Effect of Novel A2A Adenosine Receptor Agonist ATL 313 on Clostridium difficile Toxin A-Induced Murine Ileal Enteritis
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感染与免疫杂志 2006年第5期
Faculty of Medicine, Federal University of Ceará, Ceará, Brazil
University of Virginia Adenosine Therapeutics, LLC, Charlottesville, Virginia
Faculty of Medicine of Ribeiro Preto, So Paulo University, So Paulo, Brazil
ABSTRACT
Clostridium difficile is a spore-forming, anaerobic, gram-positive bacillus that releases two main virulence factors: toxins A and B. Toxin A plays an important pathogenic role in antibiotic-induced diarrhea and pseudomembranous colitis, a condition characterized by intense mucosal inflammation and secretion. Agonist activity at A2A adenosine receptors attenuates inflammation and damage in many tissues. This study evaluated the effects of a new selective A2A adenosine receptor agonist (ATL 313) on toxin A-induced injury in murine ileal loops. ATL 313 (0.5 to 5 nM) and/or the A2A adenosine receptor antagonist (ZM241385; 5 nM) or phosphate-buffered saline (PBS) were injected into ileal loops immediately prior to challenge with toxin A (1 to 10 μg/loop) or PBS. Intestinal fluid volume/length and weight/length ratios were calculated 3 h later. Ileal tissues were collected for the measurement of myeloperoxidase, adenosine deaminase activity, tumor necrosis factor alpha (TNF-) production, histopathology, and detection of cell death by the TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) method. Toxin A significantly increased volume/length and weight/length ratios in a dose-dependent fashion. ATL 313 treatment significantly (P < 0.05) reduced toxin A-induced secretion and edema, prevented mucosal disruption, and neutrophil infiltration as measured by myeloperoxidase activity. ATL 313 also reduced the toxin A-induced TNF- production and adenosine deaminase activity and prevented toxin A-induced cell death. These protective effects of ATL 313 were reversed by ZM241385. In conclusion, the A2A adenosine receptor agonist, ATL 313, reduces tissue injury and inflammation in mice with toxin A-induced enteritis. The finding of increased ileal adenosine deaminase activity following the administration of toxin A is new and might contribute to the pathogenesis of the toxin A-induced enteritis by deaminating endogenous adenosine.
INTRODUCTION
Clostridium difficile is the most common cause of nosocomial bacterial diarrhea and accounts for 10 to 20% of the cases of antibiotic-associated diarrhea (1, 19). C. difficile infection can result in asymptomatic carriage, mild diarrhea, or fulminant pseudomembranous colitis (22). This anaerobic bacterium causes intestinal damage through the actions of two large exotoxins, toxin A and toxin B. A third toxin, designated CDT, with actin-specific ADP-ribosyltransferase activity was described from C. difficile strain CD196 in 1988 (35). Strains carrying CDT genes may be associated with the severity of C. difficile disease (31). Purified toxin A (TxA) causes intestinal secretion, destruction of the intestinal epithelium, and hemorrhagic colitis when introduced in vivo to the intestinal lumen (17, 24, 25, 27, 28, 40). The mechanism of TxA-induced enteritis involves toxin binding to enterocyte receptors, leading to activation of sensory and enteric nerves that results in enhanced intestinal secretion and motility, degranulation of mast cells, and infiltration of the mucosa by neutrophils (6, 18, 37). In addition to its proinflammatory and prosecretory activities, TxA induces apoptosis and nonapoptotic cell death in human and murine cells, which could contribute to intestinal mucosal disruption (4, 5, 7, 29, 43).
Adenosine is an endogenous purine nucleoside which, after its release from cells or after being formed from the breakdown of nucleotides, diffuses to the plasma membranes of surrounding cells, where it binds to specific cell surface receptors (12, 39). There are four types of G protein-coupled adