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Regular exercise may offset age-associated increases in inflammatory cytokines and reduce the risk of developing diseases with an inflammatory etiology by exerting “anti-inflammatory” effects. Toll-like receptor 4 (TLR4) signaling stimulates inflammatory cytokine production, and may explain the “anti-inflammatory” effect attributed to regular exercise. Therefore, the purpose of the present study was to compare the effect of acute (3 sets, 9 exercises, 10 repetitions at 80% of the 1-repetition maximum) and chronic resistance exercise on TLR4 and inflammatory cytokines.Venous blood samples were collected from trained (TR, N = 10) and untrained (UT, N = 10) older (65–80 yr) postmenopausal women: before (PRE), immediately post (POST), and 2 h (2H), 6 h (6H), and 24 h (24H) after completion of exercise. Cell-surface expression of TLR4 (two-color immunofluorescent cytometry), LPS (25 μg·mL−1)-stimulated cytokine production (ELISA), plasma cytokines (ELISA), and mRNA expression of TLR4 and cytokines (RT-PCR) were determined for each sample.TR had 124% less cell-surface TLR4 expression than UT (P < 0.05). A significant time effect was found for LPS-stimulated IL-6, IL-1β, and TNF-α, where 6H was significantly greater than all other samples. No significant effects were found for plasma (IL-6 and TNF-α) or mRNA expression (IL-6, TNF-α, and IL-1β) of inflammatory cytokines. When subjects were grouped according to cell-surface TLR4 expression (HI and LO), LPS-stimulated TNF-α (302%), IL-1β (209%), and IL-6 (167%) production was greater for HI than LO (P < 0.05).Regularly exercising older women expressed less cell-surface TLR4 but did not have lower plasma levels or produce less LPS-stimulated inflammatory cytokines at rest or in response to a single bout of resistance exercise. TLR4 changes may explain the “anti-inflammatory” effect that has recently been attributed to chronic (2× wk−1 for previous 24 months) resistance exercise training.