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Purpose Corneal fibroblasts contribute to collagen remodeling in the corneal stroma in part by mediating collagen degradation. Given that corneal structure is influenced by sex hormone status, we examined the effects of sex hormones on collagen degradation by corneal fibroblasts. Methods Rabbit corneal fibroblasts were cultured in three-dimensional collagen gels with or without sex hormones including 17β-estradiol, progesterone, testosterone, and dehydroepiandrosterone (DHEA). Collagen degradation was determined by measurement of hydroxyproline after acid hydrolysis. The expression and activity of matrix metalloproteinases (MMPs) were evaluated by immunoblot analysis and gelatin zymography. The phosphorylation of mitogen-activated protein kinases (MAPKs) and the nuclear factor-kappa B (NF-κB) inhibitor NF kappa B Inhibitor-alpha (IκB-α) in corneal fibroblasts was examined by immunoblot analysis. Cell proliferation and viability were evaluated by measurement of bromodeoxyuridine incorporation and the release of lactate dehydrogenase, respectively. Results 17β-Estradiol and progesterone each inhibited interleukin (IL)–1β–induced collagen degradation by corneal fibroblasts in a concentration-dependent manner, whereas testosterone and DHEA had no such effect. MMP expression and activation in corneal fibroblasts exposed to IL-1β were also inhibited by 17β-estradiol and progesterone. These female sex hormones did not affect cell proliferation or viability. Both 17β-estradiol and progesterone inhibited the IL-1β–induced phosphorylation of p38 MAPK without affecting that of the MAPKs extracellular Signal-regulated Kinase (ERK) or c-jun N-terminal kinase (JNK). 17β-Estradiol also inhibited the IL-1β–induced phosphorylation of IκB-α. Conclusions 17β-Estradiol and progesterone inhibited MMP expression and activity in IL-1β–stimulated corneal fibroblasts and thereby suppressed collagen degradation by these cells.

Purpose The polymorphisms of the Fc receptor-like 3 gene (FCRL3), a novel immunoregulatory gene, have been shown to be associated with certain autoimmune diseases. This study was designed to examine whether the polymorphisms of FCRL3 are associated with susceptibility to Vogt-Koyanagi-Harada (VKH) syndrome in a Chinese population. Methods A case-control study was performed in 230 Chinese VKH patients and 301 healthy controls. Four single nucleotide polymorphisms (SNPs; −169C/T, −110A/G, +358C/G, and +1381A/G) in FCRL3 were detected using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). human leukocyte antigen -DR4 (HLA-DR4) and HLA-DRw53 genotyping was performed using PCR techniques. Results The results showed that the frequency of haplotype CACG was significantly lower in patients when compared with that in controls (p=0.0018, corrected p [pc]=0.0288). A significantly higher frequency was found for haplotype CGGG in HLA-DR4 negative patients than in HLA-DR4 negative controls (p=9.94×10−8, Pc=1.59×10−6). There were no significant differences in the allele and genotype frequencies of the four investigated SNPs between VKH patients and controls. HLA-DR4 and HLA-DRw53 were significantly associated with VKH syndrome (p=3.21×10−16 and p=7.08×10−5, respectively). Stratification analysis according to HLA-DR4 and HLA-DRw53 did not show any association of FCRL3 polymorphisms with VKH syndrome. Conclusions Our study confirms the previous association of HLA-DR4 and HLA-DRw53 with VKH syndrome but fails to demonstrate an association between FCRL3 polymorphisms and VKH syndrome. Haplotype CACG might be a protective haplotype for VKH syndrome, and haplotype CGGG may be a risk haplotype in HLA-DR4 negative individuals.

Purpose To analyze the potential association of programmed cell death 1 (PDCD1) with Vogt-Koyanagi-Harada (VKH) syndrome in a Chinese Han population. Methods Three single nucleotide polymorphism (SNPs), PD-1.3G/A, PD-1.5C/T, and PD-1.6G/A, were genotyped in 247 VKH patients and 289 age-, sex-, and ethnically-matched healthy controls using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. The associations of genotypes and alleles with VKH syndrome were analyzed. Results All genotype distributions in healthy controls were in Hardy–Weinberg equilibrium. The genotype and allele frequencies of PD-1.3, PD-1.5, and PD-1.6 were not different between patients with VKH syndrome and healthy controls. No significant difference was observed according to the status of human leukocyte antigen (HLA)-DR4 and HLA-DRw53. Compared to the controls, lower frequencies of the PD-1.5C genotype and allele frequencies were observed in VKH patients with extraocular findings. Conclusions PD-1.3 and PD-1.6 polymorphisms are not associated with the susceptibility to VKH syndrome in the Chinese Han population. However, PD-1.5 may be negatively associated with the occurrence of extraocular manifestations of VKH syndrome.

