Priming of neutrophil [Ca2+](i) signaling and oxidative burst by human fracture fluids

Background: Patients with major fracture/soft-tissue injuries are at risk f or adult respiratory distress syndrome after secondary infection. Fracture fluids (FF) are rich in neutrophil (PMN) -specific chemokines such as inter leukin-g, PMN respond to both interleukin-8 and bacterial stimuli with calc ium ([Ca2+](i)) fluxes, which can initiate respiratory burst (RB). We hypot hesize that small amounts of FF entering the circulation could exaggerate P MN [Ca2+](i) and RE responses, potentially increasing the risk of adult res piratory distress syndrome. Methods: FF were obtained from 10 patients at open fixation of the femur 2 to 5 days postinjury. Volunteer PMN were isolated and loaded with fura dye. PMN were preincubated either in 30% autologous plasma (AP)/70% buffer, or in 5% FF/25% AP/70% buffer. Cells mere resuspended in buffer with 1,2,3-dih ydrorhodamine and stimulated with low-dose n-formylmethionyl-leucyl-phenyla lanine (fMLP), [Ca2+](i) was assayed by fura fluorescence at 505 nm after e xcitation at 340/380 mm, RE was assessed by 1,2,3-dihydrorhodamine fluoresc ence at 530 nm after 488 nm excitation. Results: PMN basal [Ca2+](i) was higher after FP incubation than AP incubat ion (94 +/- 12 vs. 61 +/- 9 nmol/L, p = 0.0002). Peak [Ca2+](i) response to fMLP was 475 +/- 47 nmol/L after FF but only 356 +/- 22 nmol/L after AP (p = 0.01). Two hundred seconds after fMLP, [Ca2+](i) remained higher after F F (172 +/- 17 vs. 145 +/- 9 nmol/L, p = 0.04), Basal RE was slightly higher after FF than AP (13.4 +/- 0.3 vs. 113 +/- 0.3 units, p = 0.051) as was th e maximal rate of extracellular oxidant release (1.10 +/- 0.17 vs. 0.76 +/- 0.16 units/s, p = 0.004) and total oxidant production (42.5 +/- 0.8 vs. 31 .7 +/- 0.8 units, p = 0.006). Conclusions: Small amounts of FF in plasma can exaggerate PMN [Ca2+](i) flu x and RE responses to subsequent bacterial stimuli. These findings are cons istent with the hypothesis that release of FF into the circulation primes P MN and, thus, may predispose to adult respiratory distress syndrome, Such P MN priming events might have important implications for both the operative and medical management of patients with major fractures.