Interleukin-10 Production by Cultured Human Keratinocytes: Regulation by Ultraviolet B and Ultraviolet A1 Radiation

Abstract

Keratinocytes are the primary cellular target for ultraviolet radiation in human skin, and ultraviolet radiation-induced therapeutical effects may thus be mediated by keratinocyte-derived, antiinflammatory mediators. Interleukin-10 is capable of exerting antiinflammatory effects by virtue of its capacity to suppress the production of interferon-gamma. The present study therefore assessed the ability of cultured human keratinocytes to produce interleukin-10 following ultraviolet irradiation. Exposure of long-term cultured normal human keratinocytes to ultraviolet B (280-320 nm) or to ultraviolet A1 (340-400 nm) radiation caused a time- and dose-dependent induction of interleukin-10 mRNA expression and interleukin-10 protein secretion, with ultraviolet A1 radiation being the strongest stimulus. Ultraviolet radiation-induced interleukin-10 production by normal human keratinocytes was enhanced by a factor of two, when cells were cultured in high- rather than low-calcium medium. Neither addition of the ultraviolet radiation-inducible cytokines tumor necrosis factor-alpha or interleukin-1 alpha to unirradiated keratinocytes nor presence of their respective neutralizing antibodies in cultures of irradiated keratinocytes induced or inhibited interleukin-10 synthesis. Modulation of eicosanoid production by addition of prostaglandin E2 to keratinocyte cultures or disturbance of cyclooxygenase activity by indomethacin did not affect interleukin-10 production in resting or irradiated cells. These studies demonstrate that cultured human keratinocytes are capable of producing interleukin-10. Human keratinocyte interleukin-10 production is dependent on the differentiation state of the cell and induced by ultraviolet B and, in particular, ultraviolet A1 radiation exposure. This novel property of ultraviolet radiation may account at least in part for the efficacy of phototherapy in inflammatory skin diseases.

In contrast to their murine counterparts, normal human keratinocytes and human epidermoid cell lines A431 and HaCaT fail to express IL-10 mRNA and protein

“…Given the facts that (i) the increase and peak of the IL-10 mRNA signal correlates with the appearance of the macrophages in the epidermis, (ii) these macrophages are potent producers of IL-10, and (iii) the purity of the magnetic bead-selected NHK suspension was stated to be > 90% (not 100%), it is tempting to believe that the IL-10 mRNA detected by Kang et al [43] must be macrophage-derived. In contrast to our negative results, Grewe et al [38] and Enk et al [44] detected IL-10 mRNA in long-term cultures of NHK and in five out of nine blister roofs from non-irradiated skin by RT-PCR analysis. Comparison of their experimental set-up with ours revealed that in essence we all used the same culture conditions, but that the RT-PCR performance and visualization of the PCR products were different.…”

“…To our surprise, four independent research groups recently reported that NHK were able to express IL-10 mRNA and protein [38,[41][42][43][44]. Although at first sight these reports appear to disagree with our findings, we can nevertheless explain how the results in these reports and our data fit together.…”

“…However, this method also did not lead to IL-10 mRNA detection in NHK (data not shown). In a very recent publication, it was suggested that UVA1 or UVB in combination with high Ca concentration would be the strongest stimulus for IL-10 production by NHK [38]. Unfortunately, we were not able to demonstrate this (data not shown).…”

“…Comparison of their experimental set-up with ours revealed that in essence we all used the same culture conditions, but that the RT-PCR performance and visualization of the PCR products were different. We used ethidium bromide to stain these products in agarose gels, whereas both other groups used other techniques: endonuclease digestion assay followed by high performance liquid chromatography [38] and liquid hybridization with a 32 P-labelled internal probe followed by autoradiography [44]. Because Grewe et al and Enk et al were able to detect IL-10 mRNA in all their NHK cultures after a single round of PCR amplification and we needed two rounds to pick up the IL-10 signal, it might be concluded that they used a more sensitive technique.…”

“…Just prior to submission of this study, four different research groups stated that NHK are able to produce significant amounts of IL-10 mRNA and protein [38,[41][42][43][44]. These findings seem to contradict our own, and this prompted us to attempt to resolve this conflict.…”