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

Animals

The Animal Care and Use Committee of the University of Virginia (Charlottesville) approved all animal experiments. We used 8–12 weeks old C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME, USA). Mice were housed in a barrier facility under specific pathogen-free conditions.

Surgical preparation and intravital microscopy

Mice were anaesthetized with an intraperitoneal (i.p.) injection of ketamine hydrochloride (125 mg kg⁻¹; Sanofi Winthrop Pharmaceuticals, New York, NY, USA), atropine sulphate (0.025 mg kg⁻¹; Fujisawa, Deerfield, IL, USA) and xylazine (12.5 mg kg⁻¹; Tranqui Ved; Phonix Scientific, San Marcos, CA, USA) and placed on a heating pad to maintain body temperature. For specificity testing, the cremaster muscle was prepared after tracheal intubation and cannulation of the carotid artery for intravital microscopy as previously described (^{Zarbock et al., 2007b}). Microscopic observations were made on postcapillary venules (diameter 20–40 μm) using an intravital microscope (Axioskop; Zeiss, Thornwood, NY, USA) with a saline immersion objective (SW 40/0.75 numerical aperture). A CCD camera (model VE-1000CD, Dage-MTI) was used for recording. Leukocyte arrest was determined before and 1 min after intravenous (i.v.) injection of 600 ng CXCL1 (PeproTech, Rocky Hill, NJ, USA) or 5 μg leukotriene B₄ (LTB₄; Cayman Chemical, Ann Arbor, MI, USA) as described previously (^{Zarbock et al., 2007a}). Arrest was defined as leukocyte adhesion longer than 30 s and expressed as number of cells per surface area. Surface area, S, was calculated for each vessel using S=π × d × l_ν, where d is the diameter and l_ν is the length of the vessel. Blood flow centreline velocity was measured using a dual photodiode sensor and a digital online cross-correlation program (Circusoft Instrumentation, Hockessin, DE, USA). Centreline velocity and wall shear stress were calculated as described previously (^{Lipowsky and Zweifach, 1978}; ^{Long et al., 2004}).

Murine model of LPS-induced pulmonary inflammation and acid-induced ALI

LPS-induced pulmonary inflammation was induced as previously described (^{Reutershan et al., 2007}). Briefly, mice were exposed to aerosolized LPS from Salmonella enteritidis (500 μg mL⁻¹, Sigma-Aldrich, St Louis, MO, USA) for 30 min and analysed 24 h later. Aerosolized saline served as a negative control.

Acid-induced ALI was induced as previously described (^{Zarbock et al., 2006}). Briefly, 2 μL g⁻¹ body weight of HCl with a pH of 1.5 was injected intratracheally, followed by a bolus of air (30 μL g⁻¹). After the induction of ALI, the trachea of each mouse was intubated with a tube (PE 90) and the mouse was ventilated with a respirator (MiniVent, Type 845; Hugo Sachs Elektronik) for 2 h (tidal volume, 10 μL g⁻¹; respiration rate, 140 per min; fraction of inspiratory oxygen (FiO₂), 0.21). Control animals received saline instead of HCl in the same manner.

Reparixin (R(–)-2-(4-isobutylphenyl)propionyl methansulphonamide) as the L-lysine salt (^{Bertini et al., 2004}) was obtained from Dompé pha.r.ma (Italy) and dissolved in saline. Where indicated, Reparixin was injected i.p. at 15 min before and 2 h after LPS-induced pulmonary inflammation. In the model of acid-induced ALI, Reparixin was injected i.p. either 15 min before or 15 min after the induction of ALI.

Neutrophil recruitment into the lung

After the mice were killed, bronchoalveolar lavage (BAL) was collected (5 × 1 mL phosphate-buffered saline (PBS)). BAL fluid was centrifuged and neutrophils were counted using Kimura stain.

