Objective: To evaluate the role of bleomycin for cutaneous fibrosis, we investigated the effects of bleomycin stimulated peripheral blood mononuclear cell (PBMC) factors on in vitro proliferation of mouse 3T3 fibroblasts.
Methods: PBMC were incubated with 10(-4) to 10 microg/ml of bleomycin, and the supernatants (conditioned media) were collected after 48 h. 3H-TdR incorporation was assayed to examine the effects of conditioned medium on 3T3 fibroblast proliferation, which was also compared between 8 patients with systemic sclerosis (SSc) and 8 control subjects.
Results: Conditioned medium stimulated 3T3 fibroblasts to proliferate in a dose dependent manner, while culture supernatant without stimulation of bleomycin showed no growth stimulatory activity. Conditioned medium derived from PBMC of patients with SSc exhibited a significantly higher growth activity than that from controls (p < 0.05). Among PBMC subpopulations, macrophages showed the most increased growth effect on 3T3 fibroblasts, although T lymphocyte populations also showed growth activity. The growth activity was inhibited up to 35% by anti-platelet derived growth factor (PDGF) antibody. In addition, growth activity was also partially blocked by antibodies against basic fibroblast growth factor (bFGF) (20%), interleukin 1beta (IL-1beta) (15%), tumor necrosis factor-alpha(TNF-alpha) (7.9%), and transforming growth factor-beta (TGF-beta)(9.5%). The cellular release of IL-1beta, TNF-alpha, and IL-6 was elevated in response to bleomycin exposure in both controls and patients with SSc. Reverse transcriptase-polymerase chain reaction revealed that conditioned medium derived from both patients with SSc and controls induced mRNA expression of several fibrogenic cytokines such as IL-6, TNF-alpha, TGF-beta, and PDGF. IL-1alpha was induced only by conditioned medium derived from patients with SSc.
Conclusion: These results suggest that bleomycin induced cutaneous fibrosis as well as pulmonary fibrosis may be induced by inflammatory cells through several fibrogenic cytokines.