Evidence that formation of vimentin mitogen-activated protein kinase (MAPK) complex mediates mast cell activation following FcεRI/CC chemokine receptor 1 cross-talk.

Accumulating evidence points to cross-talk between FcεRI and CC chemokine receptor (CCR)-mediated signaling pathways in mast cells. Here, we propose that vimentin, a protein comprising type III intermediate filament, participates in such cross-talk for CCL2/monocyte chemotactic protein 1 (MCP-1) production in mast cells, which is a mechanism for allergic inflammation. Co-stimulation via FcεRI, using IgE/antigen, and CCR1, using recombinant CCL3/macrophage inflammatory protein-1α (MIP-1α), increased expression of phosphorylated, disassembled, and soluble vimentin in rat basophilic leukemia (RBL)-2H3 cells expressing human CCR1 (RBL-CCR1 cells) and bone marrow-derived murine mast cells, both models of mucosal type mast cells. Furthermore, co-stimulation enhanced production of CCL2 as well as phosphorylation of MAPK. Treating the cells with p38 MAPK inhibitor SB203580, but not with MEK inhibitor PD98058, reduced CCL2 production, suggesting that p38 MAPK, but not ERK1/2, plays a critical role in the chemokine production. Immunoprecipitation analysis showed that vimentin interacts with phosphorylated ERK1/2 and p38 MAPKs in the co-simulated cells. Preventing disassembly of the vimentin by aggregating vimentin filaments using ß,ß'-iminodipropionitrile reduced the interaction of vimentin with phosphorylated MAPKs as well as CCL2 production in the cells. Taken together, disassembled vimentin interacting with phosphorylated p38 MAPK could mediate CCL2 production in mast cells upon FcεRI and CCR1 activation.

The role of TRB3 in mast cells sensitized with monomeric IgE.

Mast cells play a key role in immunoglobulin E (IgE)-associated allergic disorders; however, the cellular effects of sensitization remain poorly understood. Using gene microarrays and the multiplexing ELISA method, we examined the effects of sensitization on RBL-CCR1 cell transcription and chemokine/cytokine secretion. Sensitization most prominently increased transcription of Trb3, the gene for tribbles homolog 3 (TRB3), and also increased the release of most of the cytokines and chemokines tested. Knockdown of TRB3 via RNAi significantly induced the production of tumor necrosis factor-α (TNF-α), interleukin-4 (IL-4), interleukin-6 (IL-6), and the chemokine mast cell protease-1 (MCP-1). TRB3 deficiency also induced IκBα phosphorylation. TRB3 may therefore serve as a negative regulator of pro-inflammatory cytokines and chemokines, controlling the extent of the inflammatory response.

CCR1 expression and signal transduction by murine BMMC results in secretion of TNF-alpha, TGFbeta-1 and IL-6.

Chemokine receptors (CCRs) are important co-stimulatory molecules found on many blood cells and associated with various diseases. The expression and function of CCRs on mast cells has been quite controversial. In this study, we report for the first time that murine bone marrow-derived mast cells (BMMC) express messenger RNA and protein for CCR1. BMMC cultured in the presence of murine recombinant stem cell factor and murine IL-3 expressed CCR1 after 5-6 weeks. We also report for the first time that mBMMC(CCR1+) cells endogenously express neurokinin receptor-1 and intercellular adhesion molecule-1. To examine the activity of CCR1 on these BMMC, we simultaneously stimulated two receptors: CCR1 by its ligand macrophage inflammatory protein-1alpha and the IgE receptor FcepsilonRI by antigen cross-linking. We found that co-stimulation enhanced BMMC degranulation compared with FcepsilonRI stimulation alone, as assessed by beta-hexosaminidase activity (85 versus 54%, P < 0.0001) and Ca(2+) influx (223 versus 183 nM, P < 0.05). We also observed significant increases in mast cell secretion of key growth factors, cytokines and chemokine mediators upon CCR1-FcepsilonRI co-stimulation. These factors include transforming growth factor beta-1, tumor necrosis factor-alpha and the cytokine IL-6. Taken together, our data indicate that CCR1 plays a key role in BMMC function. These findings contribute to our understanding of mechanisms for immune cell trafficking during inflammation.

