Accuri C6 Flow Cytometer System
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© by The Society for Leukocyte Biology
Journal of Leukocyte Biology, doi:10.1189/jlb.0707463

Received for publication July 13, 2007.
Revised March 1, 2008.
Accepted for publication March 10, 2008.

Article

Diminishment of {alpha}-MSH anti-inflammatory activity in MC1r siRNA-transfected RAW264.7 macrophages

Dayu Li and Andrew W. Taylor @

Schepens Eye Research Institute and the Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, USA

@ To whom correspondence should be addressed. E-mail: andrew.taylor{at}schepens.harvard.edu.


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Abstract

The neuropeptide {alpha}-melanocyte-stimulating hormone ({alpha}-MSH) is a powerful suppressor of inflammation mediated by macrophages, which express at least two receptors, melanocortin 1 and 3 receptors (MC1r and MC3r) that bind {alpha}-MSH. Albeit, the anti-inflammatory activity of {alpha}-MSH has been well documented in macrophages, the mechanisms of {alpha}-MSH activity in macrophages are not clearly understood. This study is to investigate which of the MCr expressed on macrophages is associated with the immunosuppressive activities of {alpha}-MSH on LPS-stimulated macrophages. To address this question, we transfected RAW264.7 macrophage cells with MC1r small interfering (si)RNA, which specifically targets mouse MC1r mRNA. The diminution of MC1r mRNA expression was 82% at 24 h and 67% at 48 h after transfection. There was a significant loss in {alpha}-MSH suppression of NO generation and TNF-{alpha} production by MC1r siRNA-transfected macrophages stimulated with LPS. There was an equally diminished {alpha}-MSH suppression of LPS-stimulated intracellular activation of NF-{kappa}B and p38 phosphorylation. In addition, the diminishment of MC1r expression by siRNA transfection had no influence on MC3r expression and function in the macrophages. These findings demonstrate that {alpha}-MSH suppression of LPS-induced inflammatory activity in macrophages requires expression of MC1r. The results imply that although all of the MCr are G-coupled proteins, they may not necessarily function through the same intracellular pathways in macrophages.

Key Words: neuroimmunomodulation • immunosuppression • neuroimmunology • neuropeptides • inflammation • innate immunity