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The Journal of Immunology, 2003, 171: 5514-5520.
Copyright © 2003 by The American Association of Immunologists

A Potent Human C5a Receptor Antagonist Protects against Disease Pathology in a Rat Model of Inflammatory Bowel Disease 1

Trent M. Woodruff*, Thiruma V. Arumugam*, Ian A. Shiels*,{ddagger}, Robert C. Reid{dagger}, David P. Fairlie{dagger} and Stephen M. Taylor2,*,{ddagger}

* Department of Physiology and Pharmacology, School of Biomedical Sciences and {dagger} Institute for Molecular Bioscience, University of Queensland, and {ddagger} Promics, Brisbane, Australia


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The complement system is implicated in the pathogenesis of human inflammatory bowel disease, but the specific role of C5a has never been examined. We have compared the efficacy of an orally active human C5a receptor antagonist (AcPhe[Orn-Pro-D-cyclohexylalanine-Trp-Arg]), prednisolone, and infliximab against trinitrobenzene sulfonic acid (TNBS)-induced colitis in rats. The drugs were administered either 2 days before or 24 h after TNBS instillation, and rats were then examined after 8 days. Drug-free colitis control rats showed severe disease pathology with significant mortality (39%). Rats pre or posttreated with the C5a antagonist (10 mg/kg/day peroral, 0.3 mg/kg/day s.c.) had reduced mortality and significantly improved macroscopic scores, colon edema, colon myeloperoxidase levels, reduced concentrations of TNF-{alpha} levels in the colon and serum, and had greater food intake resulting in greater weight gains than colitis-only rats. Rats pretreated with prednisolone (1 mg/kg/day s.c.) displayed significant improvement in parameters measured, but posttreatment was ineffective. Single dose pretreatment with the TNF-{alpha} inhibitor infliximab (3 mg/kg i.v.) also had significant improvements in the parameters measured. Rats pretreated with a combination of the C5a antagonist and prednisolone showed no greater improvements than either drug alone. These findings suggest a central role for complement, particularly C5a, in the pathology of TNBS-induced colitis in rats, indicating a possible therapeutic role for C5a antagonists in inflammatory bowel disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Ulcerative colitis and Crohn’s disease are chronic inflammatory disorders of the bowel that fall under the banner of inflammatory bowel disease (IBD)3. IBD is characterized by spontaneously occurring, chronic relapsing inflammation of unknown origin, in which current treatment options are inadequate (1). Despite extensive research into the disease in both humans and experimental animals, the precise mechanisms of pathology remain to be elucidated. Many immune and inflammatory mediators are thought to be involved, including biogenic amines, kinins, arachidonic acid metabolites, free radicals, NO, various proinflammatory cytokines, and complement proteins (2, 3, 4).

Therapies for IBD target one or more of these inflammatory mediators. Recent advances in drug development for IBD have involved the use of mAbs to inhibit specific proinflammatory cytokines, such as ILs, IFNs, and TNF-{alpha} (5, 6). In particular, the anti-TNF-{alpha} Abs CDP571 and infliximab have been used clinically to treat Crohn’s disease with some success (7, 8, 9). Major drawbacks of these new protein therapies are the narrow spectrum of inflammatory mediators that they regulate, their high costs of production, in vivo instability, poor bioavailability, limited routes of administration, and immunogenicity.

Activation of the complement system leads to production of a suite of complement activation products including the anaphylatoxins C3a and C5a, and the terminal membrane attack complex (10). These components, particularly the anaphylatoxins, are potent inflammatory mediators that are associated with pathology in various disease states (11, 12, 13). The complement system is believed to be activated in patients with IBD and is thought to play a role in disease pathogenesis (3, 14). However, the evidence for this has been relatively limited. It has been shown that activated complement products are found at the luminal face of surface epithelial cells, as well as in the muscularis mucosa and submucosal blood vessels in IBD patients (3, 10, 15, 16, 17). The multifunctional complement activation inhibitor K-76, has also been reported to produce symptomatic improvement of ulcerative colitis in a small uncontrolled clinical study, (18) as well as in a model of carrageenan-induced colitis in rabbits (19).

