CXCR2/CXCR2 Ligand Biology during Lung Transplant Ischemia-Reperfusion Injury¹

Lung transplantation is a therapeutic option for a number of end-stage pulmonary disorders. Unfortunately, early lung allograft dysfunction (ischemia-reperfusion injury) continues to be the most common cause of early mortality after transplantation (1, 2, 3). Studies suggest that up to 97% of lung transplantation recipients develop at least mild ischemia-reperfusion injury while moderate to severe ischemia-reperfusion injury occurs in up to 30% of recipients (1, 2, 3, 4). Moreover, the mortality rate for more severe episodes of ischemia-reperfusion injury can be >40% (2, 5, 6, 7).

Although the exact mechanism of cold ischemia-reperfusion injury is not fully understood, neutrophils have been strongly implicated (4, 8). For example, lung allograft biopsy showing an influx of neutrophils in the proper clinical setting within 72 h after lung transplantation is currently an accurate means for identifying patients with post-lung transplantation ischemia-reperfusion injury (8). In addition, in vivo neutrophil depletion experiments involving different animal models of warm ischemia-reperfusion injury (i.e., a simple cross clamp of the perfusing artery to the organ of interest for a period of time, “warm ischemia,” then releasing the clamp for reperfusion) have demonstrated protection against organ damage (9, 10, 11, 12, 13, 14). However, the role of the neutrophils during conventional and clinically relevant post-lung transplantation cold ischemia-reperfusion injury (i.e., orthotopic lung transplantation model using a donor lung with an ischemia time of 6 h at 4°C “cold ischemia” with reperfusion occurring after reanastomosis of the pulmonary artery, vein, and main stem bronchus) has not been determined. We hypothesized that multiple glutamic acid-leucine-arginine (ELR⁺) CXC chemokines acting through their shared G protein-coupled receptor, CXCR2, on neutrophils would be important during the pathogenesis of post-lung transplantation cold ischemia-reperfusion injury.

We found that elevated levels of three different ELR⁺ CXC chemokine ligands are associated with human lung transplantation ischemia-reperfusion injury. Similarly, proof of concept studies using a rat orthotopic single lung transplantation model of cold ischemia-reperfusion injury demonstrated increased levels of multiple ELR⁺ CXC chemokines. In addition, these chemokines paralleled lung infiltration with neutrophils and cells expressing CXCR2. Importantly, the inhibition of CXCR2/CXCR2 ligand interactions using an anti-CXCR2 Ab reduced lung neutrophil sequestration and attenuated post-lung transplantation cold ischemia-reperfusion injury.

With Institutional Review Board approval and informed written consent, we prospectively enrolled all patients undergoing lung transplantation from June 1992 to April 2000. All post-lung transplantation patients were eligible for this. Four lung transplantation recipients met rigorous criteria for ischemia-reperfusion lung injury without concomitant infection or colonization (see criteria below) and had a portion of their bronchoalveolar lavage fluid (BALF)³ sent to our laboratory for ELR⁺ CXC chemokine analysis. In addition, six lung transplantation recipients met rigorous criteria for healthy lung transplantation recipients within 72 h after lung transplantation without concomitant infection or colonization and had a portion of their BALF sent to our laboratory for ELR⁺ CXC chemokine analysis.

Patients were clinically diagnosed with post-lung transplantation ischemia-reperfusion injury based on the following criteria: PaO₂/FiO₂ <250, noncardiogenic diffuse allograft infiltrates, infectious disease ruled out, and all occurring within 72 h after lung transplantation. “Early” healthy lung transplantation recipient controls were those patients that had a bronchoscopy before extubation to examine their airway anastomosis and also had a concurrent bronchoalveolar lavage within 72 h after lung transplantation.

Our experimental protocol was designed to reduce bias related to infectious episodes and/or colonization. Our protocol excludes any BALF performed at a time when infection and/or colonization was diagnosed with the following criteria: a positive BALF microbiology Gram stain and/or culture for bacterial, acid-fast bacillus, fungus, CMV culture by shell vial or other molecular techniques, transbronchial or cytological evidence of CMV, other virus, or Pneumocystis carinii pneumonia.