Intravascular and interstitial neutrophils in the lungs were distinguished by a flow-cytometry-based method as previously described (^{Zarbock et al., 2006}). Briefly, an Alexa 633-labelled GR-1 antibody (clone RB6-8C5, staining kit: Invitrogen Corp., Carlsbad, CA, USA) was injected i.v. 5 min, before death. This labels only intravascular neutrophils. After performing BAL, the inferior vena cava was dissected and non-adherent neutrophils were removed from the pulmonary vasculature by flushing 10 mL of PBS at 25 cm H₂O through the spontaneously beating right ventricle. Lungs were removed, minced and digested with enzyme cocktail at 37 °C for 60 min. In order to produce a cell suspension, the digested lungs were passed through a 70 μm cell strainer (BD Falcon, Bedford, MA, USA), erythrocytes were lysed and the remaining leukocytes were counted. The fraction of neutrophils in the suspension was determined by flow cytometry (FACS Calibur; Becton Dickinson, San Jose, CA, USA). Neutrophils were identified by their typical appearance in the forward/sideward scatter and their expression of CD45 (clone 30-F11), 7/4 (clone 7/4; both BD Biosciences-Pharmingen, San Diego, CA, USA) and GR-1 (clone RB6-8C5). The i.v. injected labelled GR-1 Ab was used to differentiate between intravascular (CD45⁺7/4⁺GR-1⁺) and interstitial (CD45⁺7/4⁺GR-1⁻) neutrophils.

In acid-induced ALI, vascular permeability was determined by measuring protein concentration in the supernatant (Lowry's method).

Pulmonary microvascular permeability

Pulmonary microvascular permeability in LPS- and saline-treated mice pretreated with Reparixin or vehicle was determined by Evans blue dye extravasation as described previously (^{Reutershan et al., 2006}). Briefly, Evans blue (20 mg kg⁻¹; Sigma-Aldrich) was injected i.v. 30 min prior to death. Lungs were perfused, removed and Evans blue was extracted. The absorption of Evans blue was measured, corrected for haemoglobin and calculated in the different animal groups, 6 h after LPS or saline inhalation.

Pulmonary function: oxygenation

Blood was obtained from an arterial catheter to measure arterial blood gas (Rapidlab 800 System; Bayer HealthCare). Partial pressure of arterial oxygen (PaO₂) was normalized to the fraction of inspiratory oxygen (FiO₂) to obtain the oxygenation index.

Statistics

Statistical analysis was performed with SPSS (version 9.0, Chicago, IL, USA) and included one-way analysis of variance, Student–Newman–Keuls test and t-test where appropriate. All data are presented as mean±s.e.m.; P<0.05 was considered significant.

Reparixin, a non-competitive allosteric CXCR2 inhibitor, specifically blocks CXCL1-induced leukocyte arrest in vivo

It has been shown that Reparixin reduces ligand binding to human CXCR1 and CXCR2, calcium influx and downstream signalling in response to human CXCL8 and neutrophil recruitment into the liver in a mouse model of ischaemia-reperfusion injury (^{Bertini et al., 2004}). In order to investigate whether Reparixin specifically blocks CXCR2-induced leukocyte arrest in mice, we performed intravital microscopy of the cremaster muscle and investigated leukocyte arrest in response to CXCL1 and LTB₄. LTB₄, which binds to and activates the Gα_i-coupled receptor BLTR1 (^{Wettschureck and Offermanns, 2005}) was used as a positive control. Mice pretreated with Reparixin showed the same number of adherent leukocytes under baseline conditions compared to control mice (Figure 1a). Injection of the recombinant murine chemokine CXCL1 induced immediate firm arrest in control mice (Figure 1a). The number of adherent cells per area in mice pretreated with Reparixin was significantly reduced after CXCL1 injection (Figure 1a). The treatment with Reparixin did not influence leukocyte adhesion in response to LTB₄ (Figure 1b). The venules were of similar size and had similar haemodynamic parameters (data not shown). These data suggest that inhibition of the CXCR2 receptor by Reparixin selectively blocked CXCR2-induced arrest without inhibiting other G-protein-coupled receptors.

Efficiency of Reparixin in inhibiting neutrophil recruitment in the lung

Reparixin at concentrations of 3 μg g⁻¹ and 15 μg g⁻¹ reduced neutrophil recruitment and liver damage by approximately 30% and 80% in a model of ischaemia-reperfusion injury, respectively (^{Bertini et al., 2004}). In order to investigate the necessary dosage of Reparixin to inhibit neutrophil recruitment in the inflamed lung, we tested the effects of Reparixin at 3, 15 and 30 μg g⁻¹ on neutrophil recruitment into the alveolar compartment following LPS inhalation. Twenty-four hours after LPS exposure, neutrophil recruitment into the alveolar compartment significantly increased in control mice (Figure 2). Treatment with 3 μg g⁻¹ Reparixin did not reduce neutrophil recruitment into the lung, whereas 15 μg g⁻¹ led to a significant reduction in neutrophil recruitment into the alveolar space (Figure 2). The increase of the Reparixin dose to 30 μg g⁻¹ did not further reduce neutrophil accumulation in BAL. Therefore, 15 μg g⁻¹ of Reparixin was used in all further experiments.

