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Originally published online as doi:10.1189/jlb.0408231 on July 24, 2008

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(Journal of Leukocyte Biology. 2008;84:1213-1221.)
© 2008 by Society for Leukocyte Biology

The selective nonpeptide CXCR2 antagonist SB225002 ameliorates acute experimental colitis in mice

Allisson Freire Bento*, Daniela Ferraz Pereira Leite*, Rafaela Franco Claudino*, Daniela Balz Hara*, Paulo César Leal{dagger} and João B. Calixto*,1

* Departamento de Farmacologia, Centro de Ciências Biológicas, and
{dagger} Departamento de Química, Centro de Ciências Físicas e Matemáticas, Universidade Federal de Santa Catarina, Florianópolis, SC, Brazil

1 Correspondence: Departamento de Farmacologia, Universidade Federal de Santa Catarina, Campus Universitário, Trindade, Bloco D, CCB, Caixa Postal 476, CEP 88049-900, Florianópolis, SC, Brazil. E-mail: calixto{at}farmaco.ufsc.br or calixto3{at}terra.com.br

ABSTRACT

Although neutrophils are strongly implicated in eliminating pathogens, excessive recruitment may cause tissue damage. Therefore, reducing cell influx during an inflammatory process may be a potential target for treating inflammatory bowel diseases (IBD). As CXCR2 is involved in neutrophil migration, this study aimed to evaluate whether the systemic therapeutic treatment with selective CXCR2 antagonist SB225002 ameliorates experimental colitis, which was induced in mice by 2,4,6-trinitrobenzene sulfonic acid (TNBS). After colitis establishment (24 h), mice were treated with SB225002. At later time-points, up to 72 h, mice were monitored for body weight loss and overall mortality. At the time of sacrifice, colonic tissues were scored for macro- and microscopic damage, and cytokine levels, myeloperoxidase (MPO) activity, and protein expression were analyzed. TNBS administration induced macro- and microscopic damage in colon tissue, leading in most cases to animal death. Curative treatment with SB225002 significantly reduced all of the parameters analyzed, leading to an improvement of inflammatory signs. SB225002 reduced neutrophil influx, MPO activity, IL-1β, MIP-2, and keratinocyte-derived chemokine (KC) levels and the expression of vascular endothelial growth factor, inducible NO synthase, and cyclooxygenase-2 proteins into the colon tissue. Levels of IL-4 and IL-10 were increased significantly in the colons of animals treated with SB225002. Additionally, curative treatment with mouse anti-KC significantly reduced MPO activity and colonic damage. These results taken together demonstrate that a selective blockade of CXCR2 consistently reduced TNBS-induced colitis, suggesting that the use of SB225002 is a potential therapeutic approach for the treatment of IBD and other related inflammatory disorders.

Key Words: chemokines • neutrophil migration • trinitrobenzene sulfonic acid-induced colitis • mouse

INTRODUCTION

Inflammatory bowel disease (IBD) is a generic classification for a group of inflammatory disorders of the gastrointestinal tract characterized by mucosal damage with leukocyte infiltration. The etiology of IBD is complex and is likely to involve an interaction of environmental, genetic, and immunologic factors [1 ]. Ulcerative colitis (UC) and Crohn’s disease (CD) represent the two main types of IBD. CD is a relapsing, transmural inflammatory disease of the gastrointestinal mucosa that can affect the entire gastrointestinal tract from the mouth to the anus, and UC is a relapsing, nontransmural inflammatory disease that is generally restricted to the colon [2 ].

Over the past decade, a substantial amount of evidence has been generated to support a crucial role of neutrophils in the pathogenesis of colitis [3 , 4 ]. The infiltration of polymorphonuclear leukocyte (PMN) into the colonic mucosa is believed to play a key role in mediating tissue damage and clinical symptoms in human and experimental colitis [3 4 5 ] by releasing harmful inflammatory mediators, such as cytokines, proteases, and reactive oxygen species. Therefore, inhibition of neutrophil influx might be an attractive and relevant, therapeutic strategy to treat IBD [6 ].

