Ai Yamada

Response of corneal epithelial cells to Staphylococcus aureus

Staphylococcus aureus is a leading cause of invasive infection. It also infects wet mucosal tissues including the cornea and conjunctiva. Conflicting evidence exists on the expression of Toll-like receptors by human corneal epithelial cells. It was therefore of interest to determine how epithelial cells from this immune privileged tissue respond to S. aureus. Further, it was of interest to determine whether cytolytic toxins, with the potential to cause ion flux or potentially permit effector molecule movement across the target cell membrane, alter the response. Microarrays were used to globally assess the response of human corneal epithelial cells to S. aureus. A large increase in abundance of transcripts encoding the antimicrobial dendritic cell chemokine, CCL20, was observed. CCL20 release into the medium was detected, and this response was found to be largely TLR2 and NOD2 independent. Corneal epithelial cells also respond to S. aureus by increasing the intracellular abundance of mRNA for inflammatory mediators, transcription factors, and genes related to MAP kinase pathways, in ways similar to other cell types. The corneal epithelial cell response was surprisingly unaffected by toxin exposure. Toxin exposure did, however, induce a stress response. Although model toxigenic and non-toxigenic strains of S. aureus were employed in the present study, the results obtained were strikingly similar to those reported for stimulation of vaginal epithelial cells by clinical toxic shock toxin expressing isolates, demonstrating that the initial epithelial cellular responses to S. aureus are largely independent of strain as well as epithelial cell tissue source.

Upregulation of Tumor Necrosis Factor Receptor 1 and TNF-α Converting Enzyme During Corneal Wound Healing

PurposeTo elucidate tumor necrosis factor (TNF) receptor-1 (TNFR1) and TNF-α converting enzyme (TACE) involvement during corneal wound healing.MethodsThe corneas of BALB/c mice cornea were scarred by alkali burns using filter paper dipped in 1N NaOH solution. TACE and TNFR1 expression in the alkali-burned corneas were evaluated by immunohistochemistry (total, 16 eyes). Using cultured fibroblasts (human) and macrophages (mice), we evaluated the release of soluble TNF-α (sTNF-α) and soluble TNFR1 (sTNFR1) in the supernatant by enzyme-linked immunosorbent assay after stimulating TACE activity with phorbol myristate acetate (PMA).ResultsIn alkali-burned corneas, both TACE and TNFR1 expression were observed in the stromal cells after the acute phase of wound healing response. In macrophage-cultured supernatant, both sTNF-α and sTNFR1 release were promoted by PMA stimulation. On the other hand, only sTNFR1 released by PMA stimulation was observed in fibroblast-cultured supernatant.ConclusionsTACE and TNFR1 were expressed mainly after the acute phase of corneal wound healing. The TACE-dependent extracellular release of sTNFR1 was recognized in cultured fibroblasts and macrophages.

Upregulation of matrix metalloproteinase in tear fluid of patients with recurrent corneal erosion.

PurposeTo investigate gelatinase [matrix metalloproteinase (MMP)-2 and MMP-9] expression in the tear fluid of patients with recurrent corneal erosion (RCE).MethodsEleven patients with RCE, three patients with traumatic corneal erosion, and 10 control individuals were enrolled in this study. Tear samples from RCE eyes were obtained once, either at the time of recurrence (onset-phase samples, seven samples), or during the remission period (remission-phase samples, four samples). Tear samples from the nonaffected fellow eyes of the RCE patients were also examined once (fellow-eye samples, ten samples from ten patients). In addition, three samples from three patients with traumatic corneal erosion and ten samples from ten control individuals were obtained as well. Tear samples were collected by a modified Schirmer test I method and analyzed by gelatin zymography.ResultsNeither the active form of MMP-2 nor that of MMP-9 was detected in the samples from traumatic corneal erosion patients and control individuals. Both active MMP-2 and active MMP-9 were detected in all seven onset-phase samples. Active MMP-2 and active MMP-9 were detected in three of the four remission-phase samples. Although none of the ten fellow eyes had a history of RCE, active MMP-2 and active MMP-9 were detected in three fellow-eye samples.ConclusionsGelatinase expression was upregulated in the tear fluid of RCE patients. The presence of gelatinase in the affected eye during the remission phase as well as in the nonaffected fellow eye indicates that gelatinase expression in the tear fluid may be related to the recurrence of corneal erosion. Jpn J Ophthalmol 2007;51:343–346

Release of Soluble Tumor Necrosis Factor Receptor 1 from Corneal Epithelium by TNF-α–Converting Enzyme-Dependent Ectodomain Shedding

PURPOSE An involvement of tumor necrosis factor-alpha-converting enzyme (TACE)-dependent ectodomain shedding in the release of soluble tumor necrosis factor receptor 1 (sTNFR1) from corneal epithelium was evaluated. METHODS In vitro experiments were performed using the human SV40-transformed human corneal epithelial cell (HCEC) line. Ectodomain shedding was stimulated by phorbol myristate acetate (PMA, 3 microM) or peptidoglycan (PGN, 100 microg/mL), with or without TACE inhibition, using TNF-alpha processing inhibitor-1 (TAPI-1, 250 microg/mL) or tissue inhibitor of metalloproteinase-3 (TIMP-3, 2 microg/mL) by addition to the HCEC culture medium. The concentrations of sTNFR1 in culture medium were analyzed by enzyme-linked immunosorbent assay. To induce an inflammatory response in the ocular surface, corneal alkali burn of BALB/c mice was made from a filter paper dipped in 1 N NaOH solution. TNFR1 expression in corneal and conjunctival epithelia was evaluated by immunohistochemistry 28 days after wounding. RESULTS In HCEC culture medium, sTNFR1 release was significantly increased by the addition of PMA (t-test, P < 0.01) or PGN (P < 0.01). The increased release of sTNFR1 was significantly inhibited by the addition of TAPI-1 or TIMP-3, indicating the possibility of TACE-dependent ectodomain shedding of TNFR1. In the corneal alkali burn model, TNFR1 was expressed in corneal and conjunctival epithelia. CONCLUSIONS TACE-dependent ectodomain shedding of sTNFR1 was recognized in corneal epithelium. In the inflamed ocular surface, TNFR1 was expressed in the corneal and conjunctival epithelia after alkali burn treatment. sTNFR1, released from the ocular surface, may play an anti-inflammatory role in the inflammatory condition.

Host-pathogen interactions in the cornea

The cornea is exposed to microorganisms occurring in the conjunctival sac and in the environment, yet host defense systems can usually prevent infection and maintain visual function. Virulence factors of pathogens enhance their ability to cause infection, especially when the ocular surface is perturbed such as through contact lens wear, trauma, or ocular surgery. Infectious keratitis occurs when the sum of surface perturbation plus bacterial virulence exceeds the ability of host clearance mechanisms to eliminate the microbe. Thus, understanding the dynamic between a pathogen and the host response in the cornea is central both to preventing infection and for developing new and effective treatments and prophylaxis measures.