Related Experiment Video
Updated: Oct 18, 2026
Standardized In vitro Assays to Visualize and Quantify Interactions between Human Neutrophils and Staphylococcus aureus Biofilms
Published on: June 8, 2022
Priming of neutrophil [Ca2+]i signaling and oxidative burst by human fracture fluids
C J Hauser1, N Desai, Z Fekete
1Department of Surgery, UMD-New Jersey Medical School, Newark 07103, USA. hausercj@UMDNJ.edu
Background:
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.
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 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.
Results:
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).
Conclusion:
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.
Insights
Fracture fluids (FF) entering circulation can heighten neutrophil responses, increasing the risk of adult respiratory distress syndrome (ARDS). This study shows FF primes neutrophils, potentially impacting patient management after major fractures.
Area of Science:
- Immunology
- Critical Care Medicine
- Trauma Surgery
Background:
- Patients with major fractures and soft-tissue injuries face ARDS risk post-infection.
- Fracture fluids (FF) contain neutrophil (PMN)-attracting chemokines like interleukin-8.
- PMN activation by chemokines and bacteria can trigger calcium ([Ca2+]i) fluxes and respiratory burst (RB).
Purpose of the Study:
- To investigate if small amounts of FF in circulation exaggerate PMN [Ca2+]i and RB responses.
- To determine the potential role of FF in predisposing patients to ARDS.
Main Methods:
- Neutrophils (PMN) were isolated and incubated with either autologous plasma (AP) or FF-supplemented plasma.
- Cells were stimulated with a low-dose bacterial peptide (fMLP).
- Intracellular calcium ([Ca2+]i) flux and respiratory burst (RB) were measured using fluorescence assays.
Main Results:
- PMN incubated with FF showed significantly higher basal and fMLP-stimulated [Ca2+]i levels compared to AP.
- FF incubation also resulted in elevated basal and stimulated RB, including increased oxidant release and production.
- These exaggerated responses were statistically significant (p < 0.05).
Conclusions:
- Even small amounts of FF in plasma can prime PMN, enhancing their response to bacterial stimuli.
- This FF-induced PMN priming may contribute to ARDS development in fracture patients.
- Findings suggest implications for managing patients with major fractures, both surgically and medically.
