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Nitrite production in mouse 3T3 fibroblasts by bleomycin-stimulated peripheral blood mononuclear cell factors
T Yamamoto1, I Katayama, K Nishioka
1Department of Dermatology, Tokyo Medical and Dental University, School of Medicine, Japan.
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.
Insights
Bleomycin stimulates peripheral blood mononuclear cells (PBMCs) to release nitric oxide (NO). These activated PBMCs then induce fibroblasts to produce NO, suggesting a role for NO in bleomycin-induced tissue fibrosis.
Area of Science:
- Immunology
- Cell Biology
- Pathogenesis of Fibrosis
Background:
- Bleomycin is known to induce tissue fibrosis, but its exact pathogenesis remains unclear.
- Nitric oxide (NO) is a key mediator in immune and inflammatory responses, potentially involved in autoimmune disorders.
- Peripheral blood mononuclear cells (PBMCs) stimulated by bleomycin exhibit fibroblast growth-promoting activity.
Purpose of the Study:
- To investigate whether bleomycin-stimulated PBMCs induce nitrite production in mouse 3T3 fibroblasts in vitro.
- To explore the role of nitric oxide (NO) in the interaction between PBMCs and fibroblasts following bleomycin stimulation.
Main Methods:
- Peripheral blood mononuclear cells (PBMCs) from systemic sclerosis (SSc) patients and healthy volunteers were stimulated with bleomycin.
- Culture supernatants (conditioned medium, CM) were used to treat mouse 3T3 fibroblasts.
- Nitrite production was quantified using Griess reagents, and inducible NO synthase (iNOS) mRNA expression was analyzed via RT-PCR.
Main Results:
- Bleomycin-stimulated PBMCs released significant amounts of nitrite in a time-dependent manner.
- Conditioned medium (CM) from bleomycin-stimulated PBMCs increased nitrite production and iNOS mRNA expression in 3T3 fibroblasts.
- Inhibition of interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), or iNOS significantly reduced nitrite production.
Conclusions:
- Bleomycin, IL-1 beta, and TNF-alpha induce NO release from mononuclear cells.
- These cytokines stimulate fibroblasts to produce NO.
- NO may play a role in the development of bleomycin-induced tissue fibrosis.