Human resistin stimulates the pro-inflammatory cytokines TNF-α and IL-12 in macrophages by NF-κB-dependent pathway

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Abstract

Resistin, a recently discovered 92 amino acid protein involved in the development of insulin resistance, has been associated with obesity and type 2 diabetes. The elevated serum resistin in human diabetes is often associated with a pro-inflammatory milieu. However, the role of resistin in the development of inflammation is not well understood. Addition of recombinant human resistin protein (hResistin) to macrophages (both murine and human) resulted in enhanced secretion of pro-inflammatory cytokines, TNF-α and IL-12, similar to that obtained using 5 μg/ml lipopolysaccharide. Both oligomeric and dimeric forms of hResistin were able to activate these cytokines suggesting that the inflammatory action of resistin is independent of its conformation. Heat denatured hResistin abrogated cytokine induction while treatment of recombinant resistin with polymyxin B agarose beads had no effect thereby ruling out the role of endotoxin in the recombinant hResistin mediated cytokine induction. The pro-inflammatory nature of hResistin was further evident from the ability of this protein to induce the nuclear translocation of NF-κB transcription factor as seen from electrophoretic mobility shift assays. Induction of TNF-α in U937 cells by hResistin was markedly reduced in the presence of either dominant negative IκBα plasmid or PDTC, a pharmacological inhibitor of NF-κB. A protein involved in conferring insulin resistance is also a pro-inflammatory molecule that has important implications.

Section snippets

Materials and methods

Materials. RAW 264.7 (mouse) and U937 (human) cells were obtained from National Center for Cell Science, Pune, India. Dulbecco’s modified Eagle’s medium (DMEM), RPMI-1640 media, FCS, and antibiotics were purchased from Invitrogen (Carlsbad, CA, USA). Pyrrolidine dithiocarbamate (PDTC), phorbol myristate acetate (PMA), polymyxin B beads, and bacterial lipopolysaccharide (LPS) were purchased from Sigma (St. Louis, MO, USA). Mouse recombinant interferon-γ (IFN-γ) was procured from R&D Systems

Human recombinant resistin induces secretion of TNF-α in human and mouse macrophages

Given a direct positive correlation between TNF-α (and/or) inflammation and resistin expression [20], we investigated whether resistin itself directly activates macrophages for the production of TNF-α. The in vitro maintained U937 cell line was used as a source of human monocyte/macrophage lineage. In our initial experiments, cells were stimulated with increasing concentrations of recombinant human resistin (3, 10, 30, 60, and 100 μg/ml). Based on this titration experiment, a concentration of 30 

Discussion

We describe here a novel feature of human resistin as a pro-inflammatory molecule. Incubation of both mouse and human macrophages with human recombinant resistin stimulates the production of pro-inflammatory cytokines TNF-α and IL-12. The level of TNF-α has been previously reported to be higher in different rodent models of obesity [3], [22]. Diabetic patients bearing higher plasma resistin concentration were shown to suffer from cytokine-induced acute-phase inflammation [2]. Since TNF-α is

Acknowledgments

This work was supported by grant from Indian Council of Medical Research to N.Z.E. B.A. and A.K.S. were recipients of Senior Research Fellowships from Council of Scientific and Industrial Research, New Delhi.

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    These two authors contributed equally.

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