Priming of Neutrophil [Ca2]i Signaling and Oxidative Burst by Human Fracture Fluids

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Abstract
Patients with major fracture/soft-tissue injuries are at risk for adult respiratory distress syndrome after secondary infection. Fracture fluids (FF) are rich in neutrophil (PMN) -specific chemokines such as interleukin-8. PMN respond to both interleukin-8 and bacterial stimuli with calcium ([Ca2+]i) fluxes, which can initiate respiratory burst (RB). We hypothesize that small amounts of FF entering the circulation could exaggerate PMN [Ca2+]i and RB responses, potentially increasing the risk of adult respiratory distress syndrome. 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 were resuspended in buffer with 1,2,3-dihydrorhodamine and stimulated with low-dose n-formyl-methionyl-leucyl-phenylalanine (fMLP). [Ca2+]i was assayed by fura fluorescence at 505 nm after excitation at 340/380 nm. RB was assessed by 1,2,3-dihydrorhodamine fluorescence at 530 nm after 488 nm excitation. PMN basal [Ca2+]i was higher after FF incubation than AP incubation (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 FF (172+/-17 vs. 145+/-9 nmol/L, p = 0.04). Basal RB was slightly higher after FF than AP (13.4+/-0.3 vs. 11.3+/-0.3 units, p = 0.051) as was the 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). Small amounts of FF in plasma can exaggerate PMN [Ca2+]i flux and RB responses to subsequent bacterial stimuli. These findings are consistent with the hypothesis that release of FF into the circulation primes PMN and, thus, may predispose to adult respiratory distress syndrome. Such PMN priming events might have important implications for both the operative and medical management of patients with major fractures.