Neutrophils express two CXC chemokine receptors, CXCR1 and CXCR2 [7 ], which play an important role in the pathogenesis of inflammatory responses [8 ]. In the human system, IL-8/CXCL8 and granulocyte chemotactic protein-2 (GCP-2)/CXCL6 can bind and activate CXCR1 and CXCR2, promoting neutrophil migration, and CXCL1, -2, -3, -5, and -7 are potent agonists only for CXCR2 [9 ]. Once CXCL8 is absent in the mouse, it is assumed that neutrophil migration in this specie is mediated mainly by keratinocyte-derived chemokine (KC), MIP-2, LPS-induced CXC chemokine (LIX), and GCP-2 [10 ]. Disparity between human and rodent systems is implied further by the affinity of CXCR1 and CXCR2 to chemokines. Fan and colleagues [11 ] have demonstrated recently that mouse CXCR1 binds with high affinity to mouse GCP-2 but with low affinity to mouse MIP-2 and KC. As in the human system, mouse CXCR2 is more promiscuous than CXCR1, binding to mouse KC, MIP-2 [12 ], GCP-2 [11 ], and LIX [13 ].

It is now well characterized that activation of CXCR1 and CXCR2 is able to promote neutrophil degranulation and chemotaxis at sites of inflammation [8 ]. Evidence suggests that CXCR1 is implicated in superoxide production [14 ], and CXCR2 seems to be associated with the initiation of this cell migration [6 ]. CXCR2 signaling may also act as an angiogenic factor after KC engagement, contributing in concert with vascular endothelial growth factor (VEGF) and CXCL1 to the angiogenesis, a phenomenon that is thought to be involved in the initiation and perpetuation of IBD. Of note, CXCL8, VEGF, and CXCL1 are found to be increased in CD and UC [15 , 16 ].

Previous studies have reported that selective and competitive CXCR2 antagonist SB225002 is able to reduce neutrophil recruitment in many pathophysiological states [6 , 17 ]. Likewise, studies carried out on CXCR2-knockout mice have shown limited mucosal damage and reduced inflammatory signs following dextran sulfate sodium-induced colitis [3 ]. However, it has been demonstrated by the use of CXCR2 antibody neutralization that neutrophil influx in the rat is critically dependent on CXCR2 signaling when assessed in the early phase (8 h) but not in the late phase (Day 7) after 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis [18 ].

In the present study, we have investigated the potential beneficial effects of a potent and selective nonpeptide CXCR2 antagonist SB225002 in TNBS-induced colitis in mice. We have found enhanced levels of CXCR2 ligands, namely MIP-2 and KC, in colonic tissues. Moreover, treatment with mouse anti-KC or with SB225002 was able to ameliorate TNBS-induced colitis significantly, decreasing inflammatory cell influx and tissue damage. In addition, we have further clarified other mechanisms involved in SB225002 anti-inflammatory effects such as decreased VEGF, inducible NO synthase (iNOS), and cyclooxigenase-2 (COX-2) protein expression.

MATERIALS AND METHODS

Animals
Male BALB/c mice, at 8–10 weeks of age, were obtained from the Universidade Estadual de Campinas (Campinas, SP, Brazil). Animals were kept in chambers under a 12-h light/dark cycle with controlled humidity (60–80%) and temperature (22±1°C). Food and water were available freely. Experiments were performed during the light phase of the cycle. The Ethics Committee of the Universidade Federal de Santa Catarina (Florianópolis, SC, Brazil) approved all experimental protocols used in this study.

Animal treatment
N-(2-Hydroxy-4-nitrophenyl)-N9-(2-bromophenyl) urea (SB225002) was synthesized as described before [6 ]. To evaluate the potential therapeutic effect of SB225002 in the experimental colitis, animals received different doses of SB225002 twice per day (0.1, 0.3, and 1 mg/kg, i.p.), 24 h after the colitis induction. At 24 h or 72 h following colitis induction, the macroscopic score and myeloperoxidase (MPO) activity were analyzed. The most effective dose (0.3 mg/Kg) was used for the other experiments, such as cytokine contents, COX-2, VEGF, and iNOS protein expression. Similar protocol treatments were carried out using the positive control drug dexamethasone (1 mg/kg, s.c.) and anti-mouse KC (30 µg /kg, i.v.). SB225002 was dissolved in 0.9% NaCl solution containing 0.33% Tween 80 just before use, and control mice were treated with this vehicle.