We have previously reported a potent, orally active antagonist, AcPhe[Orn-Pro-D-cyclohexylalanine-Trp-Arg] (AcF-[OPdChaWR]), for the human C5a receptor (20, 21). This compound is an effective in vitro inhibitor of C5a-induced oxidative burst, enzyme release, phagocytosis, and cytokine release from various inflammatory leukocytes (21, 22, 23). We have also shown that this compound is effective in vivo in reducing pathology in various rat models of inflammatory disease (24, 25, 26, 27, 28). Due to the current lack of knowledge of complement involvement in IBD, we aimed to test possible protective effects of AcF-[OPdChaWR] in trinitrobenzene sulfonic acid (TNBS)-induced colitis in rats. For comparison, we also examined the efficacy of the corticosteroid prednisolone and the TNF-{alpha} Ab infliximab in this model. We found strong protective effects for the C5a antagonist, suggesting a previously undefined role for complement in TNBS-induced colitis in rats.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Drug preparation

The human C5a receptor cyclic antagonist AcF-[OPdChaWR] was synthesized as previously described (21), purified by reversed phase HPLC, and fully characterized by mass spectrometry and proton nuclear magnetic resonance spectroscopy. It was prepared in olive oil (10 mg/ml) for oral dosing and in a 30% polyethylene glycol solution (0.6 mg/ml) for s.c. dosing. The glucocorticosteroid, prednisolone (Sigma-Aldrich, St. Louis, MO) was prepared in a 30% polyethylene glycol solution for s.c. dosing. The TNF-{alpha} inhibitor infliximab (Remicade) was prepared in sterile saline (3 mg/ml) as per instructions for i.v. dosing. No anti-inflammatory effects were observed for polyethylene glycol in TNBS-colitis rats (data not shown).

Animals

Pathogen-free male Wistar rats (275–300 g) were obtained from a commercial source (Central Animal Breeding House, Pinjarra Hills, Australia). This study was performed in accordance with guidelines from the National Health and Medical Research Council of Australia, and was approved by the University of Queensland Animal Ethics Committee.

Model of IBD

To induce colitis in rats, a well-established TNBS-induced colitis model was used, because this is reportedly relevant to human IBD (29). In our study, rats were starved for a period of at least 24 h with free access to water, before the induction of colitis. Rats were then anesthetized with ketamine (80 mg/kg, i.p.) and xylazine (8 mg/kg, i.p.), and a 1.7-mm outer diameter polyethylene catheter was inserted into the colon, to a distance of 8 cm from the anus. A solution containing TNBS (120 mg/kg; Sigma-Aldrich) and 250 µl of ethanol was then injected while the rats were maintained in the head-down position for at least 30 min to prevent leakage from the anus. Animals were then allowed to recover from the anesthetic and were housed in separate cages with free access to food and water. The study progressed for 8 days and body weight and food consumption were measured daily.

At completion, rats were anesthetized with zolazapam (50 mg/kg, i.p.) and xylazine (12 mg/kg, i.p.), blood was collected, and serum was stored at -20°C for later determination of TNF-{alpha} concentrations. Colons were dissected and the distal 8 cm were rinsed with saline and scored for clinical macroscopic damage by an independent blinded observer, using a scale of 0–13 previously described (30). A section of the affected colon was then collected, weighed, placed in an 80°C oven for 24 h, then reweighed, and the wet-to-dry weight ratio was determined as a measure of colon edema (31). A separate section of affected colon was also collected and homogenized with 1 ml of PBS, sonicated for 20 s, and centrifuged (14 000 x g, 10 min). The resulting supernatant was then either stored (-20°C) for later TNF-{alpha} determination, or used immediately in an assay for the determination of myeloperoxidase (MPO) levels. Briefly, the assay involved the addition of 20 µl of substrate (o-dianasidine, 2.85 mg/ml; Sigma-Aldrich; and 0.85% hydrogen peroxidase) to a 1/40 dilution of supernatant in PBS. Absorbances were then read at 450 nM, 15 min after substrate addition. Results for colon MPO content were converted to absorbance units per gram of tissue. Additional sections of colon were removed and stored in 10% formalin for histopathological analysis. Fixed colonic samples were embedded in paraffin wax, sections were stained using a H&E stain, photographed, and examined by an independent observer in a blinded fashion. Tissue and serum TNF-{alpha} levels were determined from stored samples using an ELISA as previously described (25, 26), using no dilution of samples. Results for colon TNF-{alpha} content were then converted to picograms of TNF-{alpha} per gram of tissue.