Purpose To examine whether small ubiquitin-like modifier 4 (SUMO4) polymorphisms were associated with Vogt-Koyanagi-Harada (VKH) syndrome in the Chinese Han population. Methods Genotyping for SUMO4 polymorphisms at G-847A, A-504G, A+163G, and C+438T loci was performed on 231 VKH patients and 302 controls using polymerase chain reaction restriction fragment length polymorphism. Results A decreased frequency of SUMO4 +438 TT genotype was found in VKH patients compared with healthy controls (p=0.009). However, the significance was lost after Bonferroni correction. Human leukocyte antigens (HLA)-DR4 and HLA-DRw53 were significantly associated with susceptibility to VKH syndrome (p=3.21×10−16 and 7.08×10−5, respectively). Stratification analysis based on HLA-DR4 and HLA-DRw53 did not show any associations between SUMO4 polymorphisms and VKH syndrome, although there was a big difference in the percentage of certain allele and genotype frequencies between HLA-DRw53 negative patients and controls. There was no significance in clinical findings and gender stratification analysis. Conclusions HLA-DR4 and HLA-DRw53 are strongly associated with the susceptibility to VKH syndrome in the Chinese Han population. However, none of the currently known single nucleotide polymorphisms (SNPs) of SUMO4 are associated with this syndrome.

Purpose To investigate the frequency and phenotypic and functional characteristics of S-antigen (S-Ag) specific T cells in patients with Behcet’s disease (BD). Methods Blood was taken from 23 active BD patients, 12 inactive BD patients, and 14 healthy controls. The clinical features of the patients were summarized. T cell response against 40 mixed S-Ag peptides was identified by interferon gamma (IFN-γ) enzyme-linked immunospot assay (ELISPOT). CD69 and CD45RO were used to characterize the phenotype of S-Ag specific T cells. The functional property of S-Ag specific T cells was investigated by measuring the production of cytokines. Results Response to the mixed S-Ag peptides was found in 56.5% and 25% of active and inactive BD patients, respectively. The responsiveness to S-Ag peptides was unrelated to the clinical features of the patients. About 65.8% of IFN-γ+ CD4+ T cells in active BD patients expressed CD69 and CD45RO concomitantly. S-Ag peptides significantly induced a production of IFN-γ and tumor necrosis factor (TNF)-α but not interleukin (IL)-2, IL-4, and IL-17 by peripheral blood mononuclear cells (PBMCs) in active BD patients with a response to S-Ag. Conclusions S-Ag specific T cells are present in certain active BD patients, and most of them are activated memory CD4+ T cells. These T cells may be involved in the pathogenesis of BD via producing Th1-dominant cytokines.

Purpose To examine the expression of Foxp3 on CD8+ T cells in the spleen during anterior chamber-associated immune deviation (ACAID). Methods Ovalbumin (OVA) was injected into the anterior chamber (AC) of C57BL/6 mice, and the delayed-type hypersensitivity (DTH) response was measured to evaluate the development of ACAID. The suppressive effect of CD8+ T cells in ACAID mice was determined by a local adoptive transfer (LAT) assay. Flow cytometry was used to assay the frequency of CD8+Foxp3+ T cells from normal mice, ACAID mice, and control mice receiving an AC injection of PBS (PBS-AC-injected mice). These frequencies were also tested after polyclonal or specific antigen stimulation. The mRNA level of Foxp3 in CD8+ splenocytes from different groups with or without stimulation were determined by reverse transcription-polymerase chain reaction. Results OVA injection into the AC induced ACAID, and CD8+ T cells from ACAID mice inhibited the ear-swelling response by OVA-primed responder cells in LAT assay. Flow cytometry analysis showed that the frequency of CD8+Foxp3+ cells in splenocytes was upregulated in ACAID mice following polyclonal or specific antigen stimulation. Foxp3 mRNA was only detected in CD8+ T cells from ACAID mice after polyclonal stimulation. Conclusions An upregulated Foxp3 expression in CD8+ T cells is associated with the development of ACAID, suggesting an involvement of CD8+Foxp3+ T cells in this model of immune tolerance.

Purpose To study the phenotypes, distribution, and morphologies of different antigen-presenting cells (APCs) in the murine cornea. Methods Intravitreal injection of fluorescently tagged ovalbumin (OVA) or antibodies to MHC-II (I-Ad), F4/80, CD11c, B7-1, and B7-2 was performed to label cells in the murine cornea. Light and transmission electron microscopy were used to examine corneal histology. Intravital microscopy, epifluorescence microscopy, and confocal microscopy were used to evaluate the labeled cells. In vitro staining was performed to validate the in vivo staining and localize the labeled cells. Three-dimensional rotatable images were taken to evaluate relationships between two differently labeled cells. Results Histological examination revealed no observable change in the cornea following intravitreal injection. In vivo staining showed that OVA+ cells and cells positive for MHC-II, F4/80, CD11c, B7-1, or B7-2 were noted throughout the cornea with a decreasing density from limbus toward the central cornea. Two populations with distinct morphological features were identified among these APCs. Labeled cells were found beneath the epithelium or in the shallow stroma in the central and paracentral cornea, but in all layers in the peripheral cornea. A number of F4/80+ and CD11c+ cells were also positive for OVA, MHC-II, B7-1, or B7-2. Rotatable images showed a close contact between two differently labeled cells. Conclusions Intravitreal injection of labeled antibodies can be adapted to visualize labeled cells in the cornea. APCs with distinct morphologies, phenotypes, and distribution may contribute to the immunologically privileged feature of the cornea.