Patients were placed on a standard pre- and post-lung transplantation immunosuppression protocol. Calcineurin inhibitors were started for 4 h preoperatively and then continued either by i.v. or oral administration to maintain appropriate levels thereafter. Methylprednisolone was initiated intraoperatively with a 500-mg bolus and then appropriately tapered to 20 mg/day by the third postoperative month. Azathioprine or mycophenolate mofetil was started 8 h postoperatively and then adjusted to maintain a white blood cell count of ∼4000/mm³. Transplant recipients received viral, fungal, and bacterial prophylaxis as previously described (15, 16).

Biotinylated and nonbiotinylated anti-rat CXCL1 and CXCL2/3 were purchased from R&D Systems. Polyclonal goat anti-CXCR2 was produced by the immunization of a goat with a peptide containing the ligand-binding sequence Met-Gly-Glu-Phe-Lys-Val-Asp-Lys-Phe-Asn-Ile-Glu-Asp-Phe-Phe-Ser-Gly of CXCR2 as previously described (17). Direct ELISA was used to evaluate antisera titers, and sera were used when titers reached >1/1,000,000. The CXCR2 protein sequence has been shown to contain the ligand-binding portion of the CXCR2 receptor and has been used previously to block rat CXCR2 biological function in vitro and in vivo (17). For instance, this Ab has been shown to detect CXCR2 by Western blot and FACS analysis of neutrophils in vivo (17). Furthermore, in vitro this Ab has been shown to inhibit neutrophil chemotaxis to multiple ELR⁺ CXC chemokines and in vivo to abrogate the influx of neutrophils into the peritoneum of normal rats in response to exogenous CXCL1 and CXCL2/3 (17).

We used the established and reproducible rat orthotopic left single lung transplantation model of cold ischemia-reperfusion injury as previously described (18). Briefly, donor rats were anesthetized with an i.p. injection of a mixture of ketamine and rompun and then intubated using a pediatric larygyscope with an endotracheal tube (i.e., 18-gauge i.v. catheter). Atropine was then given by i.p. injection to control airway secretions. The endotracheal tube was then connected to a volume-controlled ventilator (Harvard Rodent Ventilator, model 683; Harvard Apparatus), and the animals were ventilated in the supine position, on a warming mattress, at a rate of 70 breaths/min with a tidal volume (V_t) of 8 ml/kg, inspired fraction of oxygen of 0.21, and a positive end expiratory pressure of 2 cm H₂O. The animals were then prepped (i.e., shaven and skin sterilized with betadine followed by ethanol) and using the clamshell approach the lungs and mediastinum were exposed. The pulmonary artery, vein, and main stem bronchus were exposed and dissected free of surrounding structures (i.e., lymph nodes, thymus, ligaments, and fat). The pulmonary circulation was flushed with 500 U of heparin via the inferior vena cava followed by 50 ml of Euro-Collins solution. The pulmonary artery, vein, and main stem bronchus were ligated and the tied off, inflated left lung was placed into Euro-Collins solution at 4°C for 6 h.

Recipient animals were anesthetized, intubated, and sterilely prepped in a similar fashion as the donor animals described above. All recipients were ventilated with a halothane and oxygen mixture for adequate sedation at a rate of 70 breaths/min with a V_t of 8 ml/kg and a positive end expiratory pressure of 2 cm H₂O at a V_t = 8 ml/kg. The animals were then placed on a warming blanket in a right lateral position. Using the left thoracotomy approach the left pulmonary artery, main stem bronchus, and pulmonary vein were dissected free of surrounding structures, clamped, and ligated. The donor lung was then anastomosed with the corresponding recipient structures using multiple interrupted sutures for each anastomosis. The clamp was released and the lung was carefully placed in the left thoracic cavity followed by closure of the thoracotomy site. This operation has >85% postoperative survival and the <15% mortality is directly attributable to surgical mortality and occurs within 8 h after lung transplantation (19, 20, 21, 22, 23, 24, 25, 26, 27). Inbred and unmodified specific pathogen-free rats were used to assure immunogenetic standardization. The RT¹-incompatible combination was Brown Norway to Lewis (allograft), and Lewis to Lewis (isograft). In separate experiments, recipient animals received either 1.5 ml of anti-CXCR2 or control Ab 24 and 3 h before the start of the lung transplantation. Neutralization of CXCR2 or the administration of control Ab in vivo did not impact on postoperative survival. Sham-operated controls consisted of the same perioperative protocols as the recipient rats. Briefly, a left thoracotomy was performed with dissection of the hilar structures from the pulmonary artery, vein, and main stem bronchus. The animals’ left thoracotomy site was then closed. The allograft, isograft, and sham operation times were maintained at <240 min for each recipient animal.

