Patients
Patients suffering from chest injury with an abbreviated injury score (AIS) of 2 or more who were admitted to the trauma department of the University Medical Centre Utrecht were included. Patients with injuries with an AIS > 2 in other regions than the thorax were excluded to reduce systemic inflammation caused by tissue damage outside the thorax. Other exclusion criteria included age <18 or >70 years, death within 24 h after admission, and patients with altered immunological status (e.g., corticosteroid use or chemotherapy).
At admission, injury severity score (ISS) [
22], new injury severity score (NISS) [
23], Apache II score [
24], and leukocyte count were determined. All patients were followed until discharge. The presence of ARDS was assessed according to their clinical criteria, as determined in the consensus conferences on ARDS [
25].
Blood samples ware taken at approximately 3 (2–4), 9 (8–10) and 24 (22–26) h after the accident to investigate the relationship between chest injury and systemic neutrophil activation. In an in vivo human inflammation model, we saw previously that systemic neutrophil activation is most prominent between 2 and 4 h after the induction of inflammation [
26]. The first measurement time point was therefore set at 3 h postinjury.
The local ethics committee approved the study, and written informed consent was obtained from all patients or their legal representatives in accordance with the Helsinki Declaration.
Expression of activation markers on neutrophils determined by flowcytometry
The following commercially available mouse–antihuman monoclonal antibodies were purchased for analyzing neutrophil receptor expression by flowcytometry: fluorescein isothiocyanate (FITC)-labeled IgG1 isotype control (clone MOPC-21, BD Pharmingen, USA), Alexa Fluor® 647-labeled IgG1 isotype control (clone MOPC-21, BD Pharmingen, USA), R-phycoerythrin (RPE)-labeled IgG2a isotype control (clone MRC OX-34, Serotec, Germany), RPE-labeled IgG1 anti-αM (CD11b; clone 2LPM19c, DAKO, Denmark), FITC-labeled IgG1 anti-l-selectin (CD62L; clone Dreg56, BD Pharmingen, USA), Alexa Fluor® 647-labeled IgG1 anti-FCγRIII (CD16; clone 3G8, BD Pharmingen, USA), RPE-labeled IgG2b anti-FcγRII (CD32; clone FLI8.26, BD Pharmingen, USA), FITC-labeled IgG2a anti-CXCR1 (CD181a; clone 42705, R&D Systems Europe, UK), RPE-labeled IgG2a anti-CXCR2 (CD182b; clone 48311, R&D Systems Europe, UK), and FITC-labeled IgG2a anti-C5aR (CD88; clone P12/1, Serotec, Germany).
A FITC-labeled monoclonal phage antibody (A27), which recognizes the active configuration of FcγRII (CD32), was manufactured at the Department of Respiratory Medicine at the University Medical Centre Utrecht (MoPhab A27, UMC Utrecht, The Netherlands) [
21,
27]. The functionality and configuration of FcγRII (CD32) on granulocytes is regulated by inside-out control [
28]. Visualization of this process by the antibody A27 is a very sensitive means of monitoring the subtle activation of innate immune cells such as neutrophils in vivo.
Blood was collected in a Vacutainer
® with sodium heparin as anticoagulant and cooled immediately on melting ice. Blood samples of eight healthy volunteers served as a control values. Red cells were lysed with ice-cold isotonic NH
4Cl [
27]. After lysis, white blood cells were washed and resuspended in PBS2+ [phosphate-buffered saline supplemented with sodium citrate (0.4% wt/vol) and pasteurized plasma protein solution (10% vol/vol)]. Resuspended cells were incubated for 45 min on ice with commercially obtained directly labeled antibodies against activation molecules:
l-selectin, αM, CXCR1, CXCR2, C5aR, FCγRII and FCγRIII.
After incubation and a final wash, expression was measured on a FACScalibur flow cytometer (Becton, Dickinson & Co., Mountain View, CA, USA). The neutrophils were identified according to their specific side-scatter and forward-scatter signals.
To measure FcγRII* expression, whole blood was incubated with FITC-labeled monoclonal phage antibody A27 for 45 min on ice [
11]. Active upregulation of FcγRII* expression was measured after 5 min of stimulation of whole blood at 37°C with
N-formyl-methionyl-leucyl-phenylalanine (fMLP 10
−6 M) to evaluate the responsiveness of the cells for bacterially derived protein products/peptides. After stimulation, the samples were put on ice again and stained with phage antibody A27. After staining, red cells were lysed, and expression was measured on FACScalibur, as described above.
Data from individual experiments are depicted as the median fluorescence intensity (MFI) of at least 10,000 neutrophils.