Various members of the Toll-like receptor family contribute to the innate immune response of human epidermal keratinocytes

Abstract

Toll-like receptors (TLRs) are important pattern recognition molecules that activate the nuclear factor (NF)-kappaB pathway leading to the production of antimicrobial immune mediators. As keratinocytes represent the first barrier against exogenous pathogens in human skin, we investigated their complete functional TLR1-10 expression profile. First, reverse transcription-polymerase chain reaction (PCR) analysis revealed a very similar pattern of TLR mRNA expression when comparing freshly isolated human epidermis and cultured primary human keratinocytes. Thus, further experiments were carried out with primary keratinocytes in comparison with the spontaneously immortalized human keratinocyte cell line HaCaT. The quantitative expression of TLR1-10 mRNA in real-time PCR of primary human keratinocytes and HaCaT cells was analysed. Both cell types constitutively expressed TLR2, TLR3, TLR5, and to a lesser extent TLR10. TLR4 was only found in HaCaT cells, TLR1 to a higher degree in primary keratinocytes. In line with this, LPS induced mRNA expression of CD14 and TLR4 only in HaCaT cells. After stimulation with various TLR ligands, the NF-kappaB-activated chemokine interleukin-8 (IL-8) was measured. In primary keratinocytes and HaCaT cells the TLR3 ligand poly (I:C) was the most potent stimulator of IL-8 secretion. The TLR ligands peptidoglycan, Pam3Cys and flagellin which bind to TLR2, TLR1/TLR2 heterodimer, and TLR5, respectively, also induced IL-8 secretion, whereas no IL-8 was induced by LPS, R-848, loxoribine and cytosine guanine dinucleotide-containing oligodeoxynucleotide. A corresponding pattern was found in the RelA NF-kappaB translocation assay after ligand stimulation of primary keratinocytes. These studies provide substantial evidence for a functional TLR expression and signalling profile of normal human keratinocytes contributing to the antimicrobial defence barrier of human skin.

Figure 1

Real-time PCR for TLRs, CD14 and MD-2 in primary human keratinocytes (PK) and HaCaT cells. Cells were cultured in six-well-plates. Constitutive TLR expression of TLR1-10, CD14, and MD-2 mRNA was analysed by quantitative real-time PCR. Three different mRNA preparations of primary keratinocytes, each pooled from three independent donors, as well as three different cell preparations of HaCaT cells were used. Columns show the mean ± standard deviation of the mRNA amount. Real-time PCR was performed in duplicates.

Figure 2

IL-8 production of primary human keratinocytes and HaCaT cells after stimulation with various TLR ligands. Primary keratinocytes (a) and HaCaT cells (b) were cultured in 96-well-plates and stimulated with the following ligands at indicated final concentrations: TNF-α (50 ng/ml), LPS from E. coli 0127:B8 (100 ng/ml), Pam₃Cys (5 µg/ml), PGN (10 µg/ml), poly (I:C) and poly (A:U) (20 µg/ml each), recombinant flagellin (10 µg/ml), loxoribine (1 mm), R-848 (1 µg/ml), CpG and non-CpG-ODN (1 µm each). Normal cell culture medium was used as control. The concentration of secreted IL-8 in the medium after 24 hr of TLR ligand stimulation was measured by ELISA. Columns show the mean ± standard deviation of six independent wells. Shown is one representative IL-8 stimulation experiment out of four. Significant differences versus the control incubation (P < 0·001) were obtained for the following TLR ligands: TNF-α, Pam₃Cys, PGN, poly (I:C), flagellin, CpG and non-CpG-ODN (primary keratinocytes); TNF-α, PGN, poly (I:C) and flagellin (HaCaT cells).