Microvascular permeability related to lung injury was measured using a modification of the Evans blue dye extravasation technique as previously described (28, 29, 30). Extravasation of Evans blue (Sigma Immunochemicals) into the extravascular compartment was used as a quantitative measure of lung injury and changes in pulmonary microvasculature. Briefly, each animal received 20 mg/kg Evan blue (pH 7.34) by penile vein injection 3 h before they were sacrificed. At the time of sacrifice, a heparinized sample of blood was harvested and plasma was removed by centrifugation. The lungs were perfused free of blood with 50 ml of 0.9% NaCl via the spontaneous beating right ventricle and removed from the thoracic cavity. The trachea, main stem bronchi, and surrounding mediastinal structures were removed. Evans blue was extracted from pulmonary tissues after homogenization in 3 ml of 0.9% NaCl. This volume was added to two volumes of deionized formamide and incubated at 60°C for 12 h. The supernatant was separated by centrifugation at 2000 × g for 30 min. Evans blue in the plasma and lung tissue was quantitated by dual wavelength spectrophotometric analysis at 620 and 740 nm. This method corrects the specimen’s absorbance at 620 nm for the absorbance of contaminating heme pigments and is calculated by the following formula: corrected absorbance at 620 nm = actual absorbance at 620 nm − (1.426 (absorbance at 740) + 0.03). A permeability index was calculated by dividing the corrected pulmonary tissue Evans blue absorbance at 620 nm of lung tissue by the corrected plasma Evans blue absorbance at 620 nm which reflects the degree of extravasation of Evans blue into the extravascular pulmonary tissue compartment.

Pulmonary neutrophil sequestration was quantitated using a myeloperoxidase (MPO) assay as previously described (28, 31, 32). Briefly, at the time of sacrifice, lungs were perfused free of blood with 50 ml of 0.9% NaCl via the spontaneously beating right ventricle. The lungs were excised and placed in a 50 mM potassium phosphate buffer solution (pH 6.0) with 5% hexadecyltrimethyl ammonium bromide (Sigma-Aldrich). The lung tissue was homogenized, sonicated, and centrifuged at 12,000 × g for 15 min at 4°C. The supernatant was then assayed for MPO activity using a spectrophotometric reaction with o-dianisidine hydrochloride (Sigma-Aldrich) at 460 nm.

CXCL1 and CXCL2/3 protein was quantitated using the double-ligand method as previously described (27, 32). Briefly, flat-bottom 96-well microtiter plates (Nunc Immuno-Plate I 96-F) were coated with anti-rat CXCL1 or CXCL2/3 (1 ng/μl in 0.6 M NaCl, 0.26 M H₃B0₄, and 0.08 N NaOH, pH 9.6) for 24 h at 4°C and then washed with PBS (pH 7.5)/0.05% Tween 20 (wash buffer). Microtiter plate nonspecific binding sites were blocked with 2% BSA in PBS and incubated for 60 min at 37°C. Plates were washed three times with wash buffer and samples or standard was added, followed by incubation for 1 h at 37°C. Plates were washed three times and 50 μl/well biotinylated anti-CXCL1 and CXCL2/3 Abs was added, and plates were incubated for 45 min at 37°C. Plates were washed three times, streptavidin-peroxidase conjugate (Jackson ImmunoResearch Laboratories)was added, and the plates were incubated for 30 min at 37°C. Plates were washed three times and 3,3′,5,5′-tetramethylbenzidine) chromogen substrate (Kirkegaard & Perry Laboratories) was added. The plates were incubated at room temperature to the desired extinction, and the reaction was terminated with 3 M H₂SO₄ solution. Plates were read at 450 nm in an automated microplate reader (Bio-Tek Instruments). Standards were 1/2 log dilutions of either CXCL1 or CXCL2/3 from 100 ng to 1 pg/ml (50 μl/well). This ELISA method consistently detected specific chemokine concentrations in a linear fashion greater than 50 pg/ml. CXCL1 and CXCL2/3 were specific in our sandwich ELISA without cross-reactivity to a panel of cytokines including human, murine, and rat IL-1Ra, IL-1, IL-2, IL-6, IL-4, TNF-α, and IFN-γ and members of the CXC and CC chemokine families.

Total cellular RNA was isolated as previously described (15, 26, 33). Total RNA was determined and reversed transcribed into cDNA and amplified using TaqMan Reverse Transcription reagents (Applied Biosystems). We performed real-time quantitative PCR with specific TaqMan primers and probes using the ABI Prism 7700 Sequence Detector and SDS analysis software (Applied Biosystems). Negative control experiments were performed as follows: real-time quantitative PCR was performed without reverse transcriptase to exclude contamination and amplification of genomic DNA and without cDNA template to exclude reagent contamination with DNA. TaqMan 18s Pre-Developed Assay Reagent (PDAR) (Applied Biosystems) was used, and CXCL1, CXCL2/3, and CXCR2 primers and probe sequence (forward primer, reverse primer, TaqMan probe) were as follows: CXCL1, AGA GCT TGA CGG TGA CCC C, CCT TGA GAG TGG CTA TGA CTT CTG, CCA GGA CCG CAC TGC ACC CAT; CXCL2/3, CCA ACC ATC AGG GTA CAG GG, GTA GGG TCG TCA GGC ATT GAC, TGT TGT GGC CAG TGA GCT GCG CTA; and CXCR2, CCT TCT ACA GTG TTC TGT TGC, GCG TGG ACG ATG GCC A, AGC CTG CAT CAG CAT GGA CCG CTA. Quantitative analysis of gene expression was done using the comparative cycle threshold (C_Τ) called the ΔC_Τ method, in which C_Τ is the threshold cycle number (15, 26, 33). The formula for the ΔC_Τ method is the difference in threshold cycles for a target (i.e., CXCL1) and an endogenous reference (i.e., housekeeping gene 18S). The amount of target normalized to an endogenous reference (i.e., CXCL1 in isografts at 16 h) and relative to a calibration normalized to an endogenous reference (i.e., CXCL1 in sham-operated controls at 16 h) is given by 2^−ΔΔCT, which is the fold increase in CXCL1 of the isografts compared with the sham-operated controls, as previously described (15, 26, 33).

Data were analyzed using the Statview 4.5 statistical software package (Abacus Concepts). All human group comparisons for chemokine levels were evaluated by the nonparametric Mann-Whitney U analysis. Data are displayed using a box plot summary. The horizontal line represents the median, the box encompasses the 25th to 75th percentile, and the error bars encompass the 10th to the 90th percentile. Human group demographics were compared using the χ² analysis and human group ages and donor lung ischemia times were compared using the unpaired comparison analysis. All animal group comparisons were evaluated by the ANOVA test with Bonferroni/Dunn post hoc analysis. All data are expressed as mean ± SEM.

