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Figure 2 | Journal of Inflammation

Figure 2

From: Heat shock protein 70 down-regulates the production of toll-like receptor-induced pro-inflammatory cytokines by a heat shock factor-1/constitutive heat shock element-binding factor-dependent mechanism

Figure 2

Hsp70 regulates LPS-dependent TNF-α production through a mechanism that does not depend on the interaction between LPS and Hsp70. A. Monocytes were stimulated for 6 h in the presence of Hsp70 (3000 to 0.003 ng/ml) and 100 ng/ml of LPS. B. Monocytes were stimulated for 6 h in the presence of LPS (10000 to 1 ng/ml) and 3 μg/ml of Hsp70. LPS was pre-incubated with Hsp70 in 200 μl of binding buffer for 2 h at 37°C, before adding them to the corresponding wells. TNF-α production was determined after 6 hr in the culture supernantant. One-way ANOVA with Tukey’s post-test: ***P < 0.001 LPS vs. LPS + Hsp70. Data is representative of 3 independent assays. C. Schematic representation of the experimental design. D, top panels. Monocytes were cultured in the absence (NS) or in the presence of LPS (100 ng/ml) for 2 or 4 h. After this incubation, fresh culture medium with Hsp70 (3 μg/ml) was added (LPS + HSp70). The cells were incubated for 24 h. As controls, LPS (100 ng/ml) or Hsp70 (3 μg/ml) were added to the cells. The amount of TNF-α was measured in the supernatants at the indicated time points. D, bottom panels. Monocytes were left unstimulates (NS) or cultured with Hsp70 (3 μg/ml) for 2 or 4 h. After this incubation, fresh culture medium with LPS (100 ng/ml) was added (Hsp70 + LPS). As controls, LPS (100 ng/ml) or Hsp70 (3 μg/ml) were added to the cells. The cells were incubated for a total of 24 h. The amount of TNF-α was measured in the supernatants at the indicated time periods. Two-way ANOVA with Bonferroni post-test: ***P < 0.001 LPS vs. Hsp70 + LPS or vs. LPS + Hsp70. NS = non-stimulated cells. Data is representative of 3 independent assays.

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