Human Conjunctival Epithelial Cells Lack Lipopolysaccharide Responsiveness Due to Deficient Expression of MD2 but Respond After Interferon-γ Priming or Soluble MD2 Supplementation

Abstract

Inflammatory responses to Gram-positive and Gram-negative bacterial cell wall components are initiated by Toll-like receptor 2 (TLR2) and TLR4, respectively. Therefore, the existence of functionally active TLR2 and TLR4 in human conjunctival epithelial cells (HCEC) are critical for the effective host defense against bacterial infections in the eye. We examined the ability of HCEC to respond to TLR4 ligand, lipopolysaccharide (LPS), or TLR2 ligands, lipoteichoic acid (LTA) and peptidoglycan (PGN) using the Chang conjunctival epithelial cell line and the primary conjunctival epithelial cell line (IOBA-NHC) as in vitro models. Incubation of Chang cells with LPS (1 to 1,000 ng/ml) failed to stimulate IL-6 production where as stimulation with LTA or PGN resulted in marked increases in IL-6 production. Semi-quantitative RT-PCR and immunofluorescence analyses showed that Chang cells express TLR2 and TLR4 mRNA and proteins. However, these cells expressed little or no mRNA encoding MD2, an accessory molecule required for TLR4 signaling. Incubation of Chang epithelial cells with interferon-gamma (IFNgamma), but not TNF-alpha, stimulated MD2 mRNA expression and restored LPS responsiveness. In addition, when Chang cell cultures were supplemented with soluble MD2, LPS was able to stimulate IL-6 production. The lack of LPS response, deficient expression of MD2, and induction of MD2 expression and LPS response after IFNgamma priming, were also evident in IOBA-NHC cells. These results demonstrate that HCEC lack LPS responsiveness due to deficient expression of MD2 and that the response can be restored by IFN-gamma priming or MD2 supplementation.

Pseudomonas aeruginosa Induces Interleukin-8 (IL-8) Gene Expression in Human Conjunctiva through the Recruitment of Both RelA and CCAAT/Enhancer-binding Protein β to the IL-8 Promoter

“…3 and 8) either in Chang or in primary cell lines. The results we obtained strongly indicate that the Chang cell line responds to P. aeruginosa similarly to primary cells in accord with other studies showing that the two cell types had a similar biological behavior (30,38,39). Although JNK and c-Jun were also activated by P. aeruginosa, this pathway had little role on bacteria-stimulated IL-8 gene induction and was only responsible for its basal expression.…”

Modulation of Corneal Epithelial Innate Immune Response to Pseudomonas Infection by Flagellin Pretreatment

“…As for Gram-negative bacteria such as P. aeruginosa, we observed that HCECs do not recognize LPS as they lack MD2 (Yu, unpublished results, 2006), in line with other mucosal epithelial cells. 10,34 We showed that the initial challenge of cells with flagellin induces a proinflammatory response and proposed that TLR5 is a major sensor on the ocular surface for detecting P. aeruginosa.…”

Histamine Directly and Synergistically with Lipopolysaccharide Stimulates Cyclooxygenase-2 Expression and Prostaglandin I2 and E2 Production in Human Coronary Artery Endothelial Cells

“…It is well recognized that TLRs are critical components of the innate immune system and each of these TLRs recognizes a distinct pathogen-associated molecule to initiate the inflammatory response (38 -43). Among the TLRs identified to date, TLR4, in association with its accessory molecules, recognizes LPS (43)(44)(45)(46)(47). Recently, we demonstrated that histamine has the ability to stimulate the expression of TLR4 mRNA and protein, and amplify LPS-induced production of cytokines in HUVECs (16).…”