Tetracyclines modulate protease-activated receptor 2-mediated proinflammatory reactions in epidermal keratinocytes

Abstract

In addition to their antibiotic effects, tetracyclines have anti-inflammatory action that is often beneficial in the control of inflammatory skin disorders. In this study, we examined the effects of tetracycline (TET) and two of its derivatives, doxycycline (DOX) and minocycline (MIN), on the production of interleukin-8 (IL-8) elicited by the activation of protease-activated receptor 2 (PAR2) in normal human epidermal keratinocytes (NHEK). In NHEK, the production of IL-8 stimulated by an agonist peptide of PAR2, SLIGKIV-NH(2), at 100 microM was significantly reduced by TET, DOX, or MIN at 5 and 10 microM, concentrations that are noncytotoxic. The tumor necrosis factor alpha (TNF-alpha)-induced production of IL-8 was synergistically augmented by SLIGKIV-NH(2), and that synergistic increase in the production of IL-8 was suppressed by 100 nM PAR2-specific small interfering RNA. It was also suppressed by TET, DOX, or MIN but not by the 14-membered-ring macrolide antibiotics erythromycin, roxithromycin, and clarithromycin, which also have anti-inflammatory activities, at 10 microM. These results suggest that tetracyclines attenuate the PAR2-IL-8 axis in keratinocytes and thereby effectively modulate proinflammatory responses in the skin.

FIG. 1.

Activation and function of PAR2 in keratinocytes. Serine proteases cleave the N-terminal arm of PAR2, and the exposed new N-terminal peptide can activate the receptor. The activation of PAR2 accelerates the release of IL-8 from the cells and increases the expression of ICAM-1 and the uptake of melanin from melanocytes but suppresses the secretion of lipid lamellae, which is essential for skin barrier homeostasis.

FIG. 2.

Effects of tetracyclines on the SLIGKV-NH₂-induced production of IL-8 by NHEK and on the expression of the PAR2 gene. (A to C) In the presence or absence of the indicated concentration of TET (A), DOX (B), or MIN (C), NHEK were treated with 100 μM SLIGKV-NH₂ for 48 h, after which the concentration of IL-8 in the culture medium was measured by ELISA. Open bars, control; filled bars, SLIGKV-NH₂-treated cells. Analysis of variance followed by Scheffe's test was performed for statistical analysis of data. *, P < 0.001; ***, P < 0.05. (D) Effect of TET, DOX, or MIN on the expression of the PAR2 gene in NHEK. NHEK were incubated without any drug (open bars) or with 10 μM TET, DOX, or MIN (filled bars) for 48 h, and the levels of PAR2 transcripts were determined by qRT-PCR.

FIG. 3.

Effects of SLIGKV-NH₂ and a PAR2-specific siRNA on TNF-α- or IFN-γ-induced production of IL-8 by NHEK. NHEK were transfected with 100 nM PAR2-specific siRNA for 24 h as indicated and were then treated with 100 ng/ml TNF-α (A) or IFN-γ (B) with or without 100 μM SLIGKV-NH₂. After 24 h of incubation, the concentration of IL-8 in the culture medium was measured by ELISA. Student's two-tailed t test was performed for statistical analysis of data. *, P < 0.001. Open bars, control; filled bars, cells transfected with the PAR2-specific siRNA.

FIG. 4.

Effects of tetracyclines on the synergistic production of IL-8 induced by SLIGKV-NH₂ in IL-1β- or TNF-α-treated NHEK. In the presence or absence of 10 μM TET (A), DOX (B), or MIN (C), NHEK were treated with 100 ng/ml IL-1β or TNF-α and/or 100 μM SLIGKV-NH₂ for 48 h. The concentration of IL-8 in the culture medium was then measured by ELISA. Open bars, control; filled bars, cells treated with TET, DOX, or MIN. Student's two-tailed t test was performed for statistical analysis of data. *, P < 0.001; **, P < 0.01; ns, not significant.

FIG. 5.

Effects of the 14-membered-ring macrolides ERY, RXM, and CLR on the synergistic production of IL-8 induced by SLIGKV-NH₂ in TNF-α-treated NHEK. In the presence or absence of 10 μM ERY (A), RXM (B), or CLR (C), NHEK were treated with 100 ng/ml TNF-α and/or 100 μM SLIGKV-NH₂ for 48 h. The concentration of IL-8 in the culture medium was then measured by ELISA. Open bars, control; filled bars, cells treated with ERY, RXM, or CLR. Student's two-tailed t test was performed for statistical analysis of data. ns, not significant.