Ablation of type I hypersensitivity in experimental allergic conjunctivitis by eotaxin-1/CCR3 blockade.

The immune response is regulated, in part, by effector cells whose activation requires multiple signals. For example, T cells require signals emanating from the T cell antigen receptor and co-stimulatory molecules for full activation. Here, we present evidence indicating that IgE-mediated hypersensitivity reactions in vivo also require cognate signals to activate mast cells. Immediate hypersensitivity reactions in the conjunctiva are ablated in mice deficient in eotaxin-1, despite normal numbers of tissue mast cells and levels of IgE. To further define the co-stimulatory signals mediated by chemokine receptor 3 (CCR3), an eotaxin-1 receptor, effects of CCR3 blockade were tested with an allergic conjunctivitis model and in ex vivo isolated connective tissue-type mast cells. Our results show that CCR3 blockade significantly suppresses allergen-mediated hypersensitivity reactions as well as IgE-mediated mast cell degranulation. We propose that a co-stimulatory axis by CCR3, mainly stimulated by eotaxin-1, is pivotal in mast cell-mediated hypersensitivity reactions.

Autoimmunity in retinal degeneration: autoimmune retinopathy and age-related macular degeneration.

Autoantibody production is associated with a variety of ocular disorders, including autoimmune retinopathy (AIR) and age-related macular degeneration (AMD). A breakdown of immunologic tolerance (ocular immune privilege), including the blood-retinal barrier, anti-immune and anti-inflammatory proteins, and anterior chamber-associated immune deviation may play important roles in these disorders. Although the exact triggers for ocular autoimmunity are unknown, autoimmune targeting of retinal tissue is clearly associated with and may contribute to the pathogenesis of both AIR and AMD. Autoantibody production has long been associated with AIR, a collection of disorders that includes cancer-associated retinopathy, melanoma-associated retinopathy and non-paraneoplastic autoimmune retinopathy. A growing body of evidence indicates that AMD pathogenesis, too, involves ocular inflammation and autoimmunity. Identification and quantification of autoantibodies produced in patients with AIR and AMD may assist with diagnosis, prognosis, and choice of treatments. Animal models that allow investigation of ocular autoimmunity will also be needed to better understand the disease processes and to develop novel therapies. In this review we discuss ocular immune privilege and potential mechanisms of autoimmunity in the eye. We describe how autoimmunity relates to the pathogenesis of AIR and AMD. We explain how the antigen microarray technique is used to detect autoantibodies in patient serum samples, and discuss how current animal models for AMD can be used to investigate autoimmune pathogenesis. Finally, we outline unanswered questions and exciting areas of future study related to autoimmune retinal degeneration.

Expression of high mobility group A2 protein in retinoblastoma and its association with clinicopathologic features.

Retinoblastoma (RB) is the commonest primary intraocular tumor in children. Overexpression of the high mobility group (HMG) A2 protein has been observed in a variety of malignant tumors and often correlates with poor prognosis. We studied the expression of HMGA2 in primary tumor samples and correlated with clinicopathologic features such as invasion, differentiation, and laterality of the tumors. Among 64 tumors, there were 29 tumors with invasion of the optic nerve, choroid, and/or orbit and 35 tumors without invasion. HMGA2 immunoreactivity was evaluated on archival paraffin sections and the results confirmed by Western blotting on 12 fresh tumor samples. Among 29 tumors with invasion, HMGA2 was strongly positive (++) in 10 tumors, moderately positive (+) in 11 tumors. Among 35 tumors without invasion, HMGA2 was strongly positive (++) in 6 tumors, moderately positive (+) in 6 tumors. Tumors with invasion showed significantly higher expression of HMGA2 compared with tumors without invasion (P<0.01). Non-neoplastic retina was negative for HMGA2. There was no correlation between HMGA2 expression and differentiation/laterality. Western blotting revealed that 7 tumors were strongly positive, 2 were moderately positive, and 1 was faintly positive for HMGA2. Our study has demonstrated the HMGA2 expression in a large cohort of primary retinoblastoma tumors and its correlation with invasiveness.

