alpha-MSH modulates local and circulating tumor necrosis factor-alpha in experimental brain inflammation

Abstract

Tumor necrosis factor (TNF-alpha) underlies pathological processes and functional disturbances in acute and chronic neurological disease and injury. The neuroimmunomodulatory peptide alpha-MSH modulates actions and production of proinflammatory cytokines including TNF-alpha, but there is no prior evidence that it alters TNF-alpha induced within the brain. To test for this potential influence of the peptide, TNF-alpha was induced centrally by local injection of bacterial lipopolysaccharide (LPS). alpha-MSH given once i.c.v. with LPS challenge, twice daily intraperitoneally (i.p.) for 5 d between central LPS injections, or both i.p. and centrally, inhibited production of TNF-alpha within brain tissue. Inhibition of TNF-alpha protein formation by alpha-MSH was confirmed by inhibition of TNF-alpha mRNA. Plasma TNF-alpha concentration was elevated markedly after central LPS, indicative of an augmented peripheral host response induced by the CNS signal. The increase was inhibited by alpha-MSH treatments, in relation to inhibition of central TNF-alpha. Presence within normal mouse brain of mRNA for the alpha-MSH receptor MC-1 suggests that the inhibitory effects of alpha-MSH on brain and plasma TNF-alpha might be mediated by this receptor subtype. The inhibitory effect of alpha-MSH on brain TNF-alpha did not depend on circulating factors because the effect also occurred in brain tissue in vitro. This indicates that alpha-MSH can act directly on brain cells to inhibit their production of TNF-alpha. If central TNF-alpha contributes to pathology in CNS disease and injury, and promotes inflammation in the periphery, agents that act on brain alpha-MSH receptors should decrease the pathological TNF-alpha reaction and promote tissue survival.

Fig. 1.

α-MSH given centrally (10 μg) and/or systemically (50 μg, repeated) inhibited TNF-α induction within the brain by local injection of LPS (5 μg). Scores are means ± SEM. After ANOVA revealed significant differences among means (p < 0.001), Dunnett’s test was used for comparisons of individual means. **p < 0.01, relative to saline (i.c.v.) + saline (i.p.);⁺p < 0.01 versus other means.

Fig. 2.

Representative Northern blot of TNF-α mRNA abundance in whole mouse brains. Representative examples from two mice given LPS alone (SALINE IP, ICV) and two others from the group (α-MSH IP, ICV) that showed the greatest inhibitory effect of α-MSH on TNF-α protein. The blot was hybridized with ³²P-labeled cDNA probe for mouse TNF-α. Ethidium bromide staining of agarose gel demonstrated equal loading of samples. This is representative of two experiments.

Fig. 3.

α-MSH given centrally and/or peripherally inhibited increases in circulating TNF-α induced by central injection of LPS. Overall, ANOVA was significant (p < 0.001). Dunnett’s test: **p < 0.01 relative to saline (i.c.v.) + saline (i.p.); ⁺p < 0.05 relative to saline (i.c.v.) + α-MSH (i.p.) p< 0.01 relative to α-MSH (i.c.v.) + saline (i.p.).

Fig. 4.

Representative Southern blot of RT-PCR product for murine MC-1 receptor in normal mouse brain. No bands were observed in lanes containing reaction mixture without tissue (not shown) or without RT (−RT). Lanes containing mouse genomic DNA (1 μg) or MC-1 cDNA also contained the 529 bp product. Approximately 10 μg of cDNA (20 μl) was used in this experiment. Exposure time was 30 min. Washes (all room temperature): first washes = 3 for 5 min, 2× SSC + 0.1% SDS; second washes = 2 for 30 min, 2× SSC + 0.1% SDS.

Fig. 5.

Dose-related inhibition by α-MSH of TNF-α protein production by tissue derived from half murine brains incubated with LPS for 1 hr. Control value was the mean TNF-α production (60.1 ± 12.7 U/gm) for all of the opposite hemibrains incubated with LPS but without α-MSH. Scores are means ± SEM of percent inhibition of TNF-α production relative to control derived from three separate experiments.

Fig. 6.

Representative Northern blot showing that single acute i.c.v. injections of LPS (1) had greater effects on brain TNF-α mRNA abundance than injections that were preceded 5 d earlier by a similar i.c.v. injection (2). Ethidium bromide staining of agarose gel demonstrated equal loading of samples. Representative of two experiments: 2 d exposure, −70°C, two intensifying screens.