nHZ-bound FG induces rapid ROS, TNF, and MCP-1 release. (A) FG content of different HZ types was analyzed by Western blot. HZ was obtained from supernatants of plasma- (nHZ, lane 1) or serum- (HZ SERUM, lane 5) or Albumax- (HZ ALBUMAX, lane 6) supplemented cultures of P falciparum. nHZ-bound FG was either degraded by plasmin treatment (see “Methods”), and both pellet (HZ PLASMIN, lane 2) and supernatants (SN PLASMIN, lane 3) were analyzed for undegraded FG and FG fragments or were denatured by heating at 100°C for 5 minutes (HZ 100°C, lane 4). Proteins extracted from 50nmoles HZ (in terms of heme content), and from the supernatant of the same amount of HZ, were separated by 10% SDS-PAGE, transferred to nitrocellulose, and probed with anti-FG γ-chain. FG (15 μg) and plasmin-digested FG (FG PLASMIN) were separated and blotted as reference (lane 7 and lane 8, respectively). (B) Immediate ROS release by FG. Suspended monocytes were supplemented with differently treated HZ of equal heme content (100nmol/10⁶ monocytes) immediately after isolation at time 0, briefly spun down, resuspended, and incubated at 37°C (n = 4). Aliquots of 1.5 × 10⁵ monocytes were taken at indicated times, and ROS was quantified by luminol-enhanced luminescence. (C) Immediate TNF and MCP-1 release by FG. Human monocytes were enriched by Ficoll passage and adhesion, maintained in culture overnight with 200 U/mL IFNγ, and then fed with differently treated HZ of equal heme content (100nmol/10⁶ monocytes) 15 hours after isolation. After 3 hours supernatants were collected and analyzed for TNF and MCP-1 by ELISA. TNF and MCP-1 release values of each HZ species are expressed as percentages of control nHZ values. Medians are shown of 8 (TNF) and 4 (MCP-1) independent experiments with a 95% confidence interval (CI). Absolute values of nHZ-induced TNF and MCP-1 release were, respectively 357 pg/mL (120-644 pg/mL) and 575 pg/mL (284-877 pg/mL). P < .05 up to 30 minutes for all modified HZ forms (B); *P < .05 and #P < .01 (C).