Pharmacological inhibition of CXCR2 reduces transendothelial and transepithelial migration, but not intravascular accumulation of neutrophils in the microcirculation of the lung

In the lung, neutrophils first accumulate in the vasculature, followed by infiltration into the interstitial space and exit into BAL (^{Reutershan et al., 2005}). In order to investigate which step in the neutrophil recruitment cascade was affected by the inhibition of CXCR2, mice received a dose of 15 μg g⁻¹ Reparixin 15 min before and 2 h after LPS inhalation. Reparixin did not affect the number of neutrophils in the intravascular (Figure 3a), interstitial (Figure 3b) or alveolar compartments (Figure 3c) under baseline conditions compared to vehicle-treated control mice. After LPS exposure, Reparixin significantly reduced neutrophil numbers in the interstitial (Figure 3b) and alveolar compartments (Figure 3c). These data show that CXCR2 is not involved in neutrophil accumulation in the microcirculation of the lung under normal conditions, but this receptor is critical for neutrophil migration into other compartments. The stronger inhibition seen in BAL compared to the interstitial space suggests that inhibition of both transendothelial and transepithelial migration contributes to this effect.

Pulmonary microvascular permeability is reduced by CXCR2 blockade

In addition to neutrophil infiltration, vascular leakage is also critically involved in pulmonary inflammation and ALI. We therefore tested the role of Reparixin in LPS-induced microvascular permeability. In control mice, LPS inhalation caused a significant increase of the pulmonary vascular permeability as measured by Evans blue (Figure 4). This vascular permeability increase was reduced by approximately 65% in mice pretreated with Reparixin (Figure 4).

Reparixin protects against acid-induced acute lung injury

Based on our finding that the inhibition of CXCR2 by Reparixin reduces neutrophil recruitment and vascular permeability in response to LPS exposure, we hypothesized that this inhibitor may also improve functional and morphological parameters in a clinically relevant model of acid-induced ALI (^{Zarbock et al., 2006}). Two hours after induction of ALI with HCl, ventilated control mice showed reduced gas exchange (Figure 5a), increased neutrophil numbers in BAL (Figure 5b) and elevated protein levels in BAL (Figure 5c) compared to mice injected with saline. Application of Reparixin 15 min before the induction of ALI, significantly improved the gas exchange (Figure 5a), but did not affect that found in mice injected with saline. Reparixin treatment reduced the number of neutrophils in the alveolar space (Figure 5b), but the inhibition was not complete. The protein concentration in the BAL of control mice significantly increased 2 h following injecting HCl. Injection of Reparixin 15 min after the induction of ALI diminished the protein concentration in BAL compared to vehicle-treated control mice (Figure 5c).

As CXCR2 inhibition by Reparixin reduced neutrophil recruitment into BAL 2 h after induction of acid-induced ALI, we investigated which step in the neutrophil recruitment cascade was influenced by CXCR2 inhibition. The number of neutrophils in the intravascular (Figure 6a) and interstitial (Figure 6b) compartments significantly increased in control mice 2 h after intratracheal instillation of HCl. After induction of acid-induced ALI, Reparixin significantly reduced the numbers of neutrophils in the intravascular (Figure 6a) and interstitial compartments (Figure 6b).

Therapeutic application of Reparixin attenuates acid-induced acute lung injury

In order to investigate whether Reparixin improves functional and morphological parameters of ALI even when given after the insult, we injected Reparixin 15 min after the induction of the acid-induced lung injury and measured gas exchange, neutrophil recruitment and protein concentration in BAL. Although Reparixin is a non-competitive allosteric inhibitor, application of this inhibitor in a therapeutic approach improved gas exchange (Figure 7a), and reduced neutrophil recruitment (Figure 7b) and vascular permeability (Figure 7c).