Background: Patients with congestive heart failure (CHF) show increased serum concentrations of cytokines like interleukin-6 (IL-6) and cardiotrophin-1 (CT-1). Additionally, monocyte function is modulated in CHF. The aim of this study was to examine if CT-1 is able to induce IL-6 in human monocytes and to investigate the underlying pathway.
Methods: Separated peripheral blood monocytes of healthy volunteers were cultured with increasing concentrations of CT-1 for different periods. IL-6 mRNA was determined by RT-PCR or real-time PCR and IL-6 protein concentration in the supernatant by ELISA. Phosphorylation of signal transducer and activation of transcription (STAT) 3 was analyzed by western blot or by FACS analysis. To clarify the signalling pathway of CT-1 induced IL-6 expression various inhibitors of possible signal transducing molecules were used.
Results: CT-1 induced IL-6 mRNA in monocytes in a time- and concentration-dependent manner. Maximal mRNA induction was detectable after 6h with 100 ng/ml CT-1. IL-6 protein also increased in a time- and concentration-dependent manner with a maximum after 48 h with 100 ng/ml CT-1. AG490 as well as SB 203580 and parthenolide blocked CT-1 induced IL-6 expression completely. AG 490 was able to inhibit STAT3 phosphorylation in western blot analysis completely. This indicates that JAK2/STAT3, p38 and nuclear factor kappaB (NFkappaB) are involved in this pathway. To exclude a possible influence of plastic adherence of monocytes on CT-1 induced IL-6 expression, we determined intracellular STAT3 phosphorylation in whole blood samples by FACS analysis and observed independently of culture conditions a CT-1 concentration-dependent STAT3 phosphorylation.
Conclusion: CT-1 induces IL-6 mRNA and protein expression in a time- and concentration-dependent manner. The underlying pathway is Janus kinase (JAK)2/STAT3, p38 and NFkappaB dependent. These data may explain increased IL-6 serum concentrations and altered monocyte function found in patients with CHF. Modulation of the CT-1 pathway might be a interesting strategy in the treatment of CHF.