Repertaxin, a novel inhibitor of rat CXCR2 function, inhibits inflammatory responses that follow intestinal ischaemia and reperfusion injury

Animals

Male Wistar rats (200–220 g) obtained from the Bioscience unit of our Institution were housed in standard conditions and had free access to commercial chow and water. All procedures described here had prior approval from the local animal ethics committee.

In vitro neutrophil chemotaxis

The chemotactic activity for neutrophils obtained from the blood of rats was assayed using the 48-well microchemotaxis chamber technique, as previously described (^{Bignold & Ferrante, 1987}; ^{DeForge et al., 1992}). Briefly, the lower compartment of the chamber was loaded with aliquots of medium, CXCL8, CINC-1, fMLP, PAF or LTB₄, while the upper compartment of the chamber was loaded with the purified neutrophils (resuspended in RPMI medium). Neutrophils (>90% purity, >95 viability) were purified over a Percoll gradient, as previously described (^{Ramos et al., 2003}), and were incubated for 10 min with vehicle (saline) or Repertaxin (10⁻¹¹–10⁻⁶M) prior to addition of the chemoattractants. The two compartments were separated by a 5.0 μm polycarbonate filter (Poretics Products, Osmonics, CA, U.S.A.). Following 1 h of incubation at 37°C, the filter was removed, fixed and stained. The migrated cells in 10-high-power fields were counted by light microscopy.

Intracellular calcium measurements

PMN (10⁷ ml⁻¹) were resuspended in RPMI 1640 and incubated with 1 mM FURA-2AM at 37°C for 15 min. Next, PMN were washed and resuspended in HBSS containing 1.2 mM CaCl₂ and incubated in the presence of vehicle or 1 mM Repertaxin for 15 min at room temperature. PMN were stimulated with vehicle, CXCL8 (100 ng ml⁻¹), as previously described (^{Brandolini et al., 1996}). FURA-2 fluorescence was measured in a Jasco FP-750 spectrophotometer (Jasco Corporation, Tokyo, Japan). Samples were excited at 340 and 380 nm, and emission at 505 nm was continuously recorded. Intracellular calcium concentration was determined as previously described (^{Bizzarri et al., 1995}).

hCXCL8 binding to rat neutrophils

[¹²⁵I]CXCL8 (specific activity 2200 Ci mmol⁻¹; Amersham Pharmacia Biotech, Buckinghamshire, U.K.) binding on rat PMN was performed as previously described (^{Bizzarri et al., 2001}). Rat PMN (4 × 10⁷ cells ml⁻¹) were incubated at 37°C for 15 min in the presence of vehicle or 1 mM Repertaxin. Next, 0.8 nM [¹²⁵I]CXCL8 and serial dilutions of unlabeled CXCL8 were added to 2 × 10⁶ rat PMN in 100 ml of binding medium and incubated at 4°C for 2 h under gentle agitation, as previously described (^{Bizzarri et al., 2001}). Scatchard analysis and calculations were performed with the LIGAND program (^{Munson & Rodbard, 1980}).

I/R injury

Rats were anaesthetized with urethane (140 mg kg⁻¹, i.p.) and laparotomy was performed. This procedure was sufficient to keep the animals under anaesthesia until the end of the experiment. The superior mesenteric artery (SMA) was isolated and ischaemia was induced by totally occluding the SMA for 30 or 120 min. After ischaemia, reperfusion was initiated by removal of the occlusion. Animals made ischaemic for 30 or 120 min were allowed to reperfuse for 30 (mild injury) or 120 (severe injury) min, respectively. The durations of I/R were based upon previous experiments (^{Souza et al., 2000a}, ^2000b) and were optimal for mild and severe reperfusion injuries. Sham-operated animals were used as controls for the reperfusion-induced injury. Lethality was accompanied and, at the end of the experiment, animals were killed by cervical dislocation. Inflammatory parameters were assessed only in animals that were alive at 120 min after reperfusion.

Initial experiments were carried out in the mild reperfusion injury model to examine the dose-dependent effects of CXCR2 inhibitor (Repertaxin, 3–30 mg kg⁻¹). In these experiments, Repertaxin was administered i.v. just prior to the reperfusion of the SMA. We then tested the effects of the administration of Repertaxin (30 mg kg⁻¹, i.v., just prior to reperfusion), Repertaxin vehicle (saline, 1 ml kg⁻¹), anti-CINC (0.5 ml of hyperimmune serum/animal, s.c., 60 min before reperfusion) or non-immune serum (0.5 ml) in the model of severe I/R injury. The drug or antibodies used in the present study had no significant effects on basal parameters (data not shown), and to simplify the graphs presented basal data obtained in vehicle- or drug-treated animals have been pooled for presentation. Similarly, results obtained in reperfused animals treated with Repertaxin vehicle or nonimmune serum were not different and were pooled to simply presentation (data not shown).