Induction and assessment of TNBS-induced colitis
Colitis was induced by intracolonic administration of TNBS, as described previously [19 ]. Briefly, 1-day fasted mice were anesthetized slightly with an i.p. injection of xylazine, 10 mg/kg, and ketamine, 80 mg/kg. TNBS (1.5 mg in 100 µL 35% ethanol) was administered intrarectally using a polyethylene PE-50 catheter slowly inserted into the colon 4 cm proximal to the anus. The animals were kept in a head-down, vertical position for 2 min. Control mice received 100 µL sterile 0.9% NaCl solution. Four hours later, the animals were given free access to food and water. Throughout the experiment, mice were monitored for body-weight loss and overall mortality. At the time of sacrifice, the colon length and weight were also measured.

Twenty-four hours or 3 days following TNBS administration, mice were killed, and the colonic tissues were excised longitudinally. They were then rinsed with saline and scored for macroscopic damage as described previously [19 , 20 ]. Macroscopic damage was graded according to Table 1 . In another set of experiments, each excised portion of distal colon was fixed immediately in 10% formaldehyde solution. All tissues were embedded in paraffin and sectioned to 5 µm thickness, mounted on glass slides, and deparaffinized. For general histology and morphometric analysis, slices were stained using H&E standard techniques. Samples were analyzed by light microscopy and scored as described in Table 2 [21 ].


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  		Table 1. Macroscopic Score


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  		Table 2. Microscopic Score

MPO, eosinophil peroxidase (EPO), and N-acetylglucosaminidase (NAG) assays
Neutrophil, eosinophil, and macrophage infiltration into the colon was assessed indirectly by measuring the MPO, EPO, and NAG activities. Colon segments were homogenized at 5% in EDTA/NaCl buffer (pH 4.7) and centrifuged at 10,000 g for 15 min at 4°C. The pellet was ressuspended in 0.5% hexadecyl trimethyl ammonium bromide buffer (pH 5.4), and the samples were frozen in liquid nitrogen and thawed three times. Upon thawing, the samples were similarly centrifuged, and 25 µL of the supernatant was used for the MPO, EPO, and NAG assays. The MPO enzymatic reaction was assessed by addition of 1.6 mM tetramethylbenzidine (TMB), 80 mM NaPO4, and 0.3 mM hydrogen peroxide (H2O2). The EPO enzymatic reaction was assessed by addition of 1.5 mM O-phenylenediamine (OPD), 0.075 mM Tris-HC, and 6.6 mM H2O2. Finally, the NAG activity measurement was determined by adding 2.25 mM p-nitrophenil-2-acetamide ß-D-glucopyranoside and 100 µL 50 mM citrate buffer (pH 4.5). The absorbance was measured spectrophotometrically at 690, 490, and 405 nm for MPO, EPO, and NAG, respectively, and the results are expressed in OD/mg tissue.

Determination of cytokine levels
Briefly, colon segments were homogenized in phosphate buffer containing 0.05% Tween 20, 0.1 mM PMSF, 0.1 mM benzethonium chloride, 10 mM EDTA, and 20 UI aprotinin A. The homogenate was centrifuged at 3000 g for 10 min, and supernatants were stored at –70°C until further analysis. IL-1β, KC, MIP-2, IL-4, and IL-10 levels were evaluated using ELISA kits from R&D Systems (Minneapolis, MN, USA), according to the manufacturer’s recommendations. The amount of protein in each sample was measured using the Bradford method [22 ].

Preparation of cytosolic fractions
The colons were homogenized in lysis buffer containing 10 mM HEPES, pH 7.4, 2 mM MgCl2, 10 mM KCl, 1 mM PMSF, 1 mg/mL leupeptin, 1 mg/mL pepstatin A, 1 mg/mL leupeptin, 1 mg/mL aprotinin, 1 mM sodium orthovanadate, 10 mM b-glycerophosphate, 50 mM sodium fluoride, and 0.5 mM DTT. After centrifugation (14,000 g for 60 min), the supernatant containing the cytosolic fraction was collected and stored at –70°C until analysis.

