Chemokine expression by human keratinocyte cell lines after activation of Toll-like receptors

Abstract

Keratinocytes in the skin play an important role in innate immune responses by secreting chemokines. This study aimed to determine if keratinocyte cell lines can be used for studies of innate immune mechanisms. Human primary keratinocytes and the HaCaT, CCD 1106 KERTr (KERTr) and HEK001 cell lines were treated with a panel of Toll-like receptor (TLR)-ligands. Expression of IL-8, CCL20, CXCL9 and CXCL10 was determined. All three cell lines expressed TLR1-6 and TLR9. KERTr cells responded to the same TLR-ligands as primary keratinocytes. Overall HEK001 responded similarly, but appeared to be relatively more sensitive to flagellin. This was in agreement with increased expression of TLR5. The expression profiles were most distinct in HaCaT cells. Furthermore, our data confirm and extend previously reported TLR7 and TLR8 independent IL-8 secretion by keratinocytes after Imiquimod treatment. The different cell lines represent complementary tools for molecular studies of innate immunity of the skin.

Figure 1

TLR mRNA expression in primary keratinocytes and the KERTr, HEK001 and HaCaT cell lines. Total RNA was isolated from 6 independent batches of primary keratinocytes and KERTr, HEK001 and HaCaT cells. TLR mRNA levels were determined using real-time RT-PCR and the comparative C_T method. TLR mRNA levels were normalized against levels of GAPDH mRNA and are graphically represented relative to the levels in the primary keratinocyte Lot 88. *, P < 0.05 (compared to primary keratinocytes Lot 88). **, P < 0.01 (compared to primary keratinocytes Lot 88). #, Not determined.

Figure 2

IL-8, CCL20, CXCL9 and CXCL10 production in primary keratinocytes, KERTr, HEK001 and HaCaT cell lines after stimulation with TLR-ligands. Primary keratinocytes (a) and the keratinocyte cell lines KERTr (b), HEK001 (c) and HaCaT (d) were treated with medium only, LPS, LTA, PGN, poly(I:C), flagellin, E. coli DNA or zymosan for 24 hours. Levels of IL-8, CCL20, CXCL9 and CXCL10 in the culture medium were determined using ELISA. *, P < 0.05 (compared to cells treated with medium only). **, P < 0.01 (compared to cells treated with medium only). ND, not detected.