Figure 6

TLR4 is required for Hsp72-induced regulation of IL-8 production from HL-60 cells. Differentiated HL-60 cells were pretreated with a neutralizing antibody against TLR4 (10 μg/ml) or an isotype control antibody for 1 h prior to Hsp72 treatment (100 ng/ml). 18 h later, cell media was harvested and analyzed by ELISA for IL-8 and TNFα production. Data are expressed as mean ± SEM for 5 experiments and differences pinpointed by ANOVA. A. IL-8 ELISA. Compared to isotype antibody control *p = 0.004, compared to TLR4 antibody **p = 0.014. B. TNFα ELISA. Compared to isotype antibody control *p < 0.001, compared to TLR4 antibody **p < 0.001. Bone marrow-derived neutrophils were isolated from wild type (C3H/HeOuJ) or mice with a spontaneous mutation in TLR4 (C3H/HeJ). Primary neutrophils were treated ex vivo with Hsp72 (100 ng/ml) and supernatant was removed 18 h later for analysis by KC (the functional IL-8 in mouse) and TNFα ELISA. Data are expressed as mean ± SEM for 3 separate experiments and differences pinpointed by ANOVA. C. KC ELISA. Compared to control *p = 0.002, compared to Hsp72 **p < 0.001. D. TNFα ELISA. Compared to control *p < 0.001, compared to Hsp72 **p < 0.001.