Macrophage inflammatory protein-1alpha as a costimulatory signal for mast cell-mediated immediate hypersensitivity reactions.

Regulation of the immune response requires the cooperation of multiple signals in the activation of effector cells. For example, T cells require signals emanating from both the TCR for antigen (upon recognition of MHC/antigenic peptide) and receptors for costimulatory molecules (e.g., CD80 and CD60) for full activation. Here we show that IgE-mediated reactions in the conjunctiva also require multiple signals. Immediate hypersensitivity reactions in the conjunctiva were inhibited in mice deficient in macrophage inflammatory protein-1alpha (MIP-1alpha) despite normal numbers of tissue mast cells and no decrease in the levels of allergen-specific IgE. Treatment of sensitized animals with neutralizing antibodies with specificity for MIP-1alpha also inhibited hypersensitivity in the conjunctiva. In both cases (MIP-1alpha deficiency and antibody treatment), the degranulation of mast cells in situ was affected. In vitro sensitization assays showed that MIP-1alpha is indeed required for optimal mast cell degranulation, along with cross-linking of the high-affinity IgE receptor, FcepsilonRI. The data indicate that MIP-1alpha constitutes an important second signal for mast cell degranulation in the conjunctiva in vivo and consequently for acute-phase disease. Antagonizing the interaction of MIP-1alpha with its receptor CC chemokine receptor 1 (CCR1) or signal transduction from CCR1 may therefore prove to be effective as an antiinflammatory therapy on the ocular surface.

Invariant chain+ N2a neuroblastoma cells stably expressing the class II MHC transactivator CIITA fail to stimulate anti-tumor immunity.

A promising cancer treatment strategy involves stimulation of anti-tumor immune responses. CD4(+) T cell responses are particularly desirable, as they enhance CD8(+) T cell activity and provide immune memory. The major histocompatibility complex (MHC) class II transactivator CIITA can be used to stimulate expression of MHC II on tumor cells, thereby promoting CD4(+) T cell activation. In this study, N2a neuroblastoma cells were stably transfected with CIITA. N2aCIITA cells displayed increased expression of MHC I, MHC II and invariant chain; CD80 and CD86 were expressed by neither the parental N2a cells nor by the N2aCIITA cells. All mice injected with N2aCIITA cells developed tumors. Furthermore, no increase in the numbers of T cells, natural killer cells, macrophages, or eosinophils was observed in the spleens or tumors of mice injected with N2aCIITA cells, compared to tissues from mice injected with the parental N2a cells. This absence of an anti-tumor immune response despite MHC II expression is likely due to the presence of invariant chain, in support of the MHCII(+)/Ii(-) paradigm.

The dendritic cell in allergic conjunctivitis.

The acquired immune response in health and disease is initiated when foreign antigens are processed and presented to T lymphocytes via antigen-presenting cells as peptides in the context of Class I and II major histocompatibility complex antigens. It is now clear that there are various types of antigen-presenting cells and that the phenotype of these cells (together with the milieu of the tissue or lymphoid organ) dictates the nature of the immune response to the antigen. Very little is known about the phenotype, distribution, and roles of dendritic cell subtypes that contribute to the pathophysiology of type I hypersensitivity reaction in the ocular surface. We review what has been learned from studies of both human ocular allergy and murine models and comment on how this compares to allergic reactions in other mucosal tissues.

A specific CCR3 chemokine receptor antagonist inhibits both early and late phase allergic inflammation in the conjunctiva.