Treatment groups and study design

An 8-day time frame was chosen for this study to ensure proper development of an inflammatory bowel condition. Intracolonic TNBS administration in untreated rats was used as a drug-free colitis control group. Saline-alone (sham saline) or saline/ethanol (sham ethanol) was also instilled to rats in both time frames, and were used as sham control groups to determine baseline levels.

All drugs were administered 2 days before colitis induction; the C5a antagonist and prednisolone were also given 24 h after colitis induction. The following treatment groups (before induction), dosed daily throughout unless otherwise indicated, were used: C5a antagonist (10 mg/kg/day, peroral (p.o.)), C5a antagonist (0.3 mg/kg/day, s.c.), prednisolone (1 mg/kg/day, s.c.), C5a antagonist (10 mg/kg/day, p.o.) + prednisolone (1 mg/kg/day, p.o.) combination, and infliximab (3 mg/kg, single i.v. infusion 2 days before TNBS instillation). The following groups were used for rats treated 24 h after induction: C5a antagonist (10 mg/kg/day, p.o.) and prednisolone (1 mg/kg/day, s.c.).

Data and statistical analysis

All experimental results are expressed as means ± SEM. Data analysis was performed using GraphPad Prism 3.0 software (GraphPad, San Diego, CA). Statistical comparisons were made between drug-treated groups and colitis control animals using a one-way ANOVA followed by a Dunnett comparison posttest analysis. {chi}2 and log rank Mantel-Haenszel tests were used to determine significance for mortality rates. Statistical significance was assessed at p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Mortality rate

Induction of colitis in drug-free rats resulted in high mortality beginning 2 days after TNBS administration (39%; Fig. 1). Rats treated orally with the C5a antagonist either before induction (10 mg/kg/day p.o.) or 24 h after induction (10 mg/kg/day p.o.) had improved survival with only 1 mortality recorded over the study period, but the data were not significantly different to colitis control rats (p < 0.16; Fig. 1). Rats pretreated with the corticosteroid prednisolone (1 mg/kg/day s.c.) had a similar reduction in mortality (11%; Fig. 1), however, posttreatment with prednisolone had no effect on reducing mortality (38%; Fig. 1). Infliximab pretreatment (3 mg/kg i.v.) was less effective at reducing mortality than the C5a antagonist or prednisolone pretreatment, and was not significantly different from colitis control rats (p < 0.34; Fig. 1). Rats pretreated with the C5a antagonist (0.3 mg/kg/day s.c.) or with a combination of the C5a antagonist (10 mg/kg/day p.o.) and prednisolone (1 mg/kg/day s.c.), produced no mortalities and the results were significantly different from colitis control rats (p < 0.05; Fig. 1).



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  		FIGURE 1. Mortality rate over 8 days. Rats received a colonic injection of either TNBS or a saline or saline/ethanol solution for sham animals on day 0. All drug treatments, except for prednisolone posttreatment, resulted in a decrease in mortality (n = 8–16) over the 8-day study period, compared with drug-free colitis control rats (n = 13). Rats pretreated s.c. with the C5a antagonist (0.3 mg/kg/day s.c.) or a combination of the C5a antagonist (10 mg/kg/day p.o.) and prednisolone (1 mg/kg/day s.c.) recorded no deaths, and mortality rates were significantly decreased from colitis control rats (p < 0.05; n = 8). No deaths were recorded in saline or saline/ethanol-instilled animals (n = 4). *, p < 0.05 drug-treated rats vs colitis control. C5aA = C5a antagonist; Pred = prednisolone.

 
Food intake

Colitis induction resulted in a decrease in the consumption of food in all TNBS-administered rats compared with saline-instilled rats in the first few days following induction (data not shown). After 8 days, drug-free colitis control rats were still eating less food than saline-instilled rats (Fig. 2). Rats treated with the C5a antagonist either before induction (10 mg/kg/day p.o. and 0.3 mg/kg/day s.c.) or 24 h after induction (10 mg/kg/day p.o.) ate significantly more food than colitis control rats, and were consuming similar levels of feed to saline-instilled rats at day 8 (p < 0.05; Fig. 2). Rats pretreated with prednisolone (1 mg/kg/day s.c.) also ate significantly more food than colitis control rats after 8 days (p < 0.05; Fig. 2), however, rats posttreated with this drug had no improvement in food consumption (p > 0.05; Fig. 2). Rats pretreated with the TNF-{alpha} inhibitor, infliximab (3 mg/kg i.v.) also ate significantly more food than colitis control rats after 8 days, as did rats pretreated with a combination of the C5a antagonist (10 mg/kg/day p.o.) and prednisolone (1 mg/kg/day s.c.) (p < 0.05; Fig. 2).