Evaluation of changes in vascular permeability

The extravasation of Evans blue dye into the tissue was used as an index of increased vascular permeability (^{Souza et al., 2000a}). Evans blue (20 mg kg⁻¹) was administered i.v. (1 ml kg⁻¹) via a femoral vein 2 min prior to reperfusion of the ischaemic artery. At 30 (in the mild injury model) or 120 min (in the severe injury model) after reperfusion, fragments of the duodenum (10 cm) were cut open and allowed to dry in a petri dish for 24 h at 37°C. The dry weight of the tissue was determined and Evans blue extracted using 3 ml of formamide (24 h at room temperature). The amount of Evans blue in the tissue was obtained by comparing the optical density (OD) of the extract with that of a standard Evans blue curve read at 620 nm in an ELISA plate reader. Results are presented as the amount of Evans blue in μg per 100 mg of tissue. The right ventricle was flushed with 20 ml of phosphate-buffered saline to wash the intravascular Evans blue in the lungs. The left lung was then excised and used for Evans blue extraction. The right lung was used for the determination of myeloperoxidase (MPO) as described below.

MPO levels

The extent of neutrophil accumulation in the intestine and right lung tissue was measured by assaying MPO activity as previously described (^{Matos et al., 1999}). Briefly, a fragment of duodenum and the flushed right lungs of animals that had undergone I/R injury were removed and snap frozen in liquid nitrogen. Upon thawing, the tissue (1 g of tissue per 19 ml of buffer) was homogenized in pH 4.7 buffer (0.1 M NaCl, 0.02 M NaPO₄, 0.015 M NaEDTA), centrifuged at 260 ×g for 10 min and the pellet underwent hypotonic lysis (15 ml of 0.2% NaCl solution followed 30 s later by addition of an equal volume of a solution containing NaCl 1.6% and glucose 5%). After a further centrifugation, the pellet was then resuspended in 0.05 M NaPO₄ buffer (pH 5.4) containing 0.5% hexadecyltrimethylammonium bromide and re-homogenized. Aliquots (1 ml) of the suspension were transferred into 1.5-ml Eppendorf tubes, followed by three freeze–thaw cycles using liquid nitrogen. These were then centrifuged for 15 min at 10,000 ×g, the pellet was resuspended to 1 ml and samples of intestine and lung were diluted prior to the assay. MPO activity in the resuspended pellet was assayed by measuring the change in OD at 450 nm using tetramethylbenzidine (1.6 mM) and H₂O₂ (0.5 mM). Results were expressed as the total number of neutrophils by comparing the OD of tissue supernatant with that of rat peritoneal neutrophils processed in the same way. To this end, neutrophils were collected from the peritoneum of rats 8–12 h after injection of 3 ml of 5% casein. A standard curve of neutrophil numbers versus OD was obtained by processing casein-elicited neutrophils (>95% purity by using this methodology), as above and assaying for MPO activity.

Determination of the concentration of circulating leukocytes

The total number of circulating leukocytes and neutrophils was evaluated in blood samples obtained via a cannula in the femoral artery. Samples were collected prior to ischaemia (time 0), 120 min after ischaemia and 30 and 120 min after reperfusion. The number of total circulating leukocytes was determined by counting leukocytes in a modified Neubauer chamber after staining with Turk's solution and differential counts by evaluating the percentage of each leukocyte on blood films stained with May–Grunwald–Giemsa.

Measurement of haemoglobin levels

The levels of haemoglobin in tissue were used as an index of tissue haemorrhage. Tissues were carefully washed with excess saline to remove blood attached to the intestinal epithelia or serosa. No attempt was made to perfuse the vessels with saline as no obvious hyperaemia was present. After washing, a sample of approximately 100 mg of duodenum was removed and homogenized in Drabkin's colour reagent according to the instructions of the manufacturer (Analisa, Belo Horizonte, Brazil). The suspension was centrifuged for 15 min at 3000 ×g and filtered using 0.2 μm filters. The resulting solution was read using an ELISA plate reader at 520 nm and compared against a standard curve of haemoglobin.

