Nitrite synthesis in a murine macrophage cell line stimulated by products released from irritant-treated normal human...

Nitric oxide is produced in dermal inflammatory reactions; its role as an irritant mediator was therefore examined. Nitrite accumulation (an indicator of nitric oxide synthesis) was measured in human keratinocytes, NCTC 2544 cells and RAW 264 macrophages, following stimulation by lipopolysaccharide (LPS), sodium dodecyl sulfate (SDS) or ultraviolet irradiation (UVA). Nitrite synthesis was low in primary cultured keratinocytes (passage 3) at 0.36 +/- 0.16 nmol/well. This was minimally affected by LPS (0.58 +/- 0.1 nmol/well). SDS and UVA failed to modify keratinocyte nitrite production. No nitrite synthesis was detected in NCTC 2544 cells. RAW 264 macrophages responded to LPS (0-10 mug/ml) by increasing nitrite production from 2 +/- 0.6 to 22 +/- 4.5 nmol/well in keratinocyte serum-free media. Responses of macrophages to LPS were diminished in Dulbecco's media containing serum or dialysed serum. Human interleukin-1 (0-30 pg/ml) increased macrophage nitrite production and l-N-monomethyl arginine (l-NMMA) (0-1 mm) inhibited nitrite synthesis. Media conditioned by keratinocytes for 24 hr following treatment with SDS (0-100 mug/ml for 0-8 hr) or UVA (1.6 mW/cm(2) for 0-4 hr) enhanced macrophage nitrite synthesis. Conditioned media from UVA-treated NCTC 2544 cells failed to stimulate macrophage nitrite synthesis.