Neutralization effects of interleukin-6 (IL-6) antibodies on sulfur mustard (HD)-induced IL-6 secretion on human epidermal keratinocytes.

The proinflammatory cytokine human interleukin-6 (hIL-6) plays an important role in the early and late courses of inflammation, trauma, and wound healing caused by sulfur mustard (HD). Previously, we demonstrated that hIL-6 might be involved in the early event of structural changes of the signal transducer glycoprotein, which indirectly initiates the cascade of events, such as skin irritation and blister formation observed in the pathophysiology of HD injury. In this present work, we focus on the neutralization effect of IL-6 antibodies with regard to the modulation of hIL-6 secretion. Levels of secreted cytokine hIL-6 in normal human epidermal keratinocytes (NHEK) stimulated with HD (10(-4)M) and incubated for 24h at 37°C were determined by enzyme immunoassay, protein immunocytologic assay and reverse-transcriptase-polymerase chain reaction (RT-PCR). The ratio of HD-treated NHEK to constitutive non-stimulated NHEK controls (S/C) on the induction of hIL-6 is reported. S/C was four-fold higher than non-stimulated NHEK controls as determined by ELISA. By using a more sensitive immunocytologic assay, Luminex(100)™, the increment was verified. hIL-6 levels in NHEK stimulated with HD were 21±11ng/mL as measured by Luminex(100)™. The messenger RNA expression of the cytokine (hIL-6) gene was analyzed semiquantitatively. RT-PCR demonstrated that HD induced an increase in the transcription of hIL-6 gene. Selective immunosuppression, using IL-6 neutralizing antibodies, led to a reduction of such expression of HD-induced transcription of hIL-6 in human keratinocytes. The neutralization by pre-incubating NHEK with monoclonal anti-IL6 antibodies decreased hIL-6 secretion by 76%±1.8 ((*)P<0.05).