Age-related changes in immunity render elderly individuals more susceptible to infections than the young. Previous work by our laboratory and others showed that macrophages from aged mice are functionally impaired. Macrophages produce proinflammatory cytokines, tumor necrosis factor alpha (TNF-alpha) and interleukin (IL)-6, when stimulated with lipopolysaccharide (LPS), which signals through Toll-like receptor-4 (TLR4) and requires activation of mitogen-activated protein kinases (MAPKs). We investigated whether aging is associated with alterations in TNF-alpha and IL-6 production and MAPK expression and activation in thioglycollate-elicited peritoneal macrophages from mice. Kinetics and LPS dose-responsiveness of macrophage TNF-alpha production did not differ by age. Unstimulated macrophages did not differ by age in their cytokine production. However, LPS-stimulated (100 ng/mL) cultures from aged mice produced 100 +/- 30 pg/mL TNF-alpha and 6000 +/- 2000 pg/mL IL-6, and those from young mice produced 280 +/- 50 pg/mL and 10,650 +/- 10 pg/mL, respectively (P<0.05). Likewise, levels of activated MAPKs did not differ by age in unstimulated macrophages, and LPS-stimulated macrophages from aged mice had <70% activated p38 and c-jun NH(2)-terminal kinase (JNK) than those of young controls. Of particular interest, we observed >25% reduction of total p38 and JNK in macrophages from aged mice relative to young. In addition, surface TLR4 levels did not vary with age. We conclude that macrophages from aged mice exhibited suppressed proinflammatory cytokine production, which correlated with diminished total levels and LPS-stimulated activation of p38 and JNK. These observations suggest that decreased MAPK expression could be a mechanism responsible for age-related deterioration of the immune system.
Extensive evidence indicates that ethanol (alcohol) has immunomodulatory properties. Many of its effects on innate immune response are dose dependent, with acute or moderate use associated with attenuated inflammatory responses, and heavy ethanol consumption linked with augmentation of inflammation. Ethanol may modify innate immunity via functional alterations of the cells of the innate immune system. Mounting evidence indicates that ethanol can diversely affect antigen recognition and intracellular signaling events, which include activation of mitogen activated protein kinases, and NFkappaB, mediated by Toll-like receptors, leading to altered inflammatory responses. The mechanism(s) underlying these changes may involve dose-dependent effects of ethanol on the fluidity of cell membrane, resulting in interference with the timely assembly or disassembly of lipid rafts. Ethanol could also modify cell activation by specific interactions with cell membrane molecules.
Ethanol is known to increase susceptibility to infections, in part, by suppressing macrophage function. Through TLRs, macrophages recognize pathogens and initiate inflammatory responses. In this study, we investigated the effect of acute ethanol exposure on murine macrophage activation mediated via TLR2, TLR4, and TLR9. Specifically, the study focused on the proinflammatory cytokines IL-6 and TNF-α and activation of p38 and ERK1/2 MAPKs after a single in vivo exposure to physiologically relevant level of ethanol followed by ex vivo stimulation with specific TLR ligands. Acute ethanol treatment inhibited IL-6 and TNF-α synthesis and impaired p38 and ERK1/2 activation induced by TLR2, TLR4, and TLR9 ligands. We also addressed the question of whether ethanol treatment modified activities of serine/threonine-specific, tyrosine-specific phosphatases, and MAPK phosphatase type 1. Inhibitors of three families of protein phosphatases did not restore ethanol-impaired proinflammatory cytokine production nor p38 and ERK1/2 activation. However, inhibitors of serine/threonine protein phosphatase type 1 and type 2A significantly increased IL-6 and TNF-α levels, and prolonged activation of p38 and ERK1/2 when triggered by TLR4 and TLR9 ligands. In contrast, with TLR2 ligand stimulation, TNF-α production was reduced, whereas IL-6 levels, and p38 and ERK1/2 activation were not affected. In conclusion, acute ethanol exposure impaired macrophage responsiveness to multiple TLR agonists by inhibiting IL-6 and TNF-α production. Mechanism responsible for ethanol-induced suppression involved inhibition of p38 and ERK1/2 activation. Furthermore, different TLR ligands stimulated IL-6 and TNF-α production via signaling pathways, which showed unique characteristics.
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