Therapeutic inhibition of CXC chemokine receptor 2 by SB225002 attenuates LPS-induced acute lung injury in mice

Abstract
Introduction: Sustained neutrophilic infiltration is known to contribute to organ damage, such as acute lung injury (ALI). CXC chemokine receptor 2 (CXCR2) is the major receptor regulating inflammatory neutrophil recruitment in acute and chronic inflamed tissues. The purpose of this study was to investigate the func- tional relevance of the CXCR2 inhibitor SB225002 in LPS-induced acute lung injury. Material and methods: Male C57BL/6 mice were randomly divided into the following four experimental groups (n = 10 per group): untreated group (control group, Ctr); LPS-treated ALI group (LPS group, LPS); LPS + PBS-treat- ed group (LPS + PBS); and SB225002-treated ALI group (LPS + SB225002). Twenty-four hours after treatment, the blood, bronchoalveolar lavage fluid (BALF), and lung tissue were collected and wet/dry ratio, protein concentra- tion, myeloperoxidase (MPO) activity, neutrophil infiltration, and inflamma- tory cytokine secretion in lung tissue were measured. The pathologic chang- es in the lungs were examined using optical microscopy. Survival rates were recorded at 120 h in all four groups, in other experiments. Results: Histology findings revealed that the SB225002-treated group had significantly milder lung injury compared to the LPS-induced ALI and the PBS-treated control groups. Treatment with SB225002 significantly attenu- ated LPS-induced lung injury and suppressed the inflammatory responses in damaged lung tissue. Compared to the PBS-treated control group, treatment with SB225002 dramatically decreased the lung wet/dry ratio, protein con- centration, and infiltration of neutrophils in lung tissue. Therefore, SB225002 treatment appeared to inhibit the production of inflammatory cytokines and increase survival time compared to the PBS-treated control group. Conclusions: Together, these data demonstrated that inhibition of CXCR2 signaling by SB225002 could ameliorate LPS-induced acute lung injury, by reducing neutrophil recruitment and vascular permeability. SB225002 may be further developed as a potential novel treatment for LPS-induced ALI.

Introduction
Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are clinical syndromes characterized by hypoxemia, decreased lung com-pliance, and bilateral pulmonary infiltrates, and which have multifactorial etiologies either from direct or indirect lung injury [1, 2]. Histopatho- logically, there is an initial acute exudative phase, involving alveolar-capillary leak in conjunction with leukocyte extravasation. This is followed by a fibroproliferative phase, involving the precipita- tion of alveolar proteins with hyaline membrane formation, persistent inflammation, and prolifer- ation of alveolar epithelia and mesenchymal cells. Despite improvements in disease management, ALI and ARDS are still major causes of morbidity and mortality, in critically ill patients of all ages [3, 4]. The incidence of ALI/ARDS in the US is 138 in 100,000 persons per year, and is expected to dou- ble in the next 25 years [3]. Currently, the treat- ment of ALI/ARDS remains primarily palliative [5]. It is well known that in pulmonary inflamma- tion, recruitment of circulating polymorphonuclear leukocytes is essential for host defense and ini- tiates a specific immune response; a pathologi- cal hallmark of ALI and ARDS is the uncontrolled transmigration of neutrophils into the lung inter- stitium and alveolar space [6]. Accumulation and recruitment of pro-inflammatory leukocytes in the lung are critical events in the development of ALI [2].