Several studies have described an association between elevated levels of CXCL8 and post-lung transplantation ischemia-reperfusion injury (34, 35). However, there are multiple ELR⁺ CXC chemokines besides CXCL8 that are also potent neutrophil chemoattractants. We determined whether there were any significant elevations in (CXCL1, CXCL3, CXCL5, CXCL7, and CXCL8) from BALF in a two-group comparison between lung transplantation recipients with ischemia-reperfusion injury (n = 4) and healthy lung transplantation recipients (n = 6). Patient’s demographics and donor lung ischemia times are given in Table I. Our ischemia-reperfusion injury group consisted of lung transplantation recipients with non-cardiogenic diffuse allograft infiltrates, hypoxemia and no concomitant infection/colonization/rejection occurring within 72 h after lung transplantation. Our healthy recipients consisted of lung transplantation patients who had undergone bronchoscopy just before extubation to evaluate the airway anastomosis and at that time had a concurrent bronchoalveolar lavage and infection/colonization excluded. CXCL3, CXCL7, and CXCL8 were markedly elevated in the BALF from recipients with ischemia-reperfusion injury, as compared to the healthy group (Fig. 1, A– C). However, there were no significant differences in the levels of CXCL1 and CXCL5 in the two-group comparison (Fig. 1, D and E). These results demonstrate that other ELR⁺ CXC chemokines besides CXCL8 may be involved in the pathogenesis of ischemia-reperfusion injury.

With early allograft dysfunction being a common occurrence in the immediate post-lung transplantation period, we generated a rodent model performed in the same manner as human lung transplantation. This rat model of clinically relevant cold ischemia-reperfusion injury related to lung transplantation was used to further evaluate the role of ELR⁺ CXC chemokines during the pathogenesis of ischemia-reperfusion lung injury (26, 27, 36, 37). This rat model consisted of a cold (i.e., donor lung ischemia time of 6 h at 4°C) orthotopic single left lung transplantation followed by reperfusion with lung injury evaluated at multiple time points. Lewis rats received a lung transplantation from either donor Brown Norway (allograft) or Lewis (isograft) rats as previously described (26, 27, 36, 37). Sham-operated matched controls consisted of a left side thoracotomy with dissection of the pulmonary artery, vein, and left main stem bronchus followed by closure of the chest wall thoracotomy site.

The kinetics of lung injury during the inflammatory phase of ischemia-reperfusion injury was evaluated by Evans blue dye analysis, a marker of microvascular permeability. We chose to start our kinetic studies at 8 h since this minimized the chance of lung injury that can occur as a complication of the transplantation procedure (i.e., <15% mortality attributable to the procedure; however, if injury from the procedure occurs it usually manifests within 8 h of the transplantation) (19, 20, 21, 26, 27). Lung injury from both allografts and isografts were markedly increased at 8 h and peaked at 16 h after lung transplantation, as compared to sham-operated controls (Fig. 2,A). There was no difference in lung injury among all three groups by 24 h, a time period where the injury resolves. However, at 48 h there was an increase in lung microvascular permeability in the allograft as compared to the isografts and sham-operated controls. The increase in allograft lung injury by Evans blue permeability is related to early acute lung allograft rejection. These results were confirmed by histopathological assessment, where patchy areas of lung injury (leukocyte extravasation, edema, and hemorrhage) are seen in the allograft and isografts at 8 and 16 h as compared to virtually normal lungs from the sham-operated controls (Fig. 2,B). In addition, all three groups (allografts, isografts, and sham-operated controls) were practically normal at 24 h (Fig. 2,B). Furthermore, at 48 h only the allografts demonstrated early acute lung allograft rejection (perivascular and peribronchial leukocyte extravasation) as compared to virtually normal lungs from the isografts and sham-operated controls (Fig. 2 B).

Neutrophils are considered to be critically involved in the pathogenesis of most forms of acute lung injury and are one of the histopathological hallmarks of ischemia-reperfusion lung injury (8). Based on this evidence, a time course of lung neutrophil sequestration during ischemia-reperfusion injury in allografts, isografts, and sham-operated controls was evaluated. Rat whole lungs were harvested and processed for MPO, an indirect measurement of neutrophil infiltration into the lungs over the 48-h time course. Whole lung homogenates had a marked increase in MPO levels from both allografts and isografts at 8 and 16 h and then fell thereafter to levels equivalent to those of sham-operated controls (Fig. 3 A).