Allergic inflammation manifests as one of a number of diseases, including asthma, dermatitis, food allergy, vernal keratoconjunctivitis, and systemic anaphylaxis. Together these diseases affect nearly 25% of the Western world and are a leading health-care problem. The diseases are often biphasic, with an early phase driven primarily by mast cell degranulation and a late phase characterized by leukocyte recruitment. While chemokines are well known to be critical for leukocyte recruitment, their importance in early-phase reactions is poorly defined. We show here that administration of a single oral dose of a high affinity and highly selective CCR3 antagonist ablates both the early and late phase reactions in a mouse model of allergic conjunctivitis. A direct analysis of mast cells in the conjunctiva demonstrates that antagonism of the CCR3 receptor stabilizes the mast cell in vivo, thereby leading to the impaired early phase reaction. The late phase reaction is also strongly inhibited as characterized by both reduced eosinophilia and neutrophilia. These results constitute the first direct evidence that antagonism of CCR3 has clear potential for the treatment of allergic diseases.

Role of CC chemokines and their receptors in multiple aspects of mast cell biology: comparative protein profiling of FcepsilonRI- and/or CCR1-engaged mast cells using protein chip technology.

Apart from the FcepsilonRI-mediated mechanism, mast cells are activated by chemokines. Evidence has accumulated indicating that there is cross-talk between the FcepsilonRI-mediated signalling pathway and CC chemokine receptor (CCR)-mediated signalling pathways in mast cells. We have found that costimulation with IgE/antigen and CC chemokine ligand 3 (CCL3) enhances degranulation but inhibits chemotaxis of rat basophilic leukaemia (RBL)-2H3 cells expressing human CCR1 (RBL-CCR1 cells). We hypothesize that this signalling cross-talk in mast cells may play important roles in the orchestration and focusing of the allergic response. In this study, we have sought information about global protein networks either enhanced or inhibited following cross-talk between the FcepsilonRI-mediated and CCR-mediated signalling pathways in mast cells. We believe this information may be useful for providing an understanding of mast cell function and in the establishment of new anti-inflammatory drugs for allergic diseases. Proteomics is a promising tool for studying protein profiles within biological samples and facilitates an understanding of the complex responses of an organism to a stimulus. Here, we show comparative data of protein profiles derived from FcepsilonRI-engaged and/or CCR1-engaged RBL-CCR1 cells using protein chip array technology, a proteomic technology. We also discuss our view of the role of CC chemokines and CCRs in regulating multiple aspects of mast cell biology.

Immunity in the eye: lessons in organ-specific responses.

Fifty-five years have past since Sir Peter Medawar first demonstrated that the fate of allografts differs in the skin, brain, subcutaneous tissue, and anterior chamber of the eye. Indeed, these and other experiments performed at the University of Birmingham and University College London not only helped define key paradigms in transplantation biology but introduced the concept of immune privilege in the eye and in other tissues. In the ensuing years, the work from dozens of laboratories has confirmed that immune responses in eye, although sharing many features with immunity in other tissues, are atypical in many respects. This has been hypothesized to stem from the requirement in the eye to hold inflammation in check to preserve visual function. Indeed, inflammatory reactions in different regions of the eye can result in visual impairment and blindness, manifesting in diseases such as vernal keratoconjunctivitis, uveitis, corneal endotheliitis, Mooren's ulcer, and pemphigoid. The investigation of the cell and molecular basis of the unique nature of immunity in the eye has revealed a remarkably complex network of interactions that controls inflammation while affording immunity. The most recent data generated in this field and the concepts arising from their interpretation were discussed at a symposium held at the University College London in late 2002. Here, we highlight some of the findings discussed at the meeting and introduce in-depth reviews published in this issue of the journal.

Novel mechanisms of class II major histocompatibility complex gene regulation.

