Differential regulation of IL-1 and IL-1 receptor antagonist in HaCaT keratinocytes by tumor necrosis factor-alpha and transforming growth factor-beta 1.

Abstract

Cytokines such as TNF alpha and TGF beta 1 have potent effects on keratinocyte differentiation and have been implicated in cutaneous injury, immunologic reactions, and wound healing. To determine whether such conditions might alter the balance of epidermal keratinocyte IL-1 and the IL-1 receptor antagonist (IL-1ra), TNF alpha and TGF beta 1 were added to HaCaT cells, a human adult keratinocyte cell line. mRNA levels of IL-1 alpha, IL-1 beta, and IL-1Ra were detected by polymerase chain reaction (PCR) on reverse transcribed RNA extracts, followed by Southern blot of the PCR products, 35S-labeled probe hybridization, and quantification against standard curves. TNF alpha (100 ng/ml) at the 3-h time point significantly induced increases in mRNA expression of II-1 alpha (9.2 +/- 2.9 fold increase) and IL-1 beta (2.5 +/- 0.7 fold increase) (n=7) which were concordant with increases in IL-1 alpha protein (7.1 +/- 1.3 fold increase) and Il-beta protein (4.4 +/- 1.0 fold increase) measured by ELISA 24 h after stimulation. By contrast, icIL-1Ra mRNA and protein levels were not affected by TNF alpha. TGF beta 1 induced a mild increase in IL-1 alpha mRNA (3.8 +/- 1.8 fold) and protein (3.5 +/- 1.2 fold). TGF beta 1 did not affect IL-1 beta mRNA levels but caused variable increases in IL-1 beta protein levels. TGF beta 1 did not alter icIL-1Ra mRNA or protein levels. Inhibition of RNA synthesis with actinomycin D demonstrated that the rate of degradation of IL-1 beta mRNA was reduced by treatment with TNF alpha. This stabilization of IL-1 beta mRNA was specific, because TGF beta 1 did not stabilize IL-1 beta mRNA, and TGF beta 1 and TNF alpha did not increase the stability of Il-1 alpha mRNA. icIL-1Ra mRNA was fairly stable over a 20 hour period and its slow degradation was not affected by treatment with either TNF alpha or TGF beta 1, indicating a higher steady state stability of icIL-1ra mRNA relative to IL-1 mRNA's. Given the high rate of degradation of IL-1 alpha and IL-1 beta mRNA, levels of these mRNAs may rapidly decrease while the icIL-1ra mRNA levels remain constant, thus allowing for rapid dampening of IL-1 activity soon after the stimuli provoking an inflammatory or reparative response have abated. In conclusion, TNF alpha and TGF beta 1, cytokines with potent effects on inflammation and differentiation, both induce keratinocyte IL-1 alpha mRNA and protein levels, but differentially regulate IL-1 beta mRNA. They both exert little effect on IL-1 Ra levels, which were constitutively highly stable. Such differential regulation provides mechanisms for separately controlling the relative activity of these cytokines under normal and disordered conditions.