Figure 1

Hsp70 decreases the production of TNF-α by LPS-activated human monocytes. A. Monocytes were left unstimulated (NS) or stimulated with Hsp70 (3 μg/ml) or LPS (100 ng/ml) for the indicated time and TNF-α concentration was measured in the supernatants. Two-way ANOVA with Bonferroni post-test: ***P < 0.001 LPS vs. Hsp70. B. Monocytes were unstimulated (NS) or stimulated with different concentrations of Hsp70 (3, 0.3 and 0.003 μg/ml) or LPS (100 ng/ml) and the amount of TNF-α in the culture supernatant was measure at the indicated time. Two-way ANOVA with Bonferroni post-test: ***P < 0.001 LPS vs. Hsp70. C. Monocytes were non stimulated (NS) or stimulated for 6 h with LPS-purified Hsp70 (3 μg/ml), LPS (100 ng/ml), Hsp70 and LPS or LPS and heat-denatured Hsp70 (Hsp70 D) as described in material and methods. TNFα production was determined after 6 hr in the culture supernantant. Kruskal-Wallis test with Dunn’s multiple comparison test: *P < 0.05. D. Monocytes were non stimulated (NS) or stimulated for 6 h with LPS-purified Hsp70 (3 μg/ml), LPS (100 ng/ml) or non-LPS-purified Hsp70 (HE/Hsp70, 3 μg/ml) and TNFα production was determined after 6 hr in the culture supernantant. Kruskal-Wallis test with Dunn’s multiple comparison test: *P < 0.05. E. Monocytes were cultured non treated (NS) or cultured with LPS (100 ng/ml) in the presence or absence of Hsp70 (3 μg/ml) for 2 or 6 hrs, and the expression levels of TNF-α mRNA were evaluated. β-actin was amplified simultaneously to verify RNA integrity and to ensure that equivalent amounts of templates were used. F. TNF-α production by the monocytes in E at four different times 1, 2, 4 and 6 hrs. Kruskal-Wallis test with Dunn’s multiple comparison test: *P < 0.05. NS = non-stimulated cells. Data is representative of 3 independent assays.