3. Results Application of IFN-  and IFN-  significantly reduced IL-6 and IL-8 expression both in CVB3 infected and mock infected myocardial fibroblasts. Moreover, MCP-1 expression was increased by high concentrations of IFN-  (1000 IU/ml). The reduction of IL-8 expression by IFN-  was concentration-dependent. For example, IL-8 concentrations were reduced to 508 pg/ml (SD 70) by 100 IU/ml IFN- , and to 109 pg/ml (SD 28) by 1000 IU/ml IFN-  (untreated controls: 1063 pg/ml (SD 299)). By contrast, IFN-  had no significant effect on IL- 6, IL-8, and MCP-1 expression. As interferons have antiproliferative activities, which are related to a general reduction of protein synthesis in many cell lines, we investigated whether the reduction of IL-8 expression by IFN-  is caused by “non-specific” antiproliferative effects. However, antiproliferative effects of IFN-  on human myocardial fibroblasts were very low (<30% with 1000 IU/ml) indicating that the suppression IL-8 expression is specific and not related to cytotoxicity. Suppression of interleukin 8 expression: an indirect antiviral activity of interferon-  and interferon- , but not interferon-  in coxsackievirus B3 infected human myocardial fibroblasts A. Heim, S. Weiss, S. Zeuke Institut für Virologie, Medizinische Hochschule Hannover, Hannover 1. Background Interferon  (IFN-  ) has a more than 120-fold higher antiviral activity than the closely related type-I interferon-  (IFN-  ) in human myocardial fibroblasts infected with coxsackievirus B3 (CVB3) [Heim et al., 1996], whereas in most cell/virus systems the antiviral activities of IFN-  and IFN-  are almost identical. Moreover, the “immune” interferon-  (IFN-  ) is also highly active against CVB3 replication in human myocardial fibroblasts. CVB3 replication induces interleukin 6 (IL-6) and interleukin 8 (IL-8) expression in cultures of human myocardial fibroblasts [Heim et al. 2000], and suppresses the expression of monocyte chemoattractant protein-1 (MCP-1). As IL-8 stimulates enterovirus replication [Khabar et al., 1997], we investigated whether the higher antiviral activity of IFN-  (and IFN-  compared to IFN-  may be caused by autocrine “immunomodulating” effects, e.g. a suppression of IL-8 expression by IFN-  and IFN- . 2. Material and Methods Human myocardial fibroblasts were treated for 9 days with either IFN- , IFN-  or IFN-  (0, 10, 100, and 1000 IU/ml), and concentrations of IL-6, IL-8 and MCP-1 were measured in culture supernatants by enzyme-linked immunoassays. In addition, IL-8 mRNA concentrations were determined by quantitative reverse transcription/ polymerase chain reaction (qPCR) using competitive internal standards (IS). All samples were diluted to equal concentrations of a house- keeping gene (GAPDH) before performing IL-6 and IL-8 qPCR. In order to evaluate the antiviral effects of a reduction of “autocrine” IL-8, CVB3 infected myocardial fibroblasts were treated with a monoclonal antibody (MAB) neutralizing IL-8, and virus yields were quantified by plaque-assays. Furthermore, antiproliferative effects of the three interferons were evaluated by EZ4U staining Moreover, IFN-  reduced IL-8 mRNA concen- trations in human myocardial fibroblasts. As all samples were prediluted to equal concentrations of GAPDH (a "house-keeping gene") mRNA, a specific reduction of IL-8 mRNA can be concluded. CVB3 infected human myocardial fibroblasts were incubated with an IL-8 neutralizing monoclonal antibody to determine the contribution of IL-8 reduction to the high antiviral activity of IFN-  and IFN-  eutralization of IL-8 resulted in a significant, but minor antiviral effect compared to the antiviral effects of IFN-  and IFN-  4. Conclusions In contrast to IFN- , IFN-  has ”immuno- modulating” properties (e.g. IL-8 suppression) similar to the type II interferon (IFN-  ) in human myocardial fibroblasts. These activities contribute to the high antienteroviral activity of IFN- , and may be useful in the treatment of enteroviral heart disease. However, the reduction of IL-8 expression is not sufficient to explain the 120-fold higher antiviral activity of IFN-  compared to IFN- . Recently discovered differences in the activation of JAK-1 kinase by IFN-  and IFN-  may also contribute to the high antiviral activity of IFN-  [Grumbach 1999]. 5. References Grumbach IM, Fish EN, Uddin S, Majchrzak B, Colamonici OR, Figulla HR, Heim A, Platanias LC (1999) Activation of the Jak-Stat pathway in cells that exhibit selective sensitivity to the antiviral effects of IFN-beta compared with IFN-alpha. J Interf Cytok Res 19, Heim A, Stille-Siegener M, Pring-Åkerblom P, Grumbach I, Brehm C, Kreuzer H, Figulla HR (1996) Recombinant interferons-β and γ have a higher antiviral activity than interferon-α in coxackievirus B3-infected carrier state cultures of human myokardial fibroblasts. J Interf Cytok Res 16, Heim A, Zeuke S, Weiss S, Ruschewski W, Grumbach IM (2000) Transient induction of cytokine production in human myocardial fibroblasts by Coxsackievirus B3, Circ Res 86, Khabar, K. S. A., Al-Zoghaibi, F., Murayama, T., Matsushima, K., Mukaida, N., Siddiqui, Y., Dhalla, M., Al-Ahdal, M. N. (1997). Interleukin-8 selektively enhances cytopathic effect (CPE) induced by positive strand RNA viruses in the human wish cell line. Biochem Biophys Res Comm 235,