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  		FIGURE 2. Food consumption at 8 days. Rats received a colonic injection of either TNBS or a saline or saline/ethanol solution for sham animals on day 0. The amount of food consumed in a 24-h period was then measured at the 8-day time point. All drug-treated rats, except for prednisolone posttreated rats, ate significantly more food than drug-free colitis control rats. Data represent the mean ± SEM (n = 4–12). *, p < 0.05 drug-treated rats vs colitis control. C5aA = C5a antagonist; Pred = prednisolone.

 
Body weight

All rats receiving TNBS lost weight in the first days following induction compared with saline-instilled rats (data not shown). Over the 8-day study period, drug-free colitis control rats continued to lose weight with the mean weight loss at day 8 being -21 ± 8 g (n = 8; see Fig. 3). Rats treated with the C5a antagonist either before colitis induction (10 mg/kg/day p.o. and 0.3 mg/kg/day s.c.) or 24 h after induction (10 mg/kg/day p.o.) gained weight after 8 days with significantly higher weights compared with colitis control rats (p < 0.05; Fig. 3). In contrast, rats either pretreated or posttreated with prednisolone (1 mg/kg/day s.c.), or rats pretreated with a combination of the C5a antagonist (10 mg/kg/day p.o.) and prednisolone (1 mg/kg/day s.c.), lost weight over the 8 days, with no significant difference compared with colitis control rats on day 8 (p > 0.05; Fig. 3). Rats pretreated with infliximab (3 mg/kg i.v.) had only a small weight gain at day 8, however, these levels were not significantly higher than colitis control rats (p > 0.05; Fig. 3). The saline-instilled rats gained a substantial degree of weight during the experiment, (+75 g over 8 days). Because the mean weight loss in rats during the 24-h starvation period before TNBS instillation was -25.6 ± 1.4 g (n = 13), some of the weight gain over 8 days reflects this reduced baseline value.



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  		FIGURE 3. Body weight loss/gain after 8 days. Rats were intracolonically administered with either TNBS or a saline or saline/ethanol solution for sham animals on day 0. The change in body weight following TNBS administration was then measured after 8 days. Only rats treated with the C5a antagonist gained significant weight compared with drug-free colitis control rats. Data represent the mean ± SEM (n = 4–12). *, p < 0.05 drug-treated rats vs colitis control. C5aA = C5a antagonist; Pred = prednisolone.

 
Macroscopic score

The colons of the rats were examined macroscopically for signs of hemorrhage and ulceration by an independent observer, in a blinded fashion, using a previously established scoring system (30). After 8 days, colons from TNBS-administered rats displayed considerable damage. Ulcer formation was extensive, and adhesions and diarrhea were common. Colitis control rats had a mean macroscopic score of 11.5 ± 0.8 (n = 12; Fig. 4). Rats treated with the C5a antagonist either before induction (10 mg/kg/day p.o. and 0.3 mg/kg/day s.c.) or 24 h after induction (10 mg/kg/day p.o.) had significantly improved macroscopic scores compared with colitis control rats, as did prednisolone pretreated rats (1 mg/kg/day, s.c.) (p < 0.05; Fig. 4). Rats pretreated with infliximab (3 mg/kg i.v.) also had significantly decreased scores (p < 0.05; Fig. 4). Rats posttreated with prednisolone (1 mg/kg/day, s.c.) did not have any improvement in macroscopic scores (p > 0.05; Fig. 4).



