Modulation of the IL-1 cytokine network in keratinocytes by intracellular IL-1α and IL-1 receptor antagonist

Abstract

The IL-1 cytokine network in epidermal cells was studied in vitro, using the spontaneously transformed HaCAT human keratinocyte line. Intracellular (ic) IL-1α and IL-1 receptor antagonist protein (IL-1Ra) following cell lysis were readily identified assayed using a capture ELISA; whereas in culture supernatants IL-1Ra was not detected, and IL-1α was present at only very low levels. Confluent cultures of HaCAT cells were shown to provide optimal conditions for the study, since confluence increased the icIL-1Ra:IL-1α ratio to a level as seen in vivo, which was independent of Ca2+ concentration in the culture medium. The IL-1Ra extracted from HaCAT cell lysates was functionally active, as demonstrated in the mouse thymocyte co-proliferation assay which could be blocked using a rabbit anti-IL-1Ra antibody. Transforming growth factor-beta (TGF-β1) stimulated a dose-dependent increase in HaCAT cell IL-1α without changing IL-1Ra concentration, with a resultant reduction in the icIL-1Ra:IL-1α ratio from 320:1 to 100:1. Similarly, TGF-α, interferon-gamma (IFN-γ), IL-6, and tumour necrosis factor-alpha (TNF-α) substantially increased HaCAT cell IL-1α, but had no effect on the IL-1Ra, with a concomitant reduction in the icIL-1Ra:IL-1α ratio. In contrast to their effects on monocytes, IL-4 and IL-10 at biologically active levels had no effect on IL-1α, IL-1Ra or the icIL-1Ra:IL-1α ratio in confluent HaCAT cells. Hydrocortisone reduced IL-1α to below the limit of sensitivity of the ELISA, and induced a small increase in IL-1Ra of questionable biological significance. Thus, regulation of the IL-1 cytokine network in keratinocytes involves modulation of icIL-1α rather than of icIL-1Ra levels, and is markedly different from that noted in monocytes.