Western blot analysis
Equivalent amounts of proteins (35 µg) were mixed in a buffer containing 200 mM Tris, 10% glycerol, 2% SDS, 2.75 mM β-ME, and 0.04% bromophenol blue and boiled for 5 min. Proteins were resolved in a 10% SDS gel by electrophoresis. After transfer to a polyvinylidene fluoride membrane, the blots were blocked with 5% fat-free dry milk-TBS buffer overnight at 4°C and then washed with TBS and 5% Tween-20 (TBST). The membranes were incubated for 2 h at room temperature with 1:1000 dilutions of primary antibodies for COX-2 or β-actin, 1:2000 for VEGF, and 1:150 for iNOS. Blots were washed four times with TBST for 5 min, followed by incubation with 1:80,000-adjusted, peroxidase-coupled biotinylated secondary antibodies for 1 h. The membranes were washed four times with TBST for 5 min and then incubated for 30 min with streptavidin–HRP reagent. The transferred proteins were visualized with an ECL detection kit according to the manufacturer’s instructions.

Drugs and reagents
Dexamethasone, H2O2, Tween 20, Tween 80, EDTA, aprotinin, PBS, H&E, TMB, H2O2, OPD, and TNBS were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Formaldehyde was obtained from Merck (Frankfurt, Darmstadt, Germany). Bradford reagent was purchased from Bio-Rad Laboratories (Richmond, CA, USA). Anti mouse-KC, mouse KC, IL-4, MIP-2, IL-10, and IL-1β/IL-1F2 DuoSet kits were obtained from R&D Systems. COX-2, β-actin, VEGF (C-1), and iNOS primary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CAa, USA). Secondary antibody Envision Plus, streptavidin–HRP reagent, and 3,3-diaminobenzidine chromogen were purchased from Dako Cytomation (Carpinteria, CA, USA).

Statistical analysis
All data are expressed as means ± SEM. For nonparametric data, Kruskal-Wallis followed by Dunn’s test was used. For parametric data, the statistical significance of differences between the groups was determined by one-way ANOVA, followed by the Student Newman-Keuls test. The {chi}2 method was used for the survive curve. Statistical analyses were performed using GraphPad Prism 4 software (GraphPad Software Inc., San Diego, CA, USA). A P value of less than 0.05 (P<0.05) was considered to be statistically significant.

RESULTS

SB225002 ameliorates TNBS-induced colitis
Colitis was induced in BALB/c mice by the administration of TNBS into the colon. At 24 h after TNBS administration, severe macroscopic damage allied to increased MPO activity was observed, suggesting that colitis was well established (Fig. 1 A and B ). At later time-points up to 72 h after TNBS administration, mice developed signs of a severe illness characterized by bloody diarrhea, rectal prolapse, and a profound and sustained weight loss, which resulted in a high mortality rate (65%), whereas control mice rapidly recovered weight after the starvation period and did not die (Fig. 2 A and B ). To evaluate the potential therapeutic effect of SB225002 24 h after TNBS administration (i.e., after colitis establishment), mice were treated twice per day with SB225002 i.p. SB225002 (0.3 mg/kg)-treated mice rapidly recovered the lost body weight, regained a healthy appearance similar to control mice, and had a survival rate of 88% (Fig. 2A) . In contrast, mice treated with the positive control drug dexamethasone (1 mg/kg, s.c.) did not recover the body weight significantly (Fig. 2A) .


Figure 1
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  		Figure 1. Twenty-four hours after TNBS administration, colitis was already established. Twenty-four hours following colitis, induction of severe macroscopic damage (A) and a high MPO activity (B) was observed. Each column represents the mean ± SEM of five to seven mice per group. ##, P < 0.01, versus control group.


Figure 2
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  		Figure 2. Pharmacological blockage of CXCR2 ameliorates TNBS-induced colitis. Throughout the experiment, mice were monitored for body weight loss and overall mortality, and at 72 h after TNBS administration, colitis severity was assessed. Treatment with SB225002 (SB) recovered mouse weight (A), increased survival (B), improved colon length (C), decreased colon weight (D), and reduced the macroscopic score (E). Dex, Dexamethasone. Each column represents the mean ± SEM of five to seven mice per group. ###, P < 0.001, versus control group; *, P < 0.05, **, P < 0.01, and ***, P < 0.001, versus TNBS-treated group.

