The Anti-Inflammatory Effects of Adiponectin Are Mediated via a Heme Oxygenase-1–Dependent Pathway in Rat Kupffer Cells

Palash Mandal, Pil-Hoon Park, Megan R. McMullen, Brian T. Pratt, and Laura E. Nagy
Source: Hepatology
Publication Date: (2009)
Issue: 9999: xxxxx
Research Area:
Cells used in publication:
Kupffer cells, Rat
Species: rat
Tissue Origin: blood
Nucleofector® I/II/2b
Nucleofection in Rat Kupffer Cells. Freshly isolated Kupffer cells were transfected using the Amaxa mouse macrophage Nucleofector kit according to the instructions of the manufacturer using the Y-001 program (Lonza, Cologne, Germany), except for the following modifications. Samples were processed individually, and the entire nucleofection procedure for each sample was completed in less than 5 minutes. For each nucleofection sample, 2 x 10^6 Kupffer cells were centrifuged for 10 minutes at 300g. The pellet was washed with 1 mL phosphate- buffered saline (PBS), collected at 300g for 5 minutes, and then resuspended in 105 uL nucleofector solution and transferred to 1.5-mL Eppendorf tubes for a final concentration of approximately 2 x 10^6 cells/100 uL. Cells were then treated or not with 2.0 ug specific or scrambled siRNA (siRNA sequences are provided in Supplemental Materials), transferred into the electroporation cuvette, and placed in the Nucleofector device. After nucleofection, cells were immediately removed from the cuvette and plated in a 96-well plate (150 uL/well) at 0.5 x 10^6 cells/well. After 4 hours, the cell culture medium was replaced with fresh medium with or without 1 ug/mL gAcrp or 10 ng/mL IL-10 for 18 hours and then treated with or without LPS or IL-10, as described in the figure legends.
Altered expression and activity of immunomodulatory cytokines plays a major role in the pathogenesis of alcoholic liver disease. Chronic ethanol feeding increases the sensitivity of Kupffer cells, the resident hepatic macrophage, to lipopolysaccharide (LPS), leading to increased tumor necrosis factor- (TNF-) expression. This sensitization is normalized by treatment of primary cultures of Kupffer cells with adiponectin, an anti-inflammatory adipokine. Here we tested the hypothesis that adiponectin-mediated suppression of LPS signaling in Kupffer cells is mediated via an interleukin-10 (IL-10)/heme oxygenase-1 (HO-1) pathway after chronic ethanol feeding. Knock-down of IL-10 expression in primary cultures of Kupffer cells with siRNA prevented the inhibitory effect of globular adiponectin (gAcrp) on LPS-stimulated TNF- expression. gAcrp increased IL-10 mRNA and protein expression, as well as expression of the IL-10 inducible gene, HO-1; expression was higher in Kupffer cells from ethanol-fed rats compared to pair-fed controls. While IL-10 receptor surface expression on Kupffer cells was not affected by ethanol feeding, IL-10-mediated phosphorylation of STAT3 and expression of HO-1 was higher in Kupffer cells after ethanol feeding. Inhibition of HO-1 activity, either by treatment with the HO-1 inhibitor, zinc protoporphyrin, or by siRNA knock-down of HO-1, prevented the inhibitory effect of gAcrp on LPS-stimulated TNF- expression in Kupffer cells. LPS-stimulated TNF- expression in liver was increased in mice after chronic ethanol exposure. When mice were treated with cobalt protoporphyrin to induce HO-1 expression, ethanol-induced sensitivity to LPS was ameliorated. Conclusion: gAcrp prevents LPS-stimulated TNF- expression in Kupffer cells via the activation of the IL-10/STAT3/HO-1 pathway. Kupffer cells from ethanol-fed rats are highly sensitive to the anti-inflammatory effects of gAcrp; this sensitivity is associated with both increased expression and sensitivity to IL-10. (HEPATOLOGY 2009.)