TLR4 and CD11b/CD18 mediate rapid ROS, TNF, and MCP-1 release induced by nHZ. (A) Inhibition of nHZ-elicited ROS release (oxidative burst) by TLR4- and CD11b/CD18-blocking antibodies from human monocytes. Suspended nonprimed monocytes were supplemented with nHZ immediately after isolation from healthy donors at time 0 and incubated at 37°C (nHZ, n = 8). For inhibition studies, cells were incubated for 15 minutes with blocking Abs, anti-TLR4 (nHZ αTLR4), and anti-CD11b (nHZ αCD11b), or nonspecific IgG2a (nHZ IgG2a isotype) and IgG1 (nHZ IgG1 isotype) Abs as isotype controls for anti-TLR4 and anti-CD11b, respectively, before nHZ addition. nHZ-elicited ROS release was quantified 4 minutes after nHZ addition by luminol-enhanced luminescence. (B) TNF and (C) MCP-1 release from monocytes pretreated or not with TLR4- and CD11b/CD18-blocking Abs and subsequent addition of nHZ. Monocytes obtained from healthy donors were enriched by adhesion, maintained in culture overnight with 200 U/mL IFNγ, and then supplemented with nHZ (100nmol/10⁶ monocytes in terms of heme content). After 3 hours supernatants were collected and analyzed for TNF and MCP-1 by ELISA. For inhibition studies, cells were treated as indicated in panel A. ROS, TNF, and MCP-1 release values after Ab treatment are expressed as the percentage of control nHZ values. Four and 7 independent experiments were performed for TNF and MCP-1 analysis, respectively. Results are expressed as medians with 95% CI; *P < .05 and #P < .01. Absolute values of nHZ-induced TNF, MCP-1, and ROS release were, respectively, 357 pg/mL (120-644 pg/mL), 575 pg/mL (284-877 pg/mL), and 106 × 10³ cps/150 000 cells (65-156 × 10³ cps/150 000 cells). (D) Surface expression of TLR4 (gray full) and CD11b (open solid line) antigens in adherent human monocytes was analyzed by flow cytofluorimetry after overnight stimulation with 200 U/mL IFNγ. Background is shown as dashed line.