Three days following TNBS administration, animals were killed, and colon length and weight as well as macroscopic damage were assessed. TNBS administration resulted in decreased colon length (by 20%, Fig. 2C ), increased colon weight (by 3.42-fold, Fig. 2D ), and severe inflammation associated with hyperemia, ulceration, and sometimes adhesion. SB225002 was administered in three different doses (0.1, 0.3, and 1 mg/kg) to detect potential dose-dependent effects. Treatment with 0.3 or 1 mg/kg SB225002 but not with 0.1 mg/kg, significantly reduced macroscopic colonic damage by 89.3 ± 3.4% and 79.1 ± 7.0%, respectively (Fig. 2E) . SB225002 curative treatment also restored colon length (Fig. 2C) and decreased colon edema by 75.2 ± 5.4% (Fig. 2D) . Likewise, dexamethasone significantly reversed TNBS-induced colon reduction (Fig. 2C) and decreased colon weight by 82 ± 1.0% (Fig. 2D) .

SB225002 treatment inhibits neutrophil influx, decreasing colon damage
Colitis has been associated with the influx of neutrophils and eosinophils into the intestinal mucosa [23 , 24 ]. Moreover, CXCR2 is implicated in neutrophil migration [6 ]. Therefore, we determined whether the diminished severity of TNBS-mediated colitis in the SB225002-treated mice was associated with alterations in the composition of inflammatory cell populations in the intestinal mucosa. Three days following TNBS administration, mucosal neutrophil and eosinophil infiltration was quantified by analysis of colonic MPO and EPO activity, respectively. TNBS-treated mice displayed a marked increase in colonic MPO levels (3.3±0.3 OD/mg tissue) by 22.8-fold when compared with control animals (0.14±0.06 OD/mg tissue; Fig. 3A ). Treatment with SB225002 (0.1, 0.3, or 1 mg/kg) significantly reduced TNBS-induced colonic tissue MPO activity by 48.7 ± 14.0%, 82.5 ± 1.6%, or 63.8 ± 8.0%, respectively (Fig. 3A) . In addition, EPO activity in the colon of TNBS-treated mice (2.0±0.09 O.D./mg tissue) was enhanced by ~30% when compared with control mice (1.5±0.1 O.D./mg tissue; Fig. 3B ). However, SB225002 treatment had no effect on EPO activity. In contrast, dexamethasone significantly reduced MPO levels by 93.6 ± 6.3% compared with the TNBS and suppressed EPO levels under basal levels (Fig. 3 A and B) . Finally, mononuclear cells were also analyzed indirectly by NAG activity; nonetheless, 72 h after colitis induction, this parameter was not altered in TNBS-treated mice when compared with control mice (data not shown).


Figure 3
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  		Figure 3. Pharmacological blockage of CXCR2 inhibits cell influx and colon damage. Seventy-two hours following colitis induction, the histopathological tissue damage, the MPO, and EPO activities were determined. Treatment with SB225002 decreased MPO activity (A), although it did not alter EPO activity (B). Colon sections from control mice, TNBS, SB225002, or dexamethasone-treated mice were stained with H&E (C and D). The images are representative of at least three mice per group. Original magnifications, 50x (C) and 200x (D). The microscopic damage (E) was scored according to Table 2 . Each column represents the mean ± SEM of five to seven mice per group. ##, P < 0.01, and ###, P < 0.001, versus control group; *, P < 0.05, **, P < 0.01, and ***, P < 0.001, versus TNBS-treated group.

Colons were then processed for histological observation (the representative sections of H&E staining revealed) in the TNBS-treated colon—a dramatic infiltration of PMN into the lamina propria and colonic mucosa corroborating the increased MPO activity described above. TNBS-treated colons also appeared thickened and ulcerated, showing distortion of crypts and loss of goblet cells (Fig. 3 C and D) . Histological evaluation of bowel tissue showed a marked reduction in the inflammatory response and mucosal ulcerations in SB225002 as well as in dexamethasone-treated mice (Fig. 3 C and D) . The histological damage score of colonic tissues obtained from inflamed mice was 23-fold higher when compared with control mice, whereas treatment with SB225002 or with dexamethasone resulted in score reductions of 89.2 ± 6.7% and 93.8 ± 3.0%, respectively (Fig. 3E) .