The migration of leukocytes is regulat- ed by chemotactic cytokines called chemokines. Chemokines are a large family of small chemotac- tic proteins divided into 4 subfamilies according to the positioning of cysteines in their primary sequences. Members with their first 2 cysteines separated by one amino acid belong to the CXC subfamily and regulate the response of polymor- phonuclear (PMN) neutrophils [7, 8]. Interleukin 8 (IL-8) is the most potent member of the CXC fam- ily and has high affinity for both of its receptors, CXCR1 and CXCR2, which are co-expressed on the membrane of neutrophils. Thus, CXCR1/2 is a ma- jor element in neutrophil recruitment [9]. Rapid neutrophil migration during inflammation, such as occurs in bacterial and fungal infections, can also be mediated by CXCR1/2-binding chemok- ines, elevating blood or inflammatory sites’ neu-trophil counts [10–12].Depending on the underlying cause of ALI, dif- ferent adhesion molecules and chemokines are in- volved in neutrophil recruitment [13]. CXCR2, the chemokine receptor for CXCL1 (keratinocyte-de- rived chemokine) and CXCL2/3 (macrophage in- flammatory protein 2) in mice, is critically involved in neutrophil recruitment and the regulation of vascular permeability in different models of ALI [14, 15].In the present study, we investigated the po- tential beneficial effects of a potent and selec- tive non-peptide CXCR2 antagonist, SB225002, in LPS-induced acute lung injury. We found thattreatment with SB225002 ameliorated LPS-in- duced ALI significantly, decreasing inflammatory neutrophil infiltration and tissue damage. In ad- dition, treatment with SB225002 also appeared to reduce MPO activity, lung wet/dry ratio, protein concentration and inflammatory cytokine produc- tion in lung tissue compared to the PBS-treated control group.

Taken together, our data suggest that the potent and selective non-peptide CXCR2 antagonist SB225002 has a potential therapeutic effect on LPS-induced lung injury.Material and methodsMiceForty C57BL/6 mice were purchased from the SLRC Laboratory (Shanghai, China). The mice were randomized to the following groups: control (n = 10, untreated), ALI (n = 10, LPS-treated only), PBS-treated control (n = 10, LPS + PBS-treated) and SB225002 (n = 10, SB225002-treated). Mice were sacrificed 24 h following LPS and group-spe- cific treatments.Establishment of the ALI mouse modelFemale C57BL/6 mice (8–10 weeks old) were treated intraperitoneally (i.p.) with either 20 mg/kg LPS (Sigma-Aldrich, St. Louis, MO, USA) from Esch- erichia coli (serotype 0111:B4) in 100 µl of PBS or an equal volume of PBS as a vehicle control. Mice were euthanized by an intravenous (i.v.) injection of thiopental 24 h following ALI induction and administration of group-specific treatments, the thoraxes were opened, and blood was sampled by cardiac puncture. Simultaneously, 3 bronchoalveo- lar lavage (BAL) procedures were performed, each with 0.5 ml of normal saline. The fluid and blood were centrifuged (2000 g, for 10 min), and the su- pernatant and plasma were stored for further pro- cessing. Survival of mice following ALI induction and group-specific treatment was assessed and the cumulative survival curve was depicted using the Kaplan-Meier method.Histopathological analysis and lung injury scoresFollowing euthanasia 24 h after ALI induction and administration of group-specific treatments, the whole left lobe of the lung was fixed in a 4% formaldehyde neutral buffer solution for 24 h, de- hydrated in a graded ethanol series, embedded in paraffin, and 5 µm slices were cut. Paraffin sec- tions were stained with hematoxylin-eosin (H + E) for histopathological analysis.

Severity of the lung injury was evaluated using the semi-quantitative histological index of quan- titative assessment (IQA) of lung injury. Eight sec-tions were randomly selected from each group of mice, and 10 fields from each section were exam- ined by microscopy at 40× magnification. A pathol- ogist blinded to the study evaluated all the sec- tions. The average values of lung injury obtained were considered a semi-quantitative histological IQA of lung injury (also called lung injury scores).Pulmonary neutrophil sequestration was quan- tified using an MPO assay as previously de- scribed [16–18]. Briefly, at the time of sacrifice, lungs were perfused with 1 ml of 0.9% saline via the spontaneously beating right ventricle, until clear of blood. The lungs were excised and placed in 50 mM potassium phosphate buffer solution (pH, 6.0) with 5% hexadecyltrimethyl ammonium bromide (Sigma-Aldrich, St. Louis, Missouri, USA), followed by homogenization and centrifugation at 15 000 rpm for 20 min at 4°C. The supernatants and standard sample were added to a microtiter plate (100 µl/well) precoated with a murine anti-MPO mAb. After incubation for 1 h at 37°C, the plate was washed 6 times, followed by addition of the sub- strate and stop solution, after which the optical den- sity (OD) was measured at 450 nm using a micro- plate reader. All samples were assayed in triplicate.Lung edema during hyperoxia-induced lung injury was quantitated by determining wet : dry weight ratios as follows. A total of 10 lungs per group were ligated away from the hilum, blotted dry, weighed, and then desiccated by incubation at 130°C overnight in a vacuum oven. They were then reweighed to determine the dry weight, and the wet : dry ratio was then calculated.The concentration of protein in the BALF was measured using Bradford reagent (Bio-Rad Protein Assay kit. USA).