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  		FIGURE 4. Clinical macroscopic scores. Rats were intracolonically administered with either TNBS or a saline or saline/ethanol solution for sham animals on day 0. The colons of the rats were then scored for macroscopic damage on a scale of 0–13 after 8 days. All drug treatments, except for prednisolone posttreatment, resulted in a significant improvement in macroscopic scores compared with drug-free colitis control rats. Saline-instilled animals displayed no appreciable colon macroscopic damage. Data represent the mean ± SEM (n = 4–12). *, p < 0.05 drug-treated rats vs colitis control. C5aA = C5a antagonist; Pred = prednisolone.

 
Colon MPO and edema

Levels of MPO in the colons of drug-free colitis control rats were substantially higher than in sham saline or sham ethanol rats (Fig. 5). In rats treated with the C5a antagonist either before induction (10 mg/kg/day p.o. and 0.3 mg/kg/day s.c.) or 24 h after induction (10 mg/kg/day p.o.), or in rats pretreated with a combination of the C5a antagonist (10 mg/kg/day p.o.) and prednisolone (1 mg/kg/day s.c.), the levels of colonic MPO were reduced to similar levels to those of the saline-instilled animals (p < 0.05; Fig. 5). Rats pretreated with either prednisolone (1 mg/kg/day s.c.) or infliximab (3 mg/kg i.v.) also had significantly reduced colonic MPO levels after 8 days, although the extent of improvement was not as great as in rats treated with the C5a antagonist (p < 0.05; Fig. 5). Rats posttreated with prednisolone (1 mg/kg/day s.c.) did not show any improvement in colonic MPO levels (p > 0.05; Fig. 5).



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  		FIGURE 5. Colon MPO levels. Rats were intracolonically administered with either TNBS or a saline or saline/ethanol solution for sham animals on day 0. Levels of MPO in the colons of the rats were then measured after 8 days. All drug treatments, except for prednisolone posttreatment, resulted in a significant improvement in colon MPO levels compared with drug-free colitis control rats. Data represent the mean ± SEM (n = 4–12). *, p < 0.05 drug-treated rats vs colitis control. C5aA = C5a antagonist; Pred = prednisolone.

 
Wet-to-dry weight ratios of colons from TNBS-administered rats were considerably increased compared with saline-instilled rats, indicating edema (Fig. 6). There were significant reductions in wet-to-dry weight ratios in rats treated with the C5a antagonist either before induction (10 mg/kg/day p.o. and 0.3 mg/kg/day s.c.) or 24 h after induction (10 mg/kg/day p.o.), as well as in rats pretreated with a combination of the C5a antagonist (10 mg/kg/day p.o.) and prednisolone (1 mg/kg/day s.c.) (p < 0.05; Fig. 6). Posttreatment with prednisolone (1 mg/kg/day s.c.) had no effect on reducing edema, while prednisolone (1 mg/kg/day s.c.) and infliximab (3 mg/kg i.v.) pretreated rats displayed no significant reductions compared with colitis control rats (p > 0.05; Fig. 6).



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  		FIGURE 6. Colon edema levels. Rats were intracolonically administered with either TNBS or a saline or saline/ethanol solution for sham animals on day 0. Colon edema, determined by wet-to-dry weight ratios, was then measured after 8 days. Only rats treated with either the C5a antagonist or a combination of the C5a antagonist and prednisolone had a significant improvement in colon wet-to-dry weight ratios compared with drug-free colitis control rats. Data represent the mean ± SEM (n = 4–12). *, p < 0.05 drug-treated rats vs colitis control. C5aA = C5a antagonist; Pred = prednisolone.

 
Serum and tissue TNF-{alpha} levels

Serum TNF-{alpha} levels in colitis control rats were increased compared with saline-instilled rats (Fig. 7A). All drug-treated rats had similar serum TNF-{alpha} levels to saline-instilled rats and these levels were significantly reduced compared with colitis control levels (p < 0.05; Fig. 7A). Colon tissue homogenate TNF-{alpha} levels were also increased in drug-free colitis control rats compared with saline-instilled animals (Fig. 7B). All drug-treated rats had significantly lower tissue levels of this cytokine compared with colitis control rats (p < 0.05; Fig. 7B), except for prednisolone posttreated rats (1 mg/kg/day s.c.), which had no significant reductions (p > 0.05; Fig. 7B).



