninjected (contralateral) counterpart. In the former, a rapid intense inflammatory reaction developed in the anterior segment (cornea, anterior chamber) and anterior portion of the uveal tract (iris and ciliary body). The retina of the injected eye was spared. In contrast, in the uninjected eyes, a delayed massive destructive reaction also developed, but was limited almost exclusively to the posterior segment ( vitreous, retina and choroid); retinas of the uninjected eyes were destroyed completely. When HSV-1 was inoculated bilaterally into both anterior chambers, destructive inflammatory responses developed in both corneas and anterior segments, but the retinas were spared bilaterally. These results indicate that (1) a unique and interesting pattern of bilateral ocular disease occurs after uniocular anterior chamber injection of HSV-1 in mice; (2) the distribution of the destructive lesions differs between the injected eye and its uninjected counterpart; and (3) local factors, perhaps produced within the eye itself, modify the progression of the virus-induced reaction within the globe.