Finding increased levels of MPO that paralleled ischemia-reperfusion injury led us to hypothesize that chemotactic factors were being expressed and mediated the recruitment of neutrophils to the lung. On this basis, the kinetics of two potent ELR⁺ CXC chemokine neutrophil chemoattractants, CXCL1 and CXCL2/3, were assessed at the same time points as mentioned above (i.e., 8, 16, 24, and 48 h). Whole lung homogenates were analyzed for chemokine protein levels by ELISA. There were elevated levels of both CXCL1 and CXCL2/3 at 8 and 16 h in both allografts and isografts, as compared to sham-operated controls (Fig. 3, B and C). Thereafter, the isograft CXCL1 levels declined to levels comparable to those of sham-operated controls (Fig. 3,B). In contrast, CXCL2/3 levels were found to decrease to the levels of the sham-operated controls at 24 h and then increase again in the allograft lungs at 48 h (Fig. 3,C). In addition, levels of CXCL2/3 were markedly greater then CXCL1 throughout the entire time course (Fig. 3, B and C). These findings demonstrate that the expression of CXCL1 and CXCL2/3 at 8 and 16 h paralleled neutrophil sequestration and ischemia-reperfusion injury.

CXCR2 is the shared cellular receptor for all ELR⁺ CXC chemokine ligands and is the only ELR⁺ CXC chemokine receptor in rats (38). Finding increased levels of CXCL1 and CXCL2/3 associated with lung neutrophil sequestration and ischemia-reperfusion injury led us to evaluate the expression of CXCR2 in the allografts and isografts of these animals. We found markedly greater CXCR2 mRNA expression from allograft and isograft lungs, as compared with sham-operated controls throughout the first 16 h after lung transplantation (Fig. 4). There was no significant elevation in CXCR2 expression from the allografts or the isografts as compared to sham-operated controls at 24 h (Fig. 4). However, there was a significant increase in CXCR2 mRNA expression in allografts, as compared with sham-operated controls and isografts at 48 h (Fig. 4).

Findings that increased expression of CXCR2 and CXCR2 ligands were associated with neutrophil sequestration and lung injury posttransplantation. We next determined whether inhibiting CXCR2/CXCR2 ligand interaction could attenuate graft injury. We initially chose the allograft rather than isograft model of ischemia-reperfusion injury because this is the more clinically relevant model with regard to human early lung allograft dysfunction. Rodent recipients were passively immunized with a specific neutralizing anti-CXCR2 Ab, as compared with a control Ab at 24 and 3 h before lung transplantation. We chose to evaluate the effects of neutralizing CXCR2 at 16 h since this was the time period of maximal ischemia-reperfusion injury without any histopathological evidence of acute lung allograft rejection. We found that the lung allografts from recipients treated with the neutralizing Ab to CXCR2 had significantly lower levels of MPO at 16 h, as compared to the control Ab-treated group (Fig. 5,A). Importantly, inhibiting CXCR2/CXCR2 ligand interaction attenuated ischemia-reperfusion injury as demonstrated by a reduction in microvascular permeability (i.e., Evans blue extravasation), lung edema (i.e., wet:dry weight ratio), and preserved lung allograft architecture at 16 h after lung transplantation (Fig. 5, B–D).

We further confirmed that the effect of inhibiting CXCR2/CXCR2 ligand interaction was not simply due to an effect on early acute lung allograft rejection, but also due to a general response to the lung neutrophil sequestration of ischemia-reperfusion injury by performing the above experiment using the rat isograft model. Similar to the allografts, we found that the rodent recipients of lung isografts that were passively immunized with anti-CXCR2, as compared with control Ab given at 24 and 3 h before lung transplantation, had a significant reduction in lung neutrophil sequestration, lung microvascular permeability, lung edema, as well as preserved lung graft architecture at 16 h after lung transplantation (Fig. 6).