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  		FIGURE 7. Serum and colonic TNF-{alpha} levels. Rats were intracolonically administered with either TNBS or a saline or saline/ethanol solution for sham animals on day 0. TNF-{alpha} levels were then measured after 8 days in either (A) serum or (B) colonic tissue. Serum levels of TNF-{alpha} were increased in drug-free colitis control animals, and all drug treatments significantly reduced these levels. All drug treatments, except for prednisolone posttreatment, significantly improved colonic TNF-{alpha} levels compared with drug-free colitis control rats. Data represent the mean ± SEM (n = 4–12). *, p < 0.05 drug-treated rats vs colitis control. C5aA = C5a antagonist; Pred = prednisolone.

 
Histopathology

Stained sections of ulcerated areas of the colons of the rats were examined for signs of pathology by an independent observer in a blinded fashion. At 8 days after TNBS challenge, there was evidence of healing with affected mucosa being replaced by fibrous tissue. There was polymorphonuclear neutrophil infiltration of affected tissues. Where discrete ulcers were identified, the bordering mucosa appeared normal although there was generalized submucosal inflammation consisting of inflammatory cells and mild edema. Colon sections from drug-free colitis control rats showed the most severe pathology after 8 days (Fig. 8B). In comparison, sections of colon from saline-instilled rats showed minimal signs of damage (Fig. 8A). Sections of colons from all rats that had been pretreated with either the C5a antagonist, prednisolone, infliximab, or a C5a antagonist/prednisolone combination, showed an overall improvement in pathology compared with colitis control rats. Sections of colons from rats that had been posttreated with the C5a antagonist (10 mg/kg/day p.o.) also showed similar improvements in disease pathology, indicated by less ulcer formation, edema, inflammatory cell infiltration, no perforations, and intact mucosal layers compared with sections from colitis control rats (Fig. 8C). In contrast, sections of colons from rats that had been posttreated with prednisolone (1 mg/kg/day s.c.) showed no improvement in disease pathology compared with colitis control rats (Fig. 8D).



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  		FIGURE 8. Photomicrographs of sections of colons from rats stained with H&E. Colon microscopic image of (A) saline-instilled, sham rat with intact epithelial and mucosal layer (ML); B, TNBS drug-free colitis control rat with extensive damage including inflammatory cell (IC) infiltration, edema (E), and the complete destruction of ML architecture (indicated by arrows); C, TNBS C5a antagonist (10 mg/kg/day, p.o.) posttreated rat with IC infiltration and E, but with intact ML; and D, TNBS prednisolone (1 mg/kg/day, s.c.) posttreated rat with IC infiltration; E, extensive hemorrhage (H) and erosion of ML (indicated by arrows). Images (x40 magnification) are typical and representative of each study group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Models of IBD typically involve the intracolonic administration of an inflammatory or haptenizing agent (29, 32). The TNBS-induced colitis model is a simple, reliable model that has been widely used to examine the efficacy of various drugs aimed at treating IBD (29). In this study, we found that 8 days after colitis induction, the colons of rats showed considerable damage. A substantial proportion of rats died over the study period, and elevated levels of TNF-{alpha} in serum and colonic tissue were observed in the surviving rats. This 8-day time frame also allowed the inclusion of postinduction drug treatments to determine whether drugs could be used to treat and perhaps reverse the developing disease. This study was conducted to compare the efficacy of a newly developed antagonist of the C5a receptor with the steroid prednisolone and the TNF-{alpha} inhibitor infliximab. All drugs were effective at reducing some, or all, of the parameters measured, however, the C5a antagonist was found to be superior to the other drugs.

Corticosteroids are commonly prescribed therapies in the treatment of IBD (33, 34). Steroids, such as prednisolone, are powerful drugs that act at the genetic level resulting in the down-regulation of various proinflammatory mediators such as cytokines as well as immune pathways (35). These drugs, with nonspecific actions, possess numerous side effects, and their chronic use must be used with caution in patients (36, 37). In our study, prednisolone pretreatment was effective at reducing the majority of parameters measured, consistent with previous findings (38, 39, 40), although treated rats lost considerable body weight attributed to catabolism, a major side effect with steroid therapy (41, 42). Although steroids are widely used in colitis therapy, it is of interest that rats treated with steroid 24 h after the induction of colitis showed no appreciable therapeutic effects. This may be explained by the delayed time it takes for steroids to become effective, which may have been too late for any appreciable activity of prednisolone on disease pathology. Alternatively, steroid treatments tend to inhibit natural healing processes associated with disease pathology (41, 43).

