Background Commercial preparations of native human interferon alpha (nHuIFN-α) contain several subtypes of interferon-alpha (IFN-α) and traces of other cytokines. Recently, we described its antifibrotic potential and showed nHuIFN-α to have a greater effect than that of recombinant human IFN-α (rHuIFN-α). We hypothesized that cooperation between different cytokines in the nHuIFN-α preparation is essential for this effect. Considerable concentrations of interleukin-1β (IL-1β) and platelet-derived growth factor AB (PDGF-AB) are present in the nHuIFN-α preparations. Methods We tested the viability and the expression of procollagen type I messenger RNA (mRNA) in MRC5 fibroblasts treated with interleukin-1 beta (IL-1β) and/or PDGF-AB, or the corresponding antibodies in combination with rHuIFN-α or nHuIFN-α. Results We showed that neither IL-1β nor PDGF-AB significantly affect the viability of MRC5 cells. Furthermore, cell viability was not affected when IL-1β or PDGF-AB were applied along with rHuIFN-α, relative to the viability of cells treated with rHuIFN-α only. In contrast, both cytokines suppressed the synthesis of procollagen type I mRNA. When coadministered with rHuIFN-α, IL-1β enhanced the suppression induced by rHuIFN-α. Conversely, PDGF-AB acted as an antagonist of rHuIFN-α and restored partially the synthesis of procollagen type I mRNA. Interestingly, the addition of IL-1β to the PDGF-AB/rHuIFN-α mix not only abolished the antagonistic activity of PDGF-AB but also decreased the synthesis of procollagen type I mRNA beyond the level achieved by IL-1β/rHuIFN-α. Therefore, IL-1β was able to reverse the activity of PDGF-AB. Conclusion Our study suggests that IL-1β is an important component of nHuIFN-α preparations, acting directly and indirectly to modulate the action of other components. This study provides insight into these complex cytokine networks, which is necessary for better and safer antifibrotic therapy.