Class II MHC molecules present processed peptides from exogenous antigens to CD4+ helper T lymphocytes. In so doing, they are central to immunity, driving both the humoral and cell mediated arms of the immune response. Class II MHC molecules, and the genes encoding them, are expressed primarily in cells of the immune system (B cells, thymic epithelial cells, activated T cells and professional antigen presenting cells). The expression is also under developmental control. Research over the past 20 years have provided a clear understanding of the cis-elements and transcription factors that regulate the expression of Class II MHC genes. Perhaps the most critical advance has been the discovery of CIITA, a non- DNA binding activator of transcription that is a master control gene for class II gene expression. Current research is focused on understanding the situations where class II MHC gene expression occurs in a CIITA-independent pathway, and the molecular basis for this expression. Finally, significant emphasis is being placed on targeting class II MHC transcription factors to either inhibit or stimulate the immune response to transplanted tissue or in cell based vaccines. This communication outlines recent advances in this field and discusses likely areas for future research.

MHC class II gene expression is not induced in HPIV3-infected respiratory epithelial cells.

The major histocompatibility complex (MHC) class II transactivator (CIITA) typically is required for both constitutive and inducible expression of MHC class II genes. However, transcription of class II MHC genes has been observed in specific cell types (e.g., thymic epithelial cells) in CIITA-deficient mice as well as in specific situations (e.g., following viral infections or in natural killer [NK]/target cell interaction). These observations have been interpreted by some to indicate that a CIITA-independent pathway of class II gene expression might be germane to processes such as the acquisition of tolerance during thymic selection or in the evasion of immune surveillance by a subset of viruses. One of the most striking examples of CIITA-independent, inducible class II gene expression has involved the de novo expression of class II MHC molecules on respiratory epithelial cells following infection by human parainfluenza virus type 3 (HPIV3). We report here that despite careful analysis using multiple techniques, we have been unable to detect HPIV3-dependent, CIITA-independent (or CIITA-dependent) induction of class II MHC genes. Thus, whereas there may still be an intriguing role for CIITA-independent gene expression in facets of the immune response, this is unlikely to manifest in the analysis of HPIV3 infection of respiratory epithelial cells.

Epigenetic silencing of the CIITA gene and posttranscriptional regulation of class II MHC genes in ocular melanoma cells.

PURPOSE: Primary uveal melanocytes and many ocular melanoma cells are resistant to interferon (IFN)-gamma-mediated induction of major histocompatibility complex (MHC) class II molecule expression. This suppression of class II MHC induction is considered to be one of the ways in which the eye is able to inhibit inflammatory responses. However, the mechanism(s) of this suppression is unknown. In this study, we have probed the molecular basis of this phenotype and report two distinct mechanisms underlying this phenotype. METHODS: Primary ocular melanocytes and ocular melanoma cell lines (retaining this IFN-gamma-resistant class II MHC phenotype) were examined for the expression of class II MHC molecules on the cell surface by flow cytometry. Class II MHC gene expression was further examined using Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR) analyses. RESULTS: The IFN-gamma signal-transduction pathway was found to be intact by electrophoretic mobility shift assay (EMSA) and transfection of reporter constructs. The lack of class II MHC gene expression appears to result from at least two mechanisms: (1) a specific inhibition of CIITA (class II transactivator) gene expression (reminiscent of trophoblasts), and (2) posttranscriptional regulation of class II MHC genes. CONCLUSIONS: The inability of primary uveal melanocytes and ocular melanoma cells to express class II MHC molecules after treatment with IFN-gamma has been found to map to two distinct points in the class II MHC biosynthetic pathway. The predominant mechanism appears to involve the silencing of the endogenous gene encoding the class II transactivator (CIITA). Here, the blockade does not involve signal transduction from the IFN-gamma receptor, but rather involves a specific silencing of the CIITA gene. A second mechanism involves the posttranscriptional regulation of class II MHC genes.

Detailed criteria for the assessment of clinical symptoms in a new murine model of severe allergic conjunctivitis.

