Differential regulation of IL‐1 and IL‐1 receptor antagonist in HaCaT keratinocytes by tumor necrosis factor‐α and transforming growth factor‐β1

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Abstract
Cytokines such as TNFα and TGFβ1 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), TNFα and TGFβ1 were added to HaCaT cells, a human adult keratinocyte cell line. mRNA levels of IL‐1α, IL‐1β, and IL‐IRa 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. TNFα (100 ng/ml) at the 3‐h time point significantly induced increases in mRNA expression of Iliα (9.2±2.9 fold increase) and IL‐1β (2.5±0.7 fold increase) (n=7) which were concordant with increases in IL‐1α protein (7.1±1.3 fold increase) and II‐β protein (4.4±1.0 fold increase) measured by ELISA 24 h after stimulation. By contrast, icIL‐IRa mRNA and protein levels were not affected by TNFα. TGFβl induced a mild increase in IL‐lα mRNA (3.8±1.8 fold) and protein (3.5±1.2 fold). TGFβl did not affect IL‐1α mRNA levels but caused variable increases in IL‐1β protein levels. TGFβ1 did not alter icIL‐1Ra mRNA or protein levels. Inhibition of RNA synthesis with actinomycin D demonstrated that the rate of degradation of IL‐1β mRNA was reduced by treatment with TNFα. This stabilization of IL‐1β mRNA was specific, because TGFP I did not stabilize IL‐1β mRNA, and TGFβ1 and TNFα did not increase the stability of II‐1α mRNA. icIL‐l Ra mRNA was fairly stable over a 20 hour period and its slow degradation was not affected by treatment with either TNFα or TGFβ1, indicating a higher steady state stability of icIL‐1ra mRNA relative to IL‐1 mRNA's. Given the high rate of degradation of IL‐1α and IL‐1β 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, TNFα and TGFβl, cytokines with potent effects on inflammation and differentiation, both induce keratinocyte IL‐1α mRNA and protein levels, but differentially regulate IL‐1β 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.