CXCR2 antagonists block the N-Ac-PGP-induced neutrophil influx in the airways of mice, but not the production of the chemokine CXCL1

Abstract

Neutrophils are innate immune cells in chronic inflammatory diseases including chronic obstructive pulmonary disease (COPD) and can be attracted to the site of inflammation via the collagen breakdown product N-acetyl Proline-Glycine-Proline (N-Ac-PGP). To elucidate whether CXCR2 is involved in N-Ac-PGP-induced neutrophil migration and activation, studies using specific antagonists were performed in vivo. N-Ac-PGP and keratinocyte cell-derived chemokine (KC; CXCL1) were administered in C57Bl/6 mice via oropharyngeal aspiration. Intraperitoneal applications of CXCR2 antagonist SB225002 or SB332235 were administered 1 h prior and 1 h after oropharyngeal aspiration. Six hours after oropharyngeal aspiration mice were sacrificed. Neutrophil counts and CXCL1 levels were determined in bronchoalveolar lavage fluid, myleoperoxidase (MPO) levels were measured in lung tissue homogenates and an immunohistological staining for neutrophils was performed on lung tissue. N-Ac-PGP and CXCL1 induced a neutrophil influx in the bronchoalveolar lavage fluid and lung tissue, which was also reflected by increased MPO levels in lung tissue. The N-Ac-PGP- and CXCL1-induced neutrophil influx and the increased pulmonary tissue MPO levels were inhibited by the CXCR2 antagonists SB225002 and SB332235. Moreover, N-Ac-PGP administration enhanced the CXCL1 levels in bronchoalveolar lavage fluid, which could not be attenuated by both CXCR2 antagonists. In conclusion, neutrophil migration induced by N-Ac-PGP is mediated via direct CXCR2 interaction. The N-Ac-PGP-induced release of CXCL1 is independent of CXCR2. Related to the maximal effect of CXCL1, N-Ac-PGP is more potent at inducing neutrophil migration in the pulmonary tissue than into the bronchoalveolar lavage fluid, or N-ac-PGP may be more potent at inducing MPO levels in the lung tissue.

Introduction

Neutrophils are innate inflammatory cells in chronic inflammatory diseases including chronic obstructive pulmonary disease (COPD) and are attracted to the site of inflammation via chemoattractants, such as interleukin-8 (CXCL8) in humans and keratinocyte cell-derived chemokine (KC; CXCL1) or macrophage inflammatory protein-2 (MIP-2; CXCL2) in mice (Kobayashi, 2008). CXCL8, CXCL1 and CXCL2 bind to the CXCR2 that is predominantly expressed by neutrophils. Activated neutrophils release proteases, which contribute to collagen breakdown leading to lung emphysema. In our hypothesis, the increased protease activity leads not only to alveolar wall destruction but also to the formation of N-acetyl Pro-Gly-Pro (N-Ac-PGP) from collagen (Folkerts et al., 2008). N-Ac-PGP is chemotactic for neutrophils in vitro as well as in vivo (Weathington et al., 2006) and activates human neutrophils to release CXCL8 (Overbeek et al., in press). The importance of N-Ac-PGP in inflammatory diseases, such as COPD, is reinforced by several studies. Clinical data demonstrated that N-Ac-PGP can be detected in the bronchoalveolar lavage fluid, sputum and serum of COPD patients (O'Reilly et al., 2009, Weathington et al., 2006). Chronic airway exposure to N-Ac-PGP causes neutrophil infiltration and lung emphysema in mice (van Houwelingen et al., 2008, Weathington et al., 2006).

It has been suggested that the basis for N-Ac-PGP effects lies in its structural homology with the GP motif present in all ELR⁺ CXC chemokines, such as CXCL8. This GP motif is essential for cell activation and ligand binding to CXCR1/2 receptors on neutrophils (Clark-Lewis et al., 1994, Weathington et al., 2006). Overbeek et al., in press, Weathington et al., 2006 reported that N-Ac-PGP activity is mediated via the G-protein coupled receptor CXCR2, since CXCR2 antibodies suppressed the N-Ac-PGP-induced neutrophil chemotaxis in vitro. Furthermore, the accumulation of neutrophils seen in mice upon intratracheal administration of N-Ac-PGP, was not detected in CXCR2^−/− mice (Weathington et al., 2006).

The aim of this study was to investigate whether the N-Ac-PGP-induced neutrophil influx was mediated via CXCR2 in vivo. We investigated the effects of two different selective nonpeptide CXCR2 antagonists, SB225002 and SB332235, in a murine model where a neutrophil influx was induced via oropharyngeal administration of N-Ac-PGP or CXCL1. In this report, we demonstrate that N-Ac-PGP and CXCL1 can induce a neutrophil influx in lung tissue and bronchoalveolar lavage fluid of mice, which can be inhibited by CXCR2 antagonists. The neutrophil migration induced by N-Ac-PGP is mediated via direct interaction with CXCR2 and by a small, if any, release of CXCL1. The N-Ac-PGP-induced release of CXCL1 is independent of the CXCR2.