title: Human conjunctival epithelial cells lack lipopolysaccharide responsiveness due to deficient expression of MD2 but respond after interferon-gamma 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.
authors.affiliation: Division of Allergy, Clinical Immunology and Rheumatology, Department of Medicine, Mail Stop 2026, The University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, KS 66160, USA.
authors.forename: Jaya
authors.initials: J
authors.lastname: Talreja
authors.forename: Kavitha
authors.initials: K
authors.lastname: Dileepan
authors.forename: Sanjeev
authors.initials: S
authors.lastname: Puri
authors.forename: Mohammad H
authors.initials: MH
authors.lastname: Kabir
authors.forename: David M
authors.initials: DM
authors.lastname: Segal
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Stechschulte
authors.forename: Kottarappat N
authors.initials: KN
authors.lastname: Dileepan
mesh_terms: D002460:Cell Line; D003228:Conjunctiva / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology*; D015850:Interleukin-6 / Q000096:biosynthesis*; D008024:Ligands; D008070:Lipopolysaccharides / Q000494:pharmacology*; D051218:Lymphocyte Antigen 96 / Q000172:deficiency* / Q000494:pharmacology*; D010457:Peptidoglycan / Q000494:pharmacology; D013682:Teichoic Acids / Q000494:pharmacology; D051195:Toll-Like Receptor 2 / Q000378:metabolism; D051197:Toll-Like Receptor 4 / Q000378:metabolism
chemical_list: D015850:Interleukin-6; C495377:LY96 protein, human; D008024:Ligands; D008070:Lipopolysaccharides; D051218:Lymphocyte Antigen 96; D010457:Peptidoglycan; C495342:TLR2 protein, human; C495345:TLR4 protein, human; D013682:Teichoic Acids; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; C009900:lipoteichoic acid; D007371:Interferon-gamma