SB225002 administration altered the inflammatory profile of cytokines released during colitis
As shown in Figure 4 , TNBS instillation resulted in a marked increase in IL-1β, MIP-2, and KC levels by ~16.3-, 2.4-, and 3.7-fold, respectively (Fig. 4 A-C) . SB225002 administration resulted in a striking decrease in IL-1β, MIP-2, and KC levels (80.5±7.8%, 80.0±10.0%, and 76.8±9.0%, respectively; Fig. 4 A-C ). Moreover, colons from TNBS-treated mice showed a reduction in the anti-inflammatory cytokines, namely, IL-4 and IL-10 (51.4±8.9% and 49.9±5.6%, respectively), when compared with colons from control mice, whereas colons from mice subjected to SB225002 treatment displayed a significant enhancement in these cytokine levels (~2.6- and 2.9-fold, respectively) when compared with colons from TNBS-treated mice (Fig. 4 D and E) . Similar results were observed by treatment with dexamethasone (Fig. 4 D and E) .


Figure 4
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  		Figure 4. CXCR2 inhibition alters cytokine production. Seventy-two hours following colitis induction, the levels of IL-1β, KC, MIP-2, IL-4, and IL-10 were measured in the colon tissue. Treatment with SB225002 decreased IL-1β (A), KC (B), and MIP-2 (C) production induced by TNBS, and TNBS decreased IL-4 (D) and IL-10 (E) levels; SB225002 treatment increased the cytokine production over basal levels. Each column represents the mean ± SEM of five to seven mice per group. #, P < 0.05, and ###, P < 0.001, versus control group; *, P < 0.05, and ***, P < 0.001, versus TNBS-treated group.

Anti-mouse KC treatment inhibits neutrophil influx and decreases colon damage
Once KC is one of the main CXCR2 ligands that is most expressed 72 h after TNBS-induced colitis and as KC does not bind mouse CXCR1 with a high affinity [11 ], we next treat mice with anti-mouse KC to analyze by another strategy the possible involvement of CXCR2 in this model. Mice treated with anti-mouse KC (30 µg/kg, i.v., twice per day) after colitis establishment had a survival rate of 75% associated with a significant reduction of macroscopic colonic damage (76.3±6.6%). In addition, colons from mice treated with anti-mouse KC displayed a marked decrease in MPO levels by 75.5 ± 9.9% when compared with TNBS-treated animals (Fig. 5 A and B ).


Figure 5
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  		Figure 5. Anti-mouse KC treatment inhibits neutrophil influx and decreases colon damage. Twenty-four hours following colitis induction, mice were treated with anti-KC (30 µg/ kg, i.v.) or dexamethasone (1 mg/kg, s.c.). Treatment with anti-KC or dexamethasone decreased macroscopic damage (A) and MPO activity (B). Each column represents the mean ± SEM of five to seven mice per group. ##, P < 0.01, and ###, P < 0.001, versus control group; *, P < 0.05, and ***, P < 0.001, versus TNBS-treated group.

Mechanisms underlying the colitis improvement caused by SB225002 treatment
Colitis severity has been associated with increased iNOS and COX-2 protein expression in the bowel tissue [25 , 26 ]. Therefore, we assessed whether treatment with SB225002 diminished these protein expressions. TNBS administration induced a prominent expression of iNOS and COX-2 by ~1.7- and 2.3-fold, respectively (Fig. 6 A and B ), when compared with basal values. As revealed by densitometry analysis, iNOS and COX-2 expression was decreased by 87.5 ± 6.1% and 51.1 ± 2.2% in colons from mice treated with 0.3 mg/kg SB225002 (Fig. 6 A and B) . Dexamethasone also significantly reduced iNOS and COX-2 expression by ~90.0 ± 3.6% and 55.4 ± 1.8%, respectively (Fig. 6 A and B) .


Figure 6
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  		Figure 6. SB225002 decreased iNOS, COX-2, and VEGF expression induced by TNBS administration. Treatment with SB225002 decreased iNOS (A), COX-2 (B), and VEGF (C) expression, as observed by the representative Western blot gel and by the quantification of the ratio between those protein bands and β-actin protein bands. Each column represents the mean ± SEM of five to seven mice per group. ###, P < 0.001, versus control group; ***, P < 0.001, versus the TNBS-treated group.