PURPOSE: The aims of this research were to: (1) generate a rapid protocol for the sensitization of rodents to a defined allergen without footpad injections yet leading to both acute- and late-phase hypersensitivity reactions in the ocular surface; and (2) define detailed criteria for the assessment of clinical symptoms in the acute-phase response. METHODS: With the approved methods for the use of experimental animals in research and existing sensitization protocols as a starting point, we developed and tested a new protocol with respect to its ability to generate an acute- and late-phase response on ocular challenge. Clinical symptoms were assessed by a trained ophthalmologist under masked conditions, and late-phase responses determined by histologic analysis of conjunctival tissue sections. RESULTS: A new protocol for the rapid sensitization of mice, avoiding footpad injections, yet yielding both acute- and late-phase allergic responses, was developed. Detailed criteria for the assessment of disease severity were established and tested. CONCLUSION: This protocol establishes a murine model of allergic conjunctivitis that will be useful for both the study of the molecular and cellular basis of allergic reactions in the ocular surface and the testing of new therapies for this disease.

The murine CCR3 receptor regulates both eosinophilia and hyperresponsiveness in IgE-mediated allergic conjunctivitis.

BACKGROUND/AIMS: Allergic conjunctivitis is characterised by early-phase clinical symptoms and late-phase inflammation in the conjunctiva. The role of different chemokine receptors in allergic conjunctivitis, especially during the early-phase reaction, is still unclear. We investigated the importance of CC chemokine receptor (CCR) 3 in a murine model of IgE-mediated allergic conjunctivitis using CCR3-deficient (CCR3(-/-)) mice. METHODS: Allergic conjunctivitis was initiated in wild-type (WT) and CCR3(-/-) mice by passive transfer of ragweed (RW)-specific IgE, followed by topical challenge with RW in eye drops. Early-phase reactions including clinical symptoms and vascular leakage, as well as late-phase eosinophil infiltration of the conjunctiva were evaluated. The expression of mRNAs in the conjunctiva was quantified by real-time PCR analysis. RESULTS: The number of infiltrated eosinophils in the conjunctiva following RW challenge, was significantly higher in RW-IgE-sensitised WT mice compared with those sensitised with phosphate-buffered saline for WT, but this was not observed in similarly treated CCR3(-/-) mice. The early-phase clinical symptoms and vascular leakage were also suppressed in CCR3(-/-) mice. The number of conjunctival mast cells were not different between CCR3(-/-) mice and WT mice, and the mRNAs for FcεRІα and the connective tissue-type mast cell proteases were detected in the conjunctiva of both WT and CCR3(-/-) mice. RW-IgE-sensitised CCR3(-/-) mice displayed significantly reduced expression of CCL2, CCL3, and IL-6 compared with WT mice. CONCLUSIONS: These results demonstrate a direct contribution of CCR3 to both the early-phase reaction and late-phase inflammation during ocular allergy.

Regulatory function of CpG-activated B cells in late-phase experimental allergic conjunctivitis.

PURPOSE: To determine how CpG, an immunostimulatory sequence, affects experimental allergic conjunctivitis and to determine the mechanisms of its action. METHODS: Experimental allergic conjunctivitis was induced in mice to investigate the suppressive mechanism of CpG treatment. Cytokine profiling, fluorescence-activated cell sorting analyses, and adoptive transfer were used to analyze suppressive mechanisms after CpG treatment. RESULTS: Administration of the CpG oligonucleotide induced significant splenomegaly. Adoptive transfer of the splenocytes isolated from CpG-treated mice was able to confer resistance to allergen-induced inflammatory responses in recipient mice. CpG treatment led to a transient upregulation of IL-1ra, IL-18, IL-1alpha, and IL-12 in the spleen, draining lymph nodes, and conjunctiva. In contrast, IL-10 showed a marked and sustained induction in the inductive and effector tissues. Splenomegaly after CpG exposure was reduced in IL-10-deficient mice, indicating that IL-10 is required for immune remodeling of the spleen. Analyses of allergen-sensitized mice deficient in IL-10 exacerbated the late-phase inflammatory responses. Fluorescence-activated cell sorting analysis of the CpG-induced splenocyte subsets showed that the predominant source of IL-10 was B220(+) CD19(+) CD23(+)IgM(+)CD40(+)class II(high) follicular B cells. Adoptive transfer of IL-10-deficient B cells exacerbated eosinophilia. Transfer of an expanded population of cells after CpG treatment, including IL-10-secreting follicular B cells, protected against eosinophilia. CONCLUSIONS: CpG treatment provided B cell-mediated regulation of immune responses and B cell differentiation in CpG-induced immune remodeling with the use of IL-10.

