Expression analysis of chemokines in human oral keratinocytes and fibroblasts

Objectives: INF-gamma and TNF-alpha, Th1 cytokines, produced by activated Th1 cells are thought to determine immunological activity in oral inflammatory disease such as Lichen planus. IL-4, Th2 cytokine, is a negative regulator of Th1 cytokine action. Th2 cytokines, as well as Th1 cytokines, are expressed in T-cell-mediated inflammatory lesions. The aim of this study is to examine the effect of these cytokines on the chemokine expression in neighboring cells, including oral keratinocytes and oral fibroblasts.

Methods: We used an immortalized human oral keratinocytes (RT7) and human gingival fibroblasts (GT1) established by transfection of human telomerase transcriptase (hTERT). The time-course effect of INF-gamma and TNF-alpha on the mRNA expression of ten chemokines, CCL2, CCL5, CCL20, CXCL5, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, and CX3CL1 in RT7 and GT1 was investigated by using real time-PCR. Subsequently, we investigated the effect of IL-4, a Th2 cytokine, on chemokine expression strongly affected by INF-gamma and TNF-alpha in the two cell types by real time-PCR and ELISA.

Results: RT7 and GT1 showed different mRNA expression patterns of all the chemokines by stimulation with INF-gamma or TNF-alpha. The addition of INF-gamma to both cell cultures resulted in a much greater increase in CXCL9, CXCL10, and CXCL11, each Tcell-recruiting chemokines, than the other chemokines. TNF-alpha significantly increased CXCL9, CXCL10, and CXCL11 mRNA expressions in GT1 cells, but not in RT7 cells. IL-4 enhanced CXCL9, CXCL10, and CXCL11 mRNA and protein levels induced by INF-gamma in RT7 cells, whereas IL-4 abolished the increase in those levels induced by INF-gamma and TNF-alpha in GT1 cells.

Conclusions: Our findings suggest that INF-gamma, TNF-alpha, and IL-4 may cooperatively regulate the expression of CXCL9, CXCL10, and CXCL11 in oral keratinocytes and oral fibroblasts to promote and shape oral inflammatory disease such as Lichen planus.