It has also been demonstrated that COX-2 and iNOS are implicated in the induction of VEGF [27 ], a cytokine that has a relevant role in angiogenesis and is increased in patients with IBD [15 ]. Induction of colitis was associated with a significant increase of VEGF expression (~7.5-fold) when compared with control colons (Fig. 6C) , and colons from mice treated with SB225002 or with dexamethasone had significant reductions in VEGF expression (96.4±1.4% and 79.8±1.9%), respectively (Fig. 6C) .

DISCUSSION

Recent advances in the understanding of the mechanisms involved in neutrophil migration have indicated an important role exerted by the chemokine receptor CXCR2 [6 ]. The infiltration of these cells mediates tissue damage and inflammatory signs in experimental colitis [3 4 5 ]. In the present study, we show for the first time that SB225002, a potent and selective nonpeptide CXCR2 antagonist, is highly effective in ameliorating TNBS-induced colitis in mice.

In this model, TNBS penetrates into the colon and causes transmural lesions, ulcerations, necrosis, loss of crypt epithelium, thickening of the bowel wall, edema, colon length reduction, and a massive infiltration of PMN cells, characterizing macroscopic and histological damage [28 ]. Furthermore, diarrhea, rectal prolapse, weight loss, and a high mortality rate are observed [21 ]. These parameters resemble some features of CD in humans. Our study reports that after colitis establishment, the systemic curative therapeutic regimen of SB225002 administration significantly reduced colon edema, attenuated macroscopic and histological damage, restored colon length, and recovered mouse body weight, greatly reducing mortality. In addition, mice treated with dexamethasone did not recover body weight, unlike those that received the curative treatment with SB225002, suggesting that the CXCR2 antagonist produces less intense side-effects.

It has been shown in different inflammatory models that massive infiltration of PMN cells, characteristic of an inflamed colon [21 ], is mediated mainly by CXCR2 engagement [3 , 6 ]. In the mouse system, at least four ligands were described for CXCR2, among them KC and MIP-2 [10 ], which were associated with colitis [29 , 30 ]. Some studies have identified increased levels of CXCR2 ligands, which correlate with MPO activity increments in colon biopsies from patients with IBD [31 ]. Accordingly, the present results demonstrate a great increase in KC, MIP-2 levels, and MPO activity in colonic tissue. Similar effects allied to enhancement of CXCR2 levels have been reported in rats after TNBS administration [18 ]. In the present study, we have also demonstrated that a KC increment in colonic tissue was more prominent than MIP-2. Once KC does not bind mouse CXCR1 with a high affinity [11 ], we next analyzed the involvement of CXCR2 by treating animals with mouse anti-KC and observed that this treatment greatly reduced MPO activity and tissue damage, thus suggesting a minor role of CXCR1 in this subject. Most important, we have also demonstrated the occurrence of MPO activity reduction and minor cell infiltration in colon tissue from mice treated with the CXCR2 antagonist SB225002. Those results demonstrated that the reduction of CXCR2 ligands and the blockage CXCR2 exert a critical role in neutrophil migration induced by TNBS; however, we cannot totally discard a role exerted by CXCR1 in TNBS-induced colitis.

Although SB225002 administration inhibited MPO activity, it failed to reduce EPO activity, suggesting that eosinophil infiltration was not altered by CXCR2 antagonism. In agreement with this data, it has been shown that CXCR2 is not expressed on the eosinophil surface [32 ]. The relevance of eosinophils in gut inflammation has been described by several authors [33 ]. Nevertheless, in the time period analyzed by us in TNBS-induced colitis, neutrophils seem to have had a major involvement in tissue damage, as SB225002 treatment almost highly decreases mucosal injury, without interfering in EPO activity.

Many other studies in different inflammatory conditions have addressed the relevance of neutrophils in tissue damage [34 , 35 ]. It has been demonstrated that inhibition of neutrophil adhesion by anti-integrins (CD11b/CD18) decreases tissue damage in TNBS-induced colitis [36 ]. Upholding these data, the work of Buanne and collaborators [3 ], who use CXCR2 knockout mice, and the present results, which applied the CXCR2 antagonist SB2205002, point to a role of neutrophils in tissue damage. In contrast, it has been shown that neutrophil depletion exacerbates TNBS-induced colitis in rats [37 ]. In fact, other works suggest a dual role of neutrophil in IBD [38 ].

