Nitrite production in mouse 3T3 fibroblasts by bleomycin-stimulated peripheral blood mononuclear cell factors.

OBJECTIVE It is well known that bleomycin induces tissue fibrosis, including pulmonary fibrosis or scleroderma-like conditions. However, the pathogenesis has not been completely elucidated. We recently observed that peripheral blood mononuclear cells (PBMCs) stimulated by bleomycin show growth stimulatory activity for fibroblasts. Nitric oxide (NO) is an important mediator of immune and inflammatory responses, and has recently been suggested to play a role in the pathogenesis of autoimmune disorders. In this study, we have examined whether bleomycin-stimulated PBMCs induce nitrite production in mouse 3T3 fibroblasts in vitro. METHODS PBMCs were obtained from 6 patients with systemic sclerosis (SSc) and 6 normal volunteers, and stimulated by bleomycin; the culture supernatants were collected as conditioned medium (CM). The release of nitrite from 3T3 fibroblasts after incubation with CM was determined by Griess reagents. Induction of inducible NO synthase (iNOS) mRNA expression in 3T3 fibroblasts after incubation with CM was examined by the reverse transcriptase-polymerase chain reaction (RT-PCR) method. RESULTS Bleomycin induced significant nitrite release from PBMCs in a time-dependent manner. Stimulation with CM increased nitrite production in 3T3 fibroblasts, which was significantly inhibited by antibodies against interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), and iNOS inhibitor, L-NMMA. CM from SSc patients induced higher amounts of nitrite from 3T3 fibroblasts, compared with that from normal subjects, although the difference was not significant. CM induced iNOS mRNA expression in 3T3 fibroblasts in a time-dependent manner. CONCLUSION These results suggest that bleomycin, as well as IL-1 beta and TNF-alpha, induce NO release from mononuclear cells, and that these cytokines furthermore stimulate fibroblasts to produce NO, which raises the possibility of the involvement of NO in the development of tissue fibrosis induced by bleomycin.