Post-lung transplantation cold ischemia-reperfusion lung injury is unfortunately a common complication after lung transplantation (1, 2, 3). In addition, ischemia-reperfusion injury remains a risk factor for the development of bronchiolitis obliterans syndrome, the leading cause of a low survival rate after lung transplantation (39, 40). Early lung allograft dysfunction most likely results from a multihit mechanism involving the donor lung, which includes the complications of brain death, mechanical ventilation, hypotension, cold storage, and ischemia-reperfusion injury. Cold storage itself is associated with oxidative stress, cell death, and the release of proinflammatory mediators which stimulate/activate both donor passenger and recipient leukocytes (4). Activated leukocytes stiffen, causing local “no-reflow” areas which enhance prolonged local ischemia even during reperfusion, leading to more vascular damage with increased lung leukocyte infiltration, underscoring the need to evaluate models of cold ischemia-reperfusion injury rather than simple warm ischemia-reperfusion injury (4). These events result in a pathological process with interstitial edema and a neutrophil predominant infiltration, which ultimately has a mortality rate of >40% (2, 4, 5, 6, 7). Since ELR⁺ CXC chemokines are potent neutrophil chemoattractants, we hypothesized that multiple ELR⁺ CXC chemokines may be involved in the pathogenesis of post-lung transplantation ischemia-reperfusion injury.

Previous human lung transplantation data demonstrated elevated CXCL8 expression from lung tissue 2 h after reperfusion, which correlated negatively with lung function and positively with acute physiology and chronic health evaluation scores (34). In addition, CXCL8 expression from human donor lungs preoperatively was associated with an increased risk of death from early lung allograft dysfunction (34, 35). We have expanded upon these studies by finding elevated levels of multiple ELR⁺ CXC chemokines, including CXCL3, CXCL7, and CXCL8 in BALF from patients with post-lung transplantation ischemia-reperfusion injury. Furthermore, our results are supported by studies involving Acute Respiratory Distress Syndrome (ARDS) patients, which is also characterized by a pulmonary capillary leak with nonhydrostatic edema and a neutrophil infiltration (41). For instance, elevated levels of CXCL1, CXCL5, CXCL7, and CXCL8 in BALF have been associated with ARDS (42, 43, 44, 45, 46). In addition, ELR⁺ CXC chemokines have also been found to correlate positively with increased BALF neutrophilia and development of ARDS in at-risk patient groups (42, 43, 44, 45, 46, 47). Collectively, these studies demonstrate an association between multiple ELR⁺ CXC chemokines and acute lung injury secondary to post-lung transplantation ischemia-reperfusion injury.

Based on the importance of finding an association between multiple ELR⁺ CXC chemokines and human ischemia-reperfusion injury, we performed proof of concept studies using the rat orthotopic single lung transplantation model of cold ischemia-reperfusion injury. We chose to use cold ischemia-reperfusion injury because it is clinically relevant and simulates a true human lung transplantation experience as compared to warm ischemia-reperfusion injury. This unique system consisted of a cold (donor lung ischemia time of 6 h at 4°C) orthotopic single rat left lung transplantation followed by reperfusion injury. The donor cold ischemia of 6 h was chosen because this is considered the average maximal ischemia times presently used for human lungs undergoing lung transplantation (48). A significant neutrophil sequestration and lung injury was found to peak at 16 h after lung transplantation. Similarly to our human data, there was an increase in expression of multiple ELR⁺ CXC chemokines (i.e., CXCL1 and CXCL2/3) in both allografts and isografts as compared with sham-operated controls. These data extend previous animal studies demonstrating elevated levels of only one ELR⁺ CXC chemokine (i.e., CXCL8 or CXCL2/3) during “warm” ischemia-reperfusion injury models (49, 50). Importantly, our study also demonstrated a more robust rise in both CXCL1 and CXCL2/3 as compared to previous warm ischemia-reperfusion injury models, which emphasizes a relationship between augmented levels of multiple ELR⁺ CXC chemokines and cold ischemia-reperfusion lung injury (49, 50, 51).