Macular and retinal dysfunction of unknown origin in adults with normal fundi: evidence for an autoimmune pathophysiology.

Here we report the discovery of and phenotypic characterization of a retinal disorder of unknown origin in adults using clinical, electrophysiological and psychophysical techniques, and to seek the presence of circulating retinal autoantibodies in the sera of these patients. Sixteen patients were identified with progressive bilateral visual loss over a period of months. Ten of the patients were male, and the average age was 55.3 years (range from 43 to 76 years). Known causes such as carcinoma-associated retinopathy, acute zonal occult outer retinopathy and hereditary cone dystrophy appeared unlikely. Investigations included electrophysiology, fundus autofluorescence imaging and psychophysical tests. The sera of these patients were analyzed with indirect immunocytochemistry and Western immunoblot analysis on murine (BALB/c) retinal tissue for the presence of retinal autoantibodies. Bilateral visual loss and photophobia progressed over a period of months to years (average 28.7 months, range 3-67) and subsequently stabilized. No abnormality was observed by biomicroscopy, angiography or autofluorescence imaging. Electrophysiology indicated predominant cone-system dysfunction, either macular or generalized, and post-phototransduction involvement in 9 patients (56%). Photopic and scotopic visual fields and dark adaptation kinetics showed both cone and rod system involvement in all cases. Heterogeneous immunohistochemical staining patterns were seen with the sera of these patients as compared with controls. A majority of the affected patients (9/15) stained with an antinuclear pattern. The retinal autoantibodies from the sera of most patients reacted with the retinal proteins of molecular weight between 34 and 40 kDa. The aetiology of this distinctive retinal disorder therefore appears to be mediated through an autoimmune mechanism.

Inhibition of MHC class II gene expression in uveal melanoma cells is due to methylation of the CIITA gene or an upstream activator.

Most cells with an intact interferon-gamma receptor and signaling pathway are able to express MHC class II molecules when treated with cytokines such as interferon-gamma and tumor necrosis factor-a. Interestingly, primary uveal melanocytes and most ocular melanoma cells are resistant to interferon-gamma mediated induction of class II MHC genes. This unusual phenotype is hypothesized to be germane to the immune-privileged status to the eye. Via a series of experiments, we have probed the molecular basis of this class II MHC resistant phenotype. We have analyzed the methylation status of the gene encoding the class II transactivator (CIITA), and asked whether treatment of class II MHC resistant ocular melanoma cells with the demethylating agent 5'-azacytidine can restore interferon-gamma inducibility of these class II MHC genes in these cells. The data obtained suggest that the specific blockade in cytokine-induced class II MHC gene expression is due to a suppression of the gene encoding the class II transactivator (CIITA). Treatment with 5' azacytidine restores the ability of these cells to express class II MHC genes upon interferon-gamma treatment. Whilst this is reminiscent of what occurs in another immune-privileged tissue--the placental trophoblast--we show here that silencing of the CIITA gene in uveal melanocytes either involves methylation of distinct nucleotides from those detected in trophoblasts, or involves an upstream activator of CIITA gene expression.