Expression of Microbial Receptors and Production of Cytokine in Mouse Keratinocyte Cells in Response to Lipopolysaccharide Stimulation

Jang, Ho Sun; Kim, Cheon Ki; Jo, Ju Huyn; Lee, Jae Bong; Kim, Moon Bum; Oh, Chang Keun; Kwon, Kyung Sool

Abstract

BACKGROUND
Microbial receptors play an important role in host defense. Microbial receptors- Toll-like receptor 4 (TLR4), CD14, CD11b, and CD18-mediate the responsiveness to Gram-negative lipopolysaccharide (LPS). The host response to LPS is characterized by an influx of inflammatory cells into host tissues, which is mediated, in part, by localized production of cytokines. Although the expression of microbial receptors and production of cytokines in macrophages are well studied, the functional responses of keratinocytes to stimulation with LPS are not well known. OBJECTIVES: We investigated the expression of microbial receptors and production of cytokine in response to LPS in mouse keratinocytes. METHODS: RAW264.7 cells, mouse macrophage cell line, were used as a positive control. Balbc/mk and PAM212 cells, mouse keratinocyte cell lines, were checked for the expression of microbial receptors and production of cytokine in response to LPS stimulation by flow cytometry and ELISA. RESULTS: RAW264.7 cells constitutively expressed high levels of TLR4, CD14, CD11b, and CD18. Balbc/mk and PAM212 cells also constitutively expressed high levels of CD14 and TLR4 similarily with RAW264.7 cells, but the levels of CD11b and CD18 expression was constitutively low. LPS stimulation resulted in increased production of TNF-alpha in RAW264.7 cells and mouse KC in mouse keratinocytes. In addition, LPS stimulation induced up-regulation of TLR4, CD14, CD11b, and CD18 in RAW264.7 cells, and TLR4 and CD14 in mouse keratinocytes. Anti-receptor specific antibodies efficiently blocked mouse KC production in mouse keratinocytes stimulated by low-dose LPS (100ng/ml). CONCLUSION: Taken together, these results indicate that mouse keratinocytes constitutively express microbial receptors and produce cytokine in response to LPS stimulation. Therefore, keratinocytes can actively participate in innate immunity against pathogens by secreting inflammatory cytokines through microbial receptor recognition.