Effects of melanocortin peptides on lipopolysaccharide/interferon-gamma-induced NF-kappaB DNA binding and nitric oxide production in macrophage-like RAW 264.7 cells:: Evidence for dual mechanisms of action
Section snippets
1. Introduction
The pro-opiomelanocortin-derived peptide1 α-MSH and its C-terminal tripeptide α-MSH(11–13) are potent antipyretic and anti-inflammatory agents [1], [2]. Immunoregulatory effects of melanocortins have been demonstrated both in vivo[3], [4], [5], [6] and in vitro[7], [8]. α-MSH is known both to inhibit the production and antagonize the effect of proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α), IL-1, IL-6, and IL-8 [8], [9], [10]. α-MSH has also been shown to induce the
Reagents
LPS (from Escherichia coli 0111:B4) and mouse recombinant IFN-γ were from Sigma Chemical Co. [γ-32P]ATP, poly(dI-dC), and [3H]cAMP were from Amersham. Double-stranded oligonucleotide containing the NF-κB binding motif of the mouse macrophage iNOS promoter, 5′-CAACTGGGGACTCTCCCTTTG-3′, and a double-stranded mutated NF-κB oligonucleotide, 5′-CAACTGCTCACTCTCCCTTTG-3′, were custom-synthesized by GIBCO BRL. Peptides were synthesized in our laboratory using a Pioneer Peptide Synthesis system
Inhibition of nitrite production by melanocortin peptides
Fig. 1 shows the effect of α-MSH, α-MSH(1–10), and α-MSH(11–13) on the accumulation of nitrite (a stable NO metabolite) in RAW 264.7 cells. As can be seen, 16 hr of stimulation of the cells with LPS/IFN-γ (100 ng/mL and 5 U/mL, respectively) caused a marked increase in nitrite production compared to the unstimulated macrophages, which produced only very low amounts of nitrite. All three melanocortin peptides inhibited LPS/IFN-γ-stimulated nitrite production in a dose-dependent manner, the IC50
Discussion
The present study provides evidence that melanocortins cause their actions in a mouse macrophage cell line via at least two different pathways. All three tested melanocortin peptides, α-MSH, α-MSH(1–10), and α-MSH(11–13), inhibited NO production and translocation of NF-κB into the nucleus. However, only α-MSH and α-MSH(1–10) stimulated cAMP in the macrophage cells, while α-MSH(11–13) was devoid of cAMP stimulatory effect. Since forskolin was also able to cause an inhibition of NO production
Acknowledgements
We thank Dr. Irina Shestakova for generous advice in setting up macrophage cell cultures. This study was supported by the Swedish MRC (04X-05957) and a grant from Melacure Therapeutics.
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