Briefly, 160 µl of each standard and sample solution was pipetted into separate microtiter plate wells, and 40 µl of the dye reagent was added to each well with thorough mixing. The mixture was incubated at room temperature for at least 5 min before the OD was measured at 595 nm. Comparison to a standard curve provided a relative measurement of the protein concentra- tion.After mice were euthanized, BALF was collect- ed and centrifuged, and neutrophils were counted using the Kimura stain.Intravascular and interstitial neutrophils in the lungs were distinguished by a flow cytome- try-based method as previously described [19]. Briefly, an Alexa 633-labeled GR-1 antibody (clone RB6-8C5, staining kit: Invitrogen Corp., Carlsbad, CA, USA) was injected i.v. 5 min before euthanasia, labeling only intravascular neutrophils. After per- forming 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 ml H2O through the spontaneously beat- ing right ventricle. Lungs were removed, minced, and digested with enzyme cocktail at 37°C for 60 min. A cell suspension was prepared by passing the digested lungs through a 70 mm cell strainer (BD Falcon, Bedford, MA, USA) which lysed the erythrocytes, and the remaining leukocytes were counted. The fraction of neutrophils in the sus- pension was determined by flow cytometry using a FACSCalibur (Becton Dickinson, San Jose, CA, USA). Neutrophils were identified by their typical expression of CD45 (clone 30-F11, BD Bioscienc- es-Pharmingen, San Diego, CA, USA) and GR-1 (clone RB6-8C5). The i.v. injected labeled GR-1 Ab differentiated intravascular and interstitial neu- trophils.ELISATumor necrosis factor (TNF)-, interleukin (IL)-6, IL-1 and macrophage inflammatory protein (MIP)-2 amounts were detected using an ELISA ac- cording to the manufacturer’s protocol. The exper- iment was repeated three times and results were presented as the mean.Pulmonary microvascular permeability in the four experimental groups of mice was determined by Evans blue dye extravasation as described previously [15].

Briefly, Evans blue (20 mg/kg, Sigma-Aldrich) was injected i.v., 30 min prior to euthanasia, lungs were perfused, removed, and the Evans blue was extracted. Absorption of the dye was then measured, corrected for hemoglobin content and calculated for the different treatment groups, 24 h after LPS induction and group-specif- ic treatments.Preparation of murine peripheral blood neutrophils and BM-derived PMNsBM from mice and BM-derived PMNs were pu- rified from a 3-layer Percoll gradient (78%, 66%, and 54%) [20]. Mouse peripheral blood neutro- phils were isolated by modification of methods previously reported [21] for the purification of rab- bit peripheral neutrophils. Briefly, C57BL/6 mice were volume expanded and exsanguinated intoa 3.8% citrate solution; the blood was centrifuged at 300 g for 20 min. The cell pellet was resuspend- ed in a 6% dextran-0.9% NaCl solution (1 : 5.25) to a final volume of 150% of the original blood vol- ume and sedimented at unity gravity for 30 min. The leukocyte-rich supernatant was aspirated, washed once in Hanks’ balanced salt solution (HBSS), layered on a three-step Percoll (Pharmacia) gradient (78%, 66%, and 54%) and centrifuged at 1060 g for 30 min. Cytospin samples of the 78– 66% interface revealed 90% neutrophils.Chemotaxic migration assayChemotaxic migration of neutrophil and BM-de- rived PMNs was determined using 24-well tran- swell chambers (Corning, Acton, MA) with 3-µm pore size membranes as reported [22].