Furthermore, the expression of CXCR2 in both isografts and allografts paralleled the production of both CXCL1 and CXCL2/3 ligands and neutrophil sequestration during ischemia-reperfusion lung injury. Although CXCR2 expression has not been evaluated in either warm or cold ischemia-reperfusion lung injury, other studies of inflammatory diseases, such as acute lung injury from high-stretch mechanical ventilation, hyperoxia, and pneumonia have demonstrated an increased expression of CXCR2 mRNA during the pathogenesis of these diseases (31, 52, 53, 54, 55, 56). Collectively, these studies demonstrate that augmented levels of ELR⁺ CXC chemokines are important in the recruitment of cells expressing CXCR2 during the pathogenesis of acute inflammatory diseases. Surprisingly, we found augmented levels of CXCR2/3 and CXCR2 in allografts at 48 h, a time point were there was no significant neutrophil sequestration. This may indicate an alternative role for CXCR2/CXCR2 ligand interactions during the pathogenesis of early acute lung allograft rejection that go beyond neutrophil recruitment. For instance, ELR⁺ CXC chemokines are potent angiogenic molecules (57). More importantly, CXCR2/CXCR2 ligand biology has recently been shown to have a critical role in vascular remodeling required to support chronic inflammation during the pathogenesis of bronchiolitis obliterans syndrome (BOS) independent of neutrophil recruitment (15).

Based on our human data and rat model findings, we performed in vivo studies to evaluate the role for CXCL1 and CXCL2/3 and their interaction with CXCR2, specifically on neutrophil recruitment during the pathogenesis of post-lung transplantation ischemia-reperfusion lung injury. Using an anti-CXCR2 Ab given 24 and 3 h before lung transplantation, we found a significant reduction in lung allograft neutrophil sequestration. Furthermore, the anti-CXCR2 Ab markedly reduced lung injury as determined by a reduction in microvascular permeability, lung edema, and histopathological assessment in the allograft model, which is the most clinically relevant model we presently have with regard to human post-lung transplantation early allograft dysfunction. Importantly, we found similar effects using the anti-CXCR2 Ab in the isograft model, demonstrating that this is a specific effect on neutrophil-mediated ischemia-reperfusion injury as compared with simply a phenomenon of inhibiting very early acute lung allograft rejection. Future investigations that go beyond the scope of this study will be required to dissect out the specific role of CXCR2/CXCR2 ligand biology on acute lung allograft rejection.

Lastly, previous studies have demonstrated that neutrophil depletion will protect organs from ischemia-reperfusion lung injury (9, 10, 11, 12, 13, 14). We now have demonstrated a specific biological mechanism by which neutrophils are recruited to the lung during the pathogenesis of post-lung transplantation ischemia-reperfusion injury. Moreover, these data expand previous work, which showed that in vivo depletion of one ELR⁺ CXC chemokine could reduce warm ischemia-reperfusion injury (49, 50, 51). Specifically, we have now shown that inhibiting the interaction of multiple ELR⁺ CXC chemokines by attenuating CXCR2 during the more robust inflammatory animal model of clinically relevant cold ischemia-reperfusion injury can indeed attenuate post-lung transplantation ischemia-reperfusion injury.

In conclusion, we have demonstrated that multiple human ELR⁺ CXC chemokines (CXCL3, CXCL7, and CXCL8) are associated with human ischemia-reperfusion lung injury. Furthermore, a recent study has demonstrated that these same three ELR⁺ CXC chemokines are associated with human BOS (15). Interestingly, post-lung transplantation ischemia-reperfusion lung injury is considered a risk factor for the development of human BOS (39). Collectively, these studies suggest a role for CXCL3, CXCL7, and CXCL8 in the continuum of human early (ischemia-reperfusion injury) to late (BOS) lung allograft dysfunction. In addition, our data demonstrated that using a specific Ab to CXCR2 in an animal model system can inhibit the pathogenesis of post-lung transplantation ischemia-reperfusion injury. These studies may pave the way for future translational human studies using a human anti-CXCR2 Ab or small molecule antagonist that may be important in decreasing early mortality after lung transplantation and also decrease the incidence of bronchiolitis obliterans syndrome.

The authors have no financial conflict of interest.