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. 2012 Sep 25;18(1):1056-66.
doi: 10.2119/molmed.2011.00497.

Initial assessment of the role of CXC chemokine receptor 4 after polytrauma

Harold H Bach 4th  1 , Vikas Saini, Todd A Baker, Abhishek Tripathi, Richard L Gamelli, Matthias Majetschak
Affiliations

Initial assessment of the role of CXC chemokine receptor 4 after polytrauma

Harold H Bach 4th et al. Mol Med. .

Abstract

CXC chemokine receptor (CXCR)-4 agonists have been shown to attenuate inflammation and organ injury in various disease models, including trauma/hemorrhage. The pathophysiological role of CXCR4 during the early response to tissue injury, however, remains unknown. Therefore, we investigated the effects of AMD3100, a drug that antagonizes binding of stromal cell-derived factor (SDF)-1α and ubiquitin to CXCR4 during the initial response to polytrauma in pigs. Fifteen minutes before polytrauma (femur fractures/lung contusion; control: sham), 350 nmol/kg AMD3100, equimolar AMD3100 and ubiquitin (350 nmol/kg each) or vehicle were administered intravenously. After a 60-min shock period, fluid resuscitation was performed for 360 min. Ubiquitin binding to peripheral blood mononuclear cells was significantly reduced after intravenous AMD3100. SDF-1α plasma levels increased transiently >10-fold with AMD3100 in all animals. In injured animals, AMD3100 increased fluid requirements to maintain hemodynamics and enhanced increases in peripheral blood granulocytes, lymphocytes and monocytes, compared with its effects in uninjured animals. Cytokine release from leukocytes in response to Toll-like receptor (TLR)-2 and TLR-4 activation was increased after in vitro AMD3100 treatment of normal whole blood and after in vivo AMD3100 administration in animals subjected to polytrauma. Coadministration of AMD3100/ubiquitin reduced lactate levels, prevented AMD3100-induced increases in fluid requirements and sensitization of the tumor necrosis factor (TNF)-α and interleukin (IL)-6 release upon TLR-2/4 activation, but did not attenuate increases in leukocyte counts and SDF-1α plasma levels. Our findings suggest that CXCR4 controls leukocyte mobilization after trauma, regulates leukocyte reactivity toward inflammatory stimuli and mediates protective effects during the early phase of trauma-induced inflammation.