Briefly, iso- lated PMNs and neutrophils were placed on top of the filter above each well of the 24-well transwell chambers, and then the chamber was incubated for 1 h at 37°C and 5% CO2. After incubation, the amount of migrated cells in the bottom chamber of the transwell were counted with a hemocytom- eter. The chemotaxic migration was expressed as the percentage of cells recovered from the bottom well compared to the total cells in the top well.We determined the potential of pretreatment with SB225002 to protect against LPS-induced ALI. ALI was induced in mice with 100 mg/kg LPS in 100 µl PBS by tail vein injection, then mice were randomly divided into 4 experimental groups (n = 10 per group) as mentioned above. Survival rates were then recorded 120 h following treatments.All data were analyzed using SPSS 13.0 soft- ware and expressed as means + SD. Significant differences were assessed using one-way analysis of variance (ANOVA) followed by Fisher’s protect- ed least significant difference (LSD) test. A proba- bility value of less than 0.05 (p < 0.05) was consid- ered statistically significant.

Results
It is well known that neutrophils are essential for maintaining innate immune surveillance un- der normal conditions. They are also a major con- tributor and the first group of cells that migrate to damaged tissue during acute lung injury [23]. CXCR2 is the key chemokine receptor for neu- trophils [24–26]. We therefore hypothesized that SB225002, an inhibitor of CXCR2, could alleviate the LPS-induced acute lung injury by inhibiting theinfiltration of neutrophils into lung tissue. Mice with LPS-induced lung injury were treated with SB225002 via tail vein injection while PBS was the vehicle control. The results showed that lung specimens from the LPS-treated (Figure 1 B) and LPS + PBS-treated (Figure 1 C) groups displayed significant histological abnormalities, including in- filtration of leukocytes into the interstitial spaces, hemorrhage, and marked swelling of the alveolar walls. Meanwhile, we discovered that treatment with SB225002 (Figure 1 D) improved the histolo- gy of the lungs compared to the PBS vehicle-treat- ed and untreated ALI groups.In addition, to further assess the effects of SB225002 on LPS-induced lung damage, we scored the injury in the left lungs in each group. We found that the score of the SB225002-treated ALI group was considerably lower than that of the PBS vehicle-treated and untreated ALI groups (Fig- ure 1 E, p < 0.05).Myeloperoxidase activity, a reliable marker of neutrophil infiltration, was also determined in differently treated lung tissue. Compared to the untreated and PBS-treated ALI groups, MPO activity was significantly (p < 0.05) reduced in SB225002-treated ALI mice (Figure 1 F).

These re- sults indicated that the CXCR2 inhibitory effect of SB225002 may alleviate LPS-induced morphologic lesions in lung tissue by inhibiting migration of neutrophils there.To quantitatively analyze the effects of SB225002 on the degree of LPS-induced lung ede- ma index, the right upper lobe of the lungs was measured in each animal. The result showed that SB225002-treated ALI mice had a significantly lower wet/dry ratio compared to the untreated ALI and PBS-treated ALI groups (Figure 2 A).Additionally, the degree of lung injury was fur- ther assessed by determining protein concentra- tion in lung BALF. As shown in Figure 2 B, the con- centration of protein in SB225002-treated BALF was significantly (p < 0.05) reduced compared to the untreated ALI and PBS-treated ALI groups. These data were consistent with the results of lung injury scores in the differently treated ALI groups.Treatment with SB225002 affected transendothelial and transepithelial migration, but not intravascular accumulation of neutrophils in lung tissueFollowing lung injury, neutrophils first accu- mulate in the vasculature and then infiltrate the interstitial space and exit into the BALF [27]. Todetermine the specific steps in the neutrophil re- cruitment cascade affected by SB225002-induced inhibition of CXCR2, infiltration of neutrophils into the intravascular and interstitial space and BALF was examined.

