Therapeutic inhibition of CXCR2 by Reparixin attenuates acute lung injury in mice

Abstract

Background and purpose: Acute lung injury (ALI) remains a major challenge in critical care medicine. Both neutrophils and chemokines have been proposed as key components in the development of ALI. The main chemokine receptor on neutrophils is CXCR2, which regulates neutrophil recruitment and vascular permeability, but no small molecule CXCR2 inhibitor has been demonstrated to be effective in ALI or animal models of ALI. To investigate the functional relevance of the CXCR2 inhibitor Reparixin in vivo, we determined its effects in two models of ALI, induced by either lipopolysaccharide (LPS) inhalation or acid instillation.

Experimental approach: In two ALI models in mice, we measured vascular permeability by Evans blue and evaluated neutrophil recruitment into the lung vasculature, interstitium and airspace by flow cytometry.

Key results: Pharmacological inhibition of CXCR2 by Reparixin reduced CXCL1-induced leukocyte arrest in the microcirculation of the cremaster muscle, but did not influence arrest in response to leukotriene B4 (LTB4) demonstrating specificity. Reparixin (15 microg g(-1)) reduced neutrophil recruitment in the lung by approximately 50% in a model of LPS-induced ALI. A higher dose did not provide additional reduction of neutrophil recruitment. This dose also reduced accumulation of neutrophils in the interstitial compartment and vascular permeability in LPS-induced ALI. Furthermore, both prophylactic and therapeutic application of Reparixin improved gas exchange, and reduced neutrophil recruitment and vascular permeability in a clinically relevant model of acid-induced ALI.

Conclusions and implications: Reparixin, a non-competitive allosteric CXCR2 inhibitor attenuates ALI by reducing neutrophil recruitment and vascular permeability.

Figure 1

Effect of Reparixin on CXCL1-induced arrest from rolling in vivo. (a, b) Number of adherent leukocytes in cremaster muscle postcapillary venules of wild-type mice pretreated with the CXCR2 inhibitor Reparixin (n=4) and control mice (n=4) before (left) and after (right) injection with 600 ng CXCL1 (a) or 5 μg LTB₄ (b). Data presented are the mean±s.e.m. from four mice. ^#P<0.05. ^*P<0.05 compared to corresponding control.

Figure 2

Dose-dependent inhibition of neutrophil recruitment into the lung following lipopolysaccharide (LPS) inhalation. Mice inhaled LPS for 30 min, and neutrophil accumulation in bronchoalveolar lavage (BAL; BAL PMN) was analysed at 24 h. Other mice were pretreated with different doses of Reparixin (n=4). Mean±s.e.m. ^#P<0.05 vs LPS-treated mice (n=4).

Figure 3

Neutrophil recruitment into the lung in a model of lipopolysaccharide (LPS)-induced pulmonary inflammation. Neutrophil recruitment into the different compartments of the lung was measured in Reparixin-treated (15 μg g⁻¹) mice (n=4) and control mice (n=4) by flow cytometry 24 h after LPS inhalation. Neutrophil accumulation in the intravascular (a), interstitial (b) and alveolar (c) compartments of Reparixin-treated mice and control mice. ^#P<0.05.

Figure 4

Effect of Reparixin on pulmonary microvascular permeability. Pulmonary permeability measured by Evans blue extravasation was determined in Reparixin-treated mice (n=4) and control mice (n=4) 6 h after lipopolysaccharide (LPS) exposure. ^#P<0.05.

Figure 5

Reparixin protects from acid-induced acute lung injury (ALI). Two hours after intratracheal saline or HCl application, gas exchange (a), neutrophils in bronchoalveolar lavage (BAL) fluid (b) and vascular permeability (c) were determined in Reparixin-treated (15 μg g⁻¹; open bars, n=4 mice) and control mice (black bars, n=4 mice). ^#P<0.05.

Figure 6

Neutrophil recruitment into the lung following the induction of the acid-induced acute lung injury (ALI). Neutrophil recruitment into the different compartments of the lung was measured in Reparixin-treated (15 μg g⁻¹) mice (n=4) and control mice (n=4–6) by flow cytometry 2 h after saline or HCl application. Neutrophil accumulation was measured in the intravascular (a) and interstitial (b) compartments of Reparixin-treated mice and control mice. ^#P<0.05.

Figure 7

Therapeutic application of Reparixin attenuates acid-induced acute lung injury. Two hours after initiation of acid-induced acute lung injury (ALI), gas exchange (a), neutrophils in bronchoalveolar lavage (BAL) fluid (b) and vascular permeability (c) were determined in mice that received Reparixin (15 μg g⁻¹, n=4) at 15 min after induction of ALI and in control mice with ALI but no Reparixin (n=4). ^#P<0.05.