The proinflammatory cytokine TNF-{alpha} is thought to be a major contributor to the pathology of IBD (44, 45). Recent advances in drug development have produced various Abs that mask this cytokine (44). One of these Abs, infliximab, has been reported to be effective in reducing IBD pathology and is now currently available for the treatment of IBD in various countries (46, 47, 48). These Ab-based therapies must be given by parenteral routes in the clinic and are significantly more expensive than other drug therapies (48). The efficacy of infliximab in an animal model of IBD has not to our knowledge been previously published. Therefore, we decided to use this compound as a comparator drug for the C5a receptor antagonist. Pretreatment of rats with infliximab was found to reduce the severity of lesions, but not as effectively as prednisolone or the C5a antagonist.

The complement system involves a cascading series of proteins that act as early defense components of the immune system. The complement anaphylatoxins C3a and C5a are potent chemotactic agents that help to recruit and activate immune and inflammatory cells (49). In diseases where complement is abnormally activated, C3a and C5a induce the release of various immunoinflammatory mediators and cytokines that contribute to the disease process (11, 12, 13), and inhibitors of complement have significant protective effects on models of these diseases (50, 51). This indicates the central role played by these anaphylatoxins in inflammatory and immune pathways. In IBD, abnormal complement activity is thought to be important in disease pathology (3, 14), although there is limited evidence for this to date.

The C5a antagonist AcF-[OPdChaWR] was the most effective agent used in this study. We found that pretreatment of rats with the C5a antagonist either orally or s.c. markedly reduced all disease markers measured. Rats that were treated orally 24 h after colitis was established also had a significant reduction in all disease markers. The C5a antagonist used in our study has high affinity for the human, rat, and dog C5a receptor (52), and is highly selective for the C5a receptor over other closely related human receptors (21). It also does not bind to the C3a receptor (21), and has no effect on the formation of the membrane attack complex (27).

The greater efficacy of the C5a antagonist over infliximab may be explained by the central role of C5a in the inflammatory cascade. It is known that C5a induces the release of not only TNF-{alpha}, but also a host of other inflammatory cytokines and various mediators that are reportedly also involved in IBD (22, 23, 53). Because the C5a antagonist used in our study is also known to prevent formation of a number of these mediators (21, 22, 23), this likely explains its greater efficacy over infliximab, which solely inhibits TNF-{alpha}. The blockade of this central complement factor is proving to be an effective strategy for treating a wide range of inflammatory diseases in animal models including sepsis (24, 54), allergic dermatitis (25), arthritis (28), and reperfusion injuries (26, 27). We now show that an antagonist of human C5a receptors is also a potent and effective treatment for IBD in a rat model. When rats were pretreated with a combination of the C5a antagonist and prednisolone, no greater reduction in disease markers was observed than with the C5a antagonist alone, and the catabolic effects of prednisolone were not reduced by the C5a antagonist. This lack of increased therapeutic efficacy of the combination drug therapy perhaps suggests a common downstream regulation of inflammatory mediators.

In summary, this study demonstrates a significant role for the complement activation product C5a in the pathogenesis of TNBS-induced colitis in rats. Therefore, complement may have a pivotal role in the development of human IBD. These findings should encourage the development of anti-complement-based therapies for human IBD. The potent therapeutic effects of the orally active human C5a antagonist AcF-[OPdChaWR] support a possible role for the use of C5a antagonists in the treatment of IBD in humans.


   Acknowledgments
 
We thank Promics (Brisbane, Australia) for supplying the C5a antagonist.


   Footnotes
 
1 These studies were supported in part by a grant from the National Health and Medical Research Council of Australia. Back

2 Address correspondence and reprint requests to Dr. Stephen M. Taylor, Department of Physiology and Pharmacology, School of Biomedical Sciences, University of Queensland, Brisbane, QLD 4072, Australia. E-mail address: s.taylor{at}mailbox.uq.edu.au Back

3 Abbreviations used in this paper: IBD, inflammatory bowel disease; TNBS, trinitrobenzene sulfonic acid; MPO, myeloperoxidase; p.o., peroral. Back

Received for publication April 24, 2003. Accepted for publication September 9, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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