Figure 7. Activity of cell surface HSP90 is necessary for cytokine response, but not for target engulfment after pattern recognition by Mϕs.

(A) Monocytes were cultured in medium supplemented with 10% FCS or 10% autologous serum. hMDMs were cultured in medium supplemented with 10% HS. Cultured cells were untreated or pretreated for 30 min with GeB (20 µM) and then stimulated with PAMPs: E. coli LPS (stLPS and upLPS), PG LPS (pgLPS), Pam2CSK4 (Pam2), Pam3CSK4 (Pam3) or bacteria SA or PG. TNF production stimulated with PAMPs or bacteria in the absence of GeB was arbitrarily set at 100% and served as the positive control. Results are expressed as percentage of corresponding positive controls (means ± sem of results from at least 3 independent experiments). *P < 0.05; **P < 0.01. (B) Cultured hMDMs were untreated or pretreated for 30 min with GeB at the concentrations indicated and then stimulated with upLPS or Pam2CSK4. Data presented are means ± sem of TNF concentration in supernatants from 3 independent experiments. (C) Cultured hMDMs were untreated or pretreated for 30 min with GeB (20 µM) and then stimulated with E. coli LPS. The levels of cytokines were measured by a bead-based multiplexing immunoassay. Cytokine production stimulated in the absence of GeB was arbitrarily set at 100% and served as the positive control. Results are expressed as percentage of corresponding positive controls. Phagocytosis of SA (D) or apoptotic PMNs (E) by hMDMs untreated or pretreated for 30 min with Rad, Ge, or GeB (20 µM) or with DMAG (1 µM). (D) After 90 min incubation with fluorescently labeled SA hMDMs were nonenzymatically detached and analyzed by flow cytometry. The intensity of phagocytosis is expressed as the phagocytic index: percentage of positive cells × MFI of positive population. **P < 0.01. The representative, raw flow cytometry data are depicted in Supplemental Fig. 2. (E) After 2 h incubation with apoptotic PMNs, noningested neutrophils were removed by intensive washing of the hMDM monolayer. The cells were solubilized with detergent, and the neutrophil elastase activity was measured in lysates. The intensity of phagocytosis is expressed as mOD_405nm / min of the substrate turnover catalyzed by neutrophil-derived elastase. **P < 0.01.