Age is associated with increased TLR4 expression and enhanced sensitivity to LPS. TLR4 (A) protein expression (assessed by western immunoblot) in BMDMs was found to be increased in an age-related manner (**
P < 0.01; Student’s t-test; n = 3). LPS (100 ng/ml; 24 h) stimulation significantly increased TNFα (B), NOS2 (C), CD40 (D) and CD11b (E) mRNA in BMDMs from young and aged rats (***
P < 0.001; **
P < 0.01; ANOVA); the LPS-induced increase in mRNA expression of these markers was significantly greater in BMDMs cultured from aged, compared with young, rats (++
P < 0.01; +++
P < 0.001; ANOVA). LPS stimulation (100 ng/ml; 3 and 6 h) significantly increased supernatant concentration of TNFα (measured by ELISA) in BMDMs from young and aged rats (F, G). While significant TNFα release was identified in cells from young and aged animals (F, G; ***
P < 0.001, ANOVA), this LPS effect was significantly greater in BMDMs from aged rats at 6 h (G; +
P < 0.05, ANOVA). Values are presented as means (±SEM; n = 3) expressed as a ratio to β-actin mRNA or as pg/ml. LPS, lipopolysaccharide; NOS2, nitric oxide synthase 2; CD40, cluster of differentiation 40; CD11b, cluster of differentiation 11b; TNFα, tumour necrosis factor-α; RQ, relative quantities.