Adenoviral gene delivery of interleukin-10 inhibits kupffer cells avtivation in vitro

Aims: Kupffer cells (KC) are the resident macrophages of the liver representing 80% of the total fixed macrophage population. They are involved in several disease states such as endotoxin shock, alcoholic liver diseases, and other toxicant- induced liver injury. They release physiologically active substances, such as eicosanoids and inflammatory cytokines as IL–6 or TNFα, produce free radical species, and are activated like other macrophages by endotoxin (LPS). The multifunctional cytokine Interleukin–10 (IL–10) is known for its general ability to inhibit activation and effector function of macrophages.

AIM: Here, we investigated the effects adenoviral gene delivery on the activation of KC in vitro.

Methods: KC were isolated from Sprague Dawley rats and cultured as previously described. Uptake of fluorescein isothiocyanate (FITC)-labeled, 1 μm latex-beads was meassured and revealed that almost 100% of the isolated KC took up the latex-beads, indicating that the KC cultures were viable and free of contaminating cells. Isolated KC were infected with adenoviral vectors expressing Interleukin–10 (Ad5.IL–10) applying different multiplicities of infection (moi: 0.1–1000 pfu/cell) and were subsequently cultured for 24h before analysis. Adenoviral vectors expressing beta-Galactosidase (Ad5.LacZ) served as control. In some experiments KC were stimulated with LPS (10 μg/ml) and cytokine release was analyzed with IL –10, IL–6, and TNFα ELISAs, respectively.

Results: Transduction of isolated KC with Ad5.IL–10 resulted in a dose-dependent induction of IL–10 secretion with a maximum at moi=50 pfu/cell. In functional assays, transduction of KC with Ad5.IL10 blunted LPS-induced IL–6 and TNFα secretion as compared to Ad5.LacZ-infected and control cells. Also this inhibition of proinflammatory cytokine release reached a maximum when Ad5.IL10 were used at a moi of 50 pfu/cell.

Conclusions: These data support the hypothesis that increased IL–10 secretion plays an important antiinflammatory role in liver disease and suggest that KC-targeted therapeutic approaches may be a promising strategy affecting hepatic inflammation.