It has been shown that iNOS, COX-2 inhibition, or IL-1R blockade decreases colitis severity significantly [25 , 26 , 39 ]. Furthermore, the existence of a positive correlation among enhancement of KC, MIP-2 levels, and colitis exacerbation has been suggested [30 ]. Notably, colons from mice treated with SB2205002 show a diminished iNOS and COX-2 protein expression, besides reduction of IL-1β, MIP-2, and KC levels, suggesting that these decreases constitute one of the mechanisms involved in SB2205002 effectiveness. As there are some reports suggesting that these proteins are directly or indirectly related to cell migration [40 , 41 ], their reduction by SB2205002 treatment could contribute to a lower cell infiltration, inflammation, and consequently, damage to colonic tissue.

Evidence suggests that the influx of inflammatory cells to the colon tissue might also be facilitated by angiogenesis [15 ]. VEGF is one of the main factors involved in this phenomenon, as it is found to be enhanced significantly in patients with IBD and in experimental models of colitis [15 ]. Corroborating these findings, the present data demonstrate that VEGF expression is increased markedly in inflamed colons. It has been shown that CXCL8 elicits angiogenic responses in microvascular endothelial cells isolated from human intestine, an effect that is believed to be associated with CXCR2 engagement [7 ]. Hence, CXCR2 antagonism or decreased levels of KC might diminish the endothelial angiogenic response. Of note, SB225002 markedly decreased KC levels, as well VEGF protein expression. Furthermore, CXCL8 is able to stimulate neutrophil-derived VEGF secretion [42 ]. Therefore, SB225002 may also reduce VEGF levels by decreasing neutrophil influx.

We next investigated the role exerted by the anti-inflammatory cytokines in the effectiveness of SB225002. It is well documented that IL-4 possesses immunosuppressive and anti-inflammatory properties and that it is reduced in IBD [43 ], which supports our data. It has also been shown that IL-4 suppresses CXCL8 production by neutrophils [44 ], and indeed, our results suggest an inverse correlation between the KC and IL-4 levels. Furthermore, IL-4 induces IL-10 production by T cells and macrophages [45 ]. This cytokine is known to deactivate respiratory burst and to inhibit proinflammatory mediator production, playing a negative modulatory role, with an effect on PMN accumulation and chemokine generation [45 ]. In addition, neutrophil depletion enhances IL-10 levels [46 ]. These data substantiate our results, which demonstrated an increased level of IL-4 and IL-10 in colons from mice treated with SB225002.

Finally, the increment in the anti-inflammatory cytokine (IL-4 and IL-10), the decreased levels of IL-1β, MIP-2, and KC, as well as the reduction of iNOS, VEGF, and COX-2 protein expression were effects of SB225002 treatment that contributed greatly to the reduction of tissue damage, signs of inflammation, and most important, to the great increase in mouse survival in TNBS-induced colitis. Of high interest, SB225002 has a curative property; i.e., it was able to attenuate TNBS-induced colitis after the establishment of inflammation. Our data not only points to the CXCR2 as an interesting and attractive target for the management of IBD, for which current therapies remain inadequate, but strongly suggest that the selective nonpeptide CXCR2 antagonist SB225002 is a potential candidate for the treatment of IBD.

ACKNOWLEDGEMENTS

This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Programa de Apoio aos Grupos de Excelência (PRONEX), and Fundação de Apoio a Pesquisa do Estado de Santa Catarina (FAPESC; Brazil). A. F. B. is a postgraduate student in pharmacology and is the recipient of a MSc fellowship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). R. F. C. is a Ph.D. student in pharmacology receiving grants from CNPq. D. F. P. L. and D. B. H. hold postdoctoral fellowships from CNPq. The authors thank Aline Mariana Venâncio and Juliana Gonçalves da Cunha for their excellent technical assistance.

Received April 7, 2008; revised June 26, 2008; accepted June 27, 2008.

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