We found that treatment with SB225002 significantly (p < 0.05) reduced the in- filtration of neutrophils into the interstitial space (Figure 3 B) and BALF (Figure 3 C) compared to the untreated ALI and PBS-treated ALI groups. Therewas however no change in the intravascular accu- mulation of neutrophils (Figure 3 A).The activation and secretion of pro-inflam- matory cytokines, such as tumor necrosis factorvitro, we investigated the relationship of CXCR2 and mice neutrophils or PMN cells and the ef- fect of SB225002 on the migration of mice neu- trophils or blood PMN cells in vitro. A 24-well chemotaxis system was loaded with chemoat- tractants (CXCL1, or SB225002), and the neu- trophils from mice peripheral blood were placed on top of the filter above each well. As expected, compared with the negative control group (no CXCL1 and no SB225002) and positive control group (no SB225002), SB225002 inhibited the CXCL1-CXCR2 pathway-mediated chemotaxis (Figure 6 A), but did not affect this process in the absence of CXCL1. Meanwhile, we observed that the migration of BM-derived PMS cells was also inhibited by SB225002 (Figure 6 B). This result was consistent with the neutrophil data in vitro. So these data provide evidence that SB225002 could impair the CXCR2-mediated migration in vitro.a critical role in the development of different mod- els of ALI [14, 15, 37, 38].

Elimination of CXCR2 by gene targeting or blocking its activation with an antibody reduced neutrophil recruitment in the lung, lung edema and protein leakage [15, 37]. CXCR2 receptors on hematopoietic and non-he- matopoietic cells are both essential for neutrophil recruitment in response to LPS, each accounting for about half of neutrophil recruitment [15].Previous studies have demonstrated that blocking CXCR2 signaling attenuated ALI in differ- ent models [37, 39, 40]. Additionally, some small molecule CXCR2 antagonists have been developed as oral drugs for lung inflammation [41], including navarixin (SCH527123) [42] and SB-656933 [43].Moreover, Leaker et al. also demonstrated that AZD8309, another CXCR2 antagonist, could inhib- it the airway neutrophilic inflammation induced by LPS inhalation in human volunteers [44]. These studies suggested that a small molecule antago- nist, which could inhibit CXCR2 or CXCR2-medi- ated inflammatory cell migration, can be used as a potential drug candidate for lung inflammatory disease. In our study, we found that treatmentwith SB225002 (Figure 1 D) improved the histol- ogy of the lung compared to the untreated ALI (Figure 1 B) and PBS-treated groups (Figure 1 C). In addition, we analyzed the effects of SB225002 on the LPS-induced lung injury edema index and found that the data were similar to the results of the histological analysis (Figure 2 A).

Neutrophils traveling along the endothelium can infiltrate the lung tissue and initiate a cascade of inflammatory responses, leading to endothelial damage and subsequent development of multi- ple organ damage [23]. Our data showed that as a marker of neutrophil influx, MPO activity was decreased in SB225002-treated mice compared to the untreated ALI and PBS-treated ALI groups (Fig- ure 1 F). Meanwhile neutrophil counts in different parts of lung tissue showed that SB225002 did not affect the intravascular neutrophil count (Figure 3 A), but significantly reduced neutrophils in the in- terstitial space (Figure 3 B) and BALF (Figure 3 C). To further confirm the beneficial effect of SB225002 on lung injury, we measured the protein concentra- tion in BALF. Our results showed that treatment of LPS-induced ALI mice with SB225002 reduced pro- tein concentration in lung BALF (Figure 2 B).
In addition, inflammatory responses play an important role in the development and patho- genesis of LPS-induced lung injury. We therefore determined the production of inflammatory cyto- kines in the lung BALF and observed that treat- ment with SB225002 dramatically reduced secre- tion of TNF- (Figure 4 A), IL-6 (Figure 4 B), IL-1 (Figure 4 C) and MIP-2 (Figure 4 D). These data indicate that SB225002 may impair the inflamma- tory responses through inhibition of inflammatory cell migration.

In conclusion, our data suggest that SB225002 may be potentially beneficial in the therapy of ALI. SB225002 treatment improved the histology of lung tissue, reduced neutrophil infiltration, and de- creased secretion of pro-inflammatory cytokines in the process of lung injury. It may therefore provide a novel drug candidate for the treatment of LPS-in- duced lung injury via inhibition of migration of neu- trophils from the blood to damaged lung tissue.