Figure 3

Analysis of TLR3 expression (insert) and of IL-8 production of primary human keratinocytes after stimulation with the dsRNA analogue poly (I:C). Primary keratinocytes were tested for specificity of TLR3 activation by poly (I:C) using RNAse A digest. Medium (control), poly (I:C), and poly (A:U) were incubated overnight with RNAse A at a concentration of 20 µg/ml and were subsequently used for stimulation of keratinocytes. Medium (control), poly (I:C) and poly (A:U) were also used without prior RNAse treatment. TNF-α (50 ng/ml) was used as another positive stimulus for IL-8 induction in keratinocytes with (TNF-α + RNAse) and without RNAse treatment. The concentration of secreted IL-8 in the medium after 24 hr of ligand stimulation was measured by ELISA. Columns show the mean ± standard deviation of six independent wells. Shown is one representative IL-8 stimulation experiment out of three. Insert: expression of TLR3 in primary keratinocytes under baseline conditions (α-TLR3) and after 16 h stimulation with poly (I:C) (α-TLR3 + poly (I:C)) as detected by goat polyclonal antibody directed against the amino terminus of TLR3. Non-immune goat IgG was used as control.

Figure 4

Analysis of IL-8 production of primary human keratinocytes (PK) and HaCaT cells after stimulation with different flagellins. Two different flagellin preparations (B. burgdorferi; S. typhimurium) were tested on primary keratinocytes (a) and HaCaT cells (b). Recombinant flagellin from B. burgdorferi was used at a final concentration of 10 µg/ml, whereas native purified flagellin from S. typhimurium was used at a final concentration of 1 µg/ml. TNF-α (50 ng/ml) served as a positive stimulation control, medium alone (control) as a negative stimulation control. The concentration of secreted IL-8 in the medium after 24 hr of ligand stimulation was measured by ELISA. Columns show the mean ± standard deviation of six independent wells. Shown is one representative IL-8 stimulation experiment out of three.

Figure 5

RelA assay for NF-κB translocation of primary human keratinocytes after stimulation with various TLR ligands. For RelA staining of primary human keratinocytes, cells were incubated for 4 hr with medium (unstimulated) as a negative control, with TNF-α (50 ng/ml) as a positive control, and with the TLR ligands poly (I:C), PGN, Pam₃Cys, flagellin, and LPS at the same concentrations used for the IL-8 assay (see legend to Fig. 2). Incubation was stopped and cells were stained with a primary rabbit anti-p65 antibody followed by an FITC-labelled goat anti-rabbit antibody. Cells which are not reactive to the stimulus are represented by a cytoplasmic staining pattern, reactive cells by a nuclear one as denoted by arrows. Arrowheads point to cells representing a typical fluorescence pattern for each of the incubations.

Figure 6

Induction of TLR4, CD14 and MD-2 by LPS in primary keratinocytes (PK) and HaCaT cells. Cells were cultured in six-well-plates stimulated with LPS (100 ng/ml) from E. coli 0127:B8. The LPS-inducible expression of TLR4 (a), CD14 (b) and MD-2 (c) was quantified after 0, 2, 8 and 24 hr of LPS stimulation. Real-time PCR was performed as described in the legend to Fig. 1. The activity of the LPS preparations used was demonstrated in human monocyte-derived dendritic cells (d). After 24 h incubation with 100 ng/ml LPS (LPS from E. coli 0127:B8 and LPS from E. coli K235), the cells were stained for the activation markers CD83, CD86, and MHC class II (HLA-DR) and analysed by flow cytometry.

Figure 7

Analysis of IL-8 production of primary human keratinocytes (PK) and HaCaT cells after stimulation with LPS over a time period of 4 days. Primary keratinocytes and HaCaT cells were incubated with 100 ng/ml LPS from E. coli K235. After 24 hr, the supernatant was removed for analysis of IL-8 production. The same cells were incubated with a new preparation of LPS from E. coli K235 (100 ng/ml) for another 24 hr. This procedure was repeated over a time period of 4 days (96 hr). TNF-α (50 ng/ml) served as a positive stimulation control, medium alone (control) as a negative stimulation control. The concentration of secreted IL-8 in the medium after 24 hr of LPS stimulation was measured by ELISA. Columns show the mean ± standard deviation of six independent wells. Shown is one representative IL-8 stimulation experiment out of three.