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. 2004 Sep;143(1):132-42.
doi: 10.1038/sj.bjp.0705862. Epub 2004 Aug 9.

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

Danielle G Souza  1 , Riccardo Bertini, Angelica T Vieira, Fernando Q Cunha, Steve Poole, Marcello Allegretti, Francesco Colotta, Mauro M Teixeira
Affiliations

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

Danielle G Souza et al. Br J Pharmacol. 2004 Sep.

Abstract

1. Neutrophils are thought to play a major role in the mediation of reperfusion injury. CXC chemokines are known inducers of neutrophil recruitment. Here, we assessed the effects of Repertaxin, a novel low molecular weight inhibitor of human CXCL8 receptor activation, on the local, remote and systemic injuries following intestinal ischaemia and reperfusion (I/R) in the rat. 2. Pre-incubation of rat neutrophils with Repertaxin (10(-11)-10(-6) m) inhibited the chemotaxis of neutrophils induced by human CXCL8 or rat CINC-1, but not that induced by fMLP, PAF or LTB(4), in a concentration-dependent manner. Repertaxin also prevented CXCL8-induced calcium influx but not CXCL8 binding to purified rat neutrophils. 2. In a model of mild I/R injury (30 min of ischaemia and 30 min of reperfusion), Repertaxin dose-dependently (3-30 mg kg(-1)) inhibited the increase in vascular permeability and neutrophil influx. Maximal inhibition occurred at 30 mg kg(-1). 4. Following severe I/R injury (120 min of ischaemia and 120 min of reperfusion), Repertaxin (30 mg kg(-1)) markedly prevented neutrophil influx, the increase in vascular permeability both in the intestine and the lungs. Moreover, there was prevention of haemorrhage in the intestine of reperfused animals. 5. Repertaxin effectively suppressed the increase in tissue (intestine and lungs) and serum concentrations of TNF-alpha and the reperfusion-associated lethality. 6. For comparison, we also evaluated the effects of an anti-CINC-1 antibody in the model of severe I/R injury. Overall, the antibody effectively prevented tissue injury, systemic inflammation and lethality. However, the effects of the antibody were in general of lower magnitude than those of Repertaxin. 7. In conclusion, CINC-1 and possibly other CXC chemokines, acting on CXCR2, have an important role during I/R injury. Thus, drugs, such as Repertaxin, developed to block the function of the CXCR2 receptor may be effective at preventing reperfusion injury in relevant clinical situations.

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Figures

Figure 1
Figure 1
Chemical structure of Repertaxin (R(-)-2-(4-isobutylphenyl)propionyl methansulphonamide). As shown in the figure, Repertaxin is salified with L-lysine.
Figure 2
Figure 2
Effects of Repertaxin on the chemotaxis of neutrophils induced by LTB4, fMLP, CXCL8, CINC-1 or PAF. These experiments were assayed in a 48-well microchemotaxis chamber, as described in the Methods section. Neutrophils were incubated for 10 min with vehicle (saline) or increasing concentration of Repertaxin (10−11–10−6 M) prior to addition of chemoattractants. In (b) and (c), the concentrations of agonists were as follows: CINC-1 (50 ng ml−1), CXCL8 (50 ng ml−1), fMLP (10−6 M), PAF (10−6 M), LTB4 (10−7 M). Results are the number of neutrophils per field and are expressed the mean±s.e.m. of at least 10 fields in each group.
Figure 3
Figure 3
Effects of Repertaxin on the increase in intracellular Ca2+ in neutrophils induced by CXCL8 or fMLP. Neutrophils were incubated for 10 min with vehicle (saline) or Repertaxin (10−6 M) prior to addition of CXCL8 (100 ng ml−1) or fMLP (10−6 M). Results are representative of at least three determinations using each chemoattractant in the presence or absence of Repertaxin.
Figure 4
Figure 4
Dose-dependent effects of the treatment with Repertaxin on the increase in vascular permeability and recruitment of neutrophils in the intestine and lungs following mild ischaemia (30 min) and reperfusion (30 min) injury of the SMA. Changes in vascular permeability in the (a) intestine and (b) lungs were assessed by evaluating the extravasation of Evans blue dye. Neutrophil recruitment in the (c) intestine and (d) lungs was assessed by evaluating tissue levels of MPO. Repertaxin (3–30 mg kg−1) was given i.v. 5 min prior to reperfusion. Control animals (I/R) received drug vehicle (saline). Results are shown as μg Evans blue or as the number of neutrophils per 100 mg of tissue, and are the mean±s.e.m. of at least 5–6 animals in each group. *P<0.01 when compared to sham-operated animals; #P<0.05 when compared to mild I/R animals.
Figure 5
Figure 5
Effects of the treatment with Repertaxin or anti-CINC-1 on the increase in vascular permeability, recruitment of neutrophils and haemorrhage in the intestine and lung following severe ischaemia (120 min) and reperfusion (120 min) injury of the SMA. Changes in vascular permeability in the (a) intestine and (b) lungs were assessed by evaluating the extravasation of Evans blue dye. Neutrophil recruitment in the (c) intestine and (d) lungs was assessed by evaluating tissue levels of MPO. Haemorrhage was evaluating by haemoglobin content in the intestine (e). Repertaxin (30 mg kg−1) was given i.v. 5 min prior to reperfusion, and the anti-CINC-1 antibody (aCINC-1) was given s.c. 60 min prior reperfusion. Control animals received saline (vehicle) or nonimune serum. Results are shown as μg Evans blue, as the number of neutrophils or μg haemoglobin per 100 mg of tissue and are the mean±s.e.m. of 5–6 animals in each group. *P<0.01 when compared to sham-operated animals; # P< 0.05 when compared to vehicle I/R animals.
Figure 6
Figure 6
Effects of the treatment with Repertaxin or anti-CINC-1 on the concentrations of TNF-α and IL-10 in the intestine, lung and serum following severe ischaemia (120 min) and reperfusion (120 min) of the SMA. The concentrations of TNF-α (a, c, e) and IL-10 (b, d, f) were assessed in the intestine (a, b), lung (c, d) and serum (e, f) by using specific ELISA. Repertaxin (30 mg kg−1) was given i.v. 5 min prior to reperfusion and the anti-CINC-1 antibody (aCINC-1) was given s.c. 60 min prior to reperfusion. Control animals received saline (vehicle) or nonimune serum. Results are shown as pg TNF-α or IL-10 per ml of plasma or as pg TNF-α or IL-10 per 100 mg of tissue, and are the mean ±s.e.m. of 5–6 animals in each group. *P<0.01 when compared to sham-operated animals; # P< 0.05 when compared to severe I/R animals.
Figure 7
Figure 7
Effects of the treatment with Repertaxin or anti-CINC-1 on the lethality following severe I/R of the SMA. Repertaxin (30 mg kg−1) was given i.v. 5 min prior to reperfusion, and the anti-CINC-1 antibody was given s.c. 60 min prior reperfusion. Control animals received saline (vehicle) or non-imune serum. Survival was monitored as indicated and animals were killed after 120 min.
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