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Suppression of interleukin 8 expression: an indirect antiviral activity of interferon- g and interferon- b , but not interferon- a in coxsackievirus B3 infected human myocardial fibroblasts A. Heim, S. Weiss, S. Zeuke Institut für Virologie, Medizinische Hochschule Hannover, 30623 Hannover

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3. Results

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Suppression of interleukin 8 expression: an indirect antiviral activity of interferon-g and interferon-b, but not interferon-a in coxsackievirus B3 infected human myocardial fibroblasts

A. Heim, S. Weiss, S. Zeuke

Institut für Virologie, Medizinische Hochschule Hannover, 30623 Hannover

3. Results

Application of IFN-b and IFN-g 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-g (1000 IU/ml).

The reduction of IL-8 expression by IFN-b was concentration-dependent. For example, IL-8 concentrations were reduced to 508 pg/ml (SD 70) by 100 IU/ml IFN-b, and to 109 pg/ml (SD 28) by 1000 IU/ml IFN-b (untreated controls: 1063 pg/ml (SD 299)).

By contrast, IFN-a 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-b is caused by “non-specific” antiproliferative effects. However, antiproliferative effects of IFN-b 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.

1. Background

Interferon-b (IFN-b) has a more than 120-fold higher antiviral activity than the closely related type-I interferon-a (IFN-a) in human myocardial fibroblasts infected with coxsackievirus B3 (CVB3) [Heim et al., 1996], whereas in most cell/virus systems the antiviral activities of IFN-a and IFN-b are almost identical. Moreover, the “immune” interferon-g (IFN-g) 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-b (and IFN-g) compared to IFN-a may be caused by autocrine “immunomodulating” effects, e.g. a suppression of IL-8 expression by IFN-b and IFN-g.

2. Material and Methods

Human myocardial fibroblasts were treated for 9 days with either IFN-a, IFN-b or IFN-g (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-b 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-b and IFN-g. Neutralization of IL-8 resulted in a significant, but minor antiviral effect compared to the antiviral effects of IFN-b and IFN-g.

4. Conclusions

In contrast to IFN-a, IFN-b has ”immuno-modulating” properties (e.g. IL-8 suppression) similar to the type II interferon (IFN-g) in human myocardial fibroblasts. These activities contribute to the high antienteroviral activity of IFN-b, 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-b compared to IFN-a. Recently discovered differences in the activation of JAK-1 kinase by IFN-b and IFN-a may also contribute to the high antiviral activity of IFN-b [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, 797-801

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, 283-287

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, 753-759

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 235,774-778