Measurement of cytokine levels in serum, intestine and lungs

TNF-α, IL-1β, IL-6, IL-10 and CINC levels were measured in serum and intestine of animals using ELISA techniques previously described (^{Hagan et al., 1993}; ^{Rees et al., 1999a}, ^1999b; ^{Francischi et al., 2000}). Serum was obtained from coagulated blood (15 min at 37°C, then 30 min at 4°C) and stored at −20°C until further analysis. Serum samples were analysed at a 1 : 3 dilution in PBS. In all, 100 mg of duodenum or lung of sham-operated and reperfused animals were homogenized in 1 ml of PBS (0.4 M NaCl and 10 mM NaPO₄) containing anti-proteinases (0.1 mM PMSF, 0.1 mM benzethonium chloride, 10 mM EDTA and 20 KI aprotinin A) and 0.05% Tween 20. The samples were then centrifuged for 10 min at 3000 ×g and the supernatant immediately used for ELISA assays at a 1 : 5 dilution in PBS. ELISA plates (Nunc MaxiSorb) were coated with a sheep anti-rat TNF-α/IL-1β/IL-6 or IL-10 polyclonal antibodies (1–2 μg ml⁻¹) overnight. The plates were washed thrice and then blocked with 1% bovine serum albumin. After a further wash, plates were incubated with samples or recombinant rat cytokine and incubated overnight. The biotinylated polyclonal antibodies were used at a 1 : 1000 to 1 : 2000 dilution and the assays had a sensitivity of 16 pg ml⁻¹.

Drugs and reagents

The following drugs were obtained from Sigma (U.S.A.): urethane, Evans blue, hexadecyltrimethylammonium bromide, 3,3,5,5, tetramethyl-benzidine, Percoll. Repertaxin (R(-)-2-(4-isobutylphenyl)propionyl methansulphonamide) was from Dompé, L'Aquila, Italy (Figure 1). Anti-CINC-1 antibodies were raised in rabbits and shown to be optimally inhibitory at the dose used, as previously described (^{Lorenzetti et al., 2002}).

Statistical analysis

Results are shown as the mean±s.e.m. Percent inhibition of a given parameter was calculated by subtracting the background levels obtained in sham-operated animals. Differences were evaluated by using analysis of variance (ANOVA) followed by Student–Newman–Keuls post-hoc analysis. Results with P<0.05 were considered significant. For survival curves, differences between groups at different time points were compared using Fisher's exact test and considered significant when P<0.05.

Effects of Repertaxin on chemoattractant-induced neutrophil chemotaxis in vitro

Initial experiments were carried out in vitro with rat neutrophils to assess whether Repertaxin was able to inhibit CXC-ELR⁺ chemokine-induced neutrophil recruitment. Neutrophils purified from rat blood migrated in response to various concentrations of human IL-8 (CXCL8), rat CINC-1 (CXCL1-3), fMLP, PAF and LTB₄ (Figure 2a). Pre-incubation of neutrophils with Repertaxin inhibited the recruitment of neutrophils induced by CXCL8 or CINC-1 in a concentration-dependent manner (Figure 2b). The IC₅₀ of Repertaxin for the inhibition of CINC-1- and CXCL8-induced migration was 6 and 30 nM, respectively. In contrast, Repertaxin had no significant effect on the recruitment induced by fMLP, PAF or LTB₄ (Figure 2c). In experiments evaluating intracellular Ca²⁺ concentration in rat neutrophils, repertaxin (10⁻⁶M) effectively inhibited the response elicited by CXCL8 (100 ng ml⁻¹) (Figure 3a, b). In contrast, the drug failed to affect the elevation in intracellular Ca²⁺ concentration induced by fMLP (Figure 3c, d). In binding experiments of [¹²⁵I]-CXCL8 to rat neutrophils, the K_d values in the presence or in the absence of Repertaxin (1 mM) were 8.98±1.07 × 10⁻⁹ and 8.99±1.61 × 10⁻⁹M, respectively (mean±s.d., n=3). These results show that Repertaxin is a noncompetitive specific inhibitor of rat neutrophil migration induced by CXC-ELR⁺-chemokines.

Dose-dependent effects of Repertaxin in a model of mild I/R injury

The next experiments in a model of mild I/R injury were designed to investigate the dose-dependent effects of Repertaxin in a model of reperfusion injury and, hence, the putative role of CXCR2 in the system. As clearly observed in Figure 4, postischaemic treatment of animals with Repertaxin inhibited in a dose-dependent manner both the increase in vascular permeability and the recruitment of neutrophils in the intestine (Figure 4a, b) and lungs (Figure 4c, d) following reperfusion of the ischaemic SMA. Repertaxin appeared to be more potent against reperfusion-induced vascular permeability than neutrophil influx in the intestine, but not in the lung (Figure 4). Moreover, 50% inhibition only occurred when doses greater than 10 mg kg⁻¹ were used and the drug was equieffective and markedly prevented tissue injury when used at 30 mg kg⁻¹.

Effects of Repertaxin on the local, remote and systemic injuries in a model of severe I/R injury

The next series of experiments was carried out in a model of severe I/R injury, where, in addition to the changes in vascular permeability and neutrophil accumulation, we could observe tissue haemorrhage, leucopoenia, increase in the levels of cytokine in tissue and blood and significant lethality (^{Souza et al., 2000b}).

For the experiments evaluating the role of Repertaxin during severe I/R injury, the drug was used at a dose shown to be maximally inhibitory in the mild I/R injury model (30 mg kg⁻¹). Postischaemic treatment with Repertaxin virtually abolished the increase in vascular permeability and neutrophil recruitment in the intestine and in the lung following severe I/R injury (Figure 5). Treatment with Repertaxin also abolished the intestinal increase of haemoglobin, a marker of tissue haemorrhage (Figure 5).

We have previously shown an increase in the concentration of blood neutrophils during the ischaemic period and a rapid drop in neutrophil levels once reperfusion occurs (^{Souza et al., 2000b}). The concentration of circulating neutrophils at 120 min of ischaemia was similar and markedly greater in both Repertaxin and vehicle-treated than sham-operated animals (sham, 2.1±0.4 neutrophils × 10⁶ ml⁻¹ of blood; 120 min after ischaemia, 16.0±1.1 neutrophils; 120 min after in Repertaxin-treated animals, 15.0±1.2; n=5–6). This is consistent with the administration of Repertaxin at the end of the ischaemic period. In vehicle-treated animals, reperfusion of the ischaemic SMA induced a rapid fall of circulating neutrophils to levels observed in sham-operated animals. Pretreatment with Repertaxin reversed by approximately 40% the rapid neutropaenia that occurred 120 min after reperfusion (sham, 2.3±0.2 neutrophils × 10⁶ ml⁻¹ of blood; 120 min after reperfusion, 0.3±0.02 neutrophils; 120 min after reperfusion in Repertaxin-treated animals, 4.8±0.5; n=5–6, P<0.05).

The levels of pro-inflammatory cytokines – IL-1β, IL-6 and TNF-α– and of the anti-inflammatory cytokine IL-10 are markedly elevated in serum and tissues after severe I/R injury (Figure 6, Table 1) (^{Souza et al., 2000b}). Postischaemic treatment with Repertaxin significantly inhibited the elevations of TNF-α in tissue and serum after severe I/R injury (Figure 6a, c, e). Interestingly, pretreatment with Repertaxin was accompanied by an increase in the concentrations of IL-10 in the lung but not in intestine and serum above that observed after severe I/R injury (Figure 6b, d, f). Overall, pre-treatment with Repertaxin prevented the increase in concentrations of IL-6 in tissues and serum and augmented the increase in concentrations of IL-1β in tissues (Table 1). Repertaxin did not alter the concentrations of IL-1β in serum (Table 1).

Our previous studies have shown that severe reperfusion injury is accompanied by significant TNF-α-dependent lethality, reaching 60% in most experiments (^{Souza et al., 2001}). In the present series of experiments, 55% of animals were dead after 120 min of reperfusion (Figure 7). Treatment with Repertaxin prevented lethality and 100% of animals were alive at 120 min (Figure 7).

Effects of the treatment with antibodies anti-CINC on the local, remote and systemic injuries in a model of severe I/R injury

As tissue and systemic inflammation was suppressed and lethality abolished in Repertaxin-treated rat and CINC-1 is one of the ligands at this receptor, it was of interest to examine whether similar effects could be observed after treatment with anti-CINC-1 antibodies. The treatment with anti-CINC-1 60 min prior to the reperfusion virtually abolished the increases in vascular permeability and influx of neutrophils in the intestine and lungs following intestinal I/R (Figure 5). The reperfusion-induced intestinal haemorrhage, as assessed by extravasation of haemoglobin, was abrogated in anti-CINC-1-treated animals (Figure 5). As in mice treated with Repertaxin, pretreatment with anti-CINC-1 also reversed by approximately 40% the rapid neutropaenia that occurred 120 min after reperfusion (sham, 2.1±0.4 neutrophils × 10⁶ ml⁻¹ of blood; 120 min after reperfusion, 0.3±0.02 neutrophils; 120 min after reperfusion in anti-CINC-treated animals, 4.9±0.5; n=5–6, P<0.05).

Anti-CINC-1 also prevented the reperfusion-induced increase in TNF-α concentrations in tissue and serum (Figure 6). Our previous studies have shown a strong correlation between serum concentrations of TNF-α and lethality (^{Souza et al., 2001}, ^2002a). Consistent with these results, treatment of mice with anti-CINC prevented the lethality that followed reperfusion of the ischaemic mesenteric artery (Figure 7). Anti-CINC failed to enhance significantly the increases in IL-10 production in the lungs, intestine and serum following reperfusion of the ischaemic SMA (Figure 6). Furthermore, pretreatment with anti-CINC prevented the increase in concentrations of IL-6 in tissues and serum, whereas this treatment had little effects on the concentrations of IL-1β (Table 1).