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Figures

Figure 1
Figure 1
AMD3100 treatment prevents ubiquitin receptor binding. FITC-ubiquitin binding to PBMCs (1 min, 4°C) was measured as described (12). (A) Saturation binding curves for FITC-ubiquitin with PBMCs that were isolated before (open circles, t = −15 min) and after (black circles, t = 0 min) i.v. vehicle administration. Black triangles: nonspecific binding (NSB), assessed in the presence of 300 μmol/L native ubiquitin. NSB was identical before and after i.v. vehicle. Means ± SD from triplicate measurements are shown. (B) Saturation binding curves for FITC-ubiquitin with PBMCs that were isolated before (open circles, t = −15 min) and after (black circles, t = 0 min) i.v. AMD3100 administration. Black triangles: NSB, assessed in the presence of 300 μmol/L native ubiquitin. NSB was identical before and after i.v. AMD3100. Means ± SD from triplicate measurements are shown. (C) Bmax values from saturation binding curves for FITC-ubiquitin with PBMCs from animals after sham procedure and vehicle (open bars) or AMD3100 (gray bars) treatment at t = −15 min. Data are expressed as percent of Bmax at baseline (t = −15 min). Data are means ± SD; n = 3–5 animals per group and time point. (D) Bmax values from saturation binding curves for FITC-ubiquitin with PBMCs from animals after polytrauma and vehicle (open bars) or AMD3100 (gray bars) treatment at t = −15 min. Data are expressed as percent of Bmax at baseline (t = −15 min). Data are means ± SD; n = 3–5 animals per group and time point. *p < 0.05 versus baseline.
Figure 2
Figure 2
AMD3100 induces release of SDF-1α into the systemic circulation. The arrows indicate the time points of drug administration. (A) SDF-1α plasma concentrations (mean ± SD) after i.v. AMD3100 (gray squares, n = 6) or vehicle (open squares, n = 7) administration in uninjured animals. *p < 0.05 versus vehicle. (B) SDF-1α plasma concentrations (mean ± SD) after i.v. AMD3100 (gray circles, n = 9), AMD3100 plus ubiquitin (black circles) or vehicle (open circles, n = 8) administration in animals undergoing polytrauma at t = 0 min. Shock: simulated shock phase. EMS: simulated prehospital emergency medical services. Resuscitation: simulated in-hospital resuscitation phase. *p < 0.05 for AMD3100 versus vehicle; #p < 0.05 for AMD3100 plus ubiquitin versus vehicle.
Figure 3
Figure 3
Physiological responses after AMD3100 administration. The arrows indicate the time points of drug administration. Data are means ± SD. (A–E) Uninjured animals. Grey squares: AMD3100 treatment (n = 6). Open squares: vehicle treatment (n = 7). (F–J) Polytrauma groups. Grey circles: AMD3100 treatment (n = 9). Black circles: AMD3100 plus ubiquitin treatment (n = 9). Open circles: vehicle treatment (n = 8). (A, F) MAP (mmHg). (B, G) i.v. fluid requirements (mL/kg) to maintain a MAP of 70 mmHg. (C, H) Hematocrit (%). (D, I) Ratio of partial pressure of oxygen in arterial blood to the fraction of inspired oxygen (PaO2/FiO2). (E, J) Lactate concentrations (% of baseline). Shock: simulated shock phase. EMS: simulated prehospital emergency medical services. Resuscitation: simulated in-hospital resuscitation phase. *p < 0.05 versus vehicle.
Figure 4
Figure 4
WBC counts after AMD3100 administration. The arrows indicate the time points of drug administration. Data are mean ± SD. (A–D) Uninjured animals. Grey squares: AMD3100 treatment (n = 6). Open squares: vehicle treatment (n = 7). (E–H) Polytrauma groups. Grey circles: AMD3100 treatment (n = 9). Black circles: AMD3100 plus ubiquitin treatment (n = 9). Open circles: vehicle treatment (n = 8). Shock: simulated shock phase. EMS: simulated prehospital emergency medical services. Resuscitation: simulated in-hospital resuscitation phase. (A, E) Total WBC counts (×103 cells). (B, F) Granulocytes (×103 cells). (C, G) Lymphocytes (×103 cells). (D, H) Monocytes (×103 cells). *p < 0.05 AMD3100 versus vehicle. #p < 0.05 AMD3100 plus ubiquitin versus vehicle.
Figure 5
Figure 5
LPS stimulated whole blood TNF-α and IL-6 release after AMD3100 administration in vivo. Drugs were administered within 15 min at t = −15 min. Data are expressed as percent of baseline (t = −15 min) of the cytokine concentration in the cell culture supernatant per 103 leukocytes in the incubation mixtures (mean ± SD). (A, C) Uninjured animals. Open bars: vehicle treatment. Grey bars: AMD3100 treatment. (B, D) Polytrauma groups. Open bars: vehicle treatment. Grey bars: AMD3100 treatment. Black bars: AMD3100 and ubiquitin treatment. (A, B) TNF-α. C/D: IL-6. *p < 0.05 versus vehicle.
Figure 6
Figure 6
LTA-stimulated whole blood TNF-α and IL-6 release after AMD3100 administration in vivo. Drugs were administered within 15 min at t = −15 min. Data are expressed as percent of baseline (t = −15 min) of the cytokine concentration in the cell culture supernatant per 103 leukocytes in the incubation mixtures (mean ± SD). (A, C) Uninjured animals. Open bars: vehicle treatment. Grey bars: AMD3100 treatment. (B, D) Polytrauma groups. Open bars: vehicle treatment. Grey bars: AMD3100 treatment. Black bars: AMD3100 and ubiquitin treatment. (A, B) TNF-α. (C, D) IL-6. *p < 0.05 versus vehicle.
Figure 7
Figure 7
LPS- and LTA-stimulated whole blood release after AMD3100 administration in vitro. Normal whole blood was stimulated with LPS (left) or LTA (right) in the absence (−) or presence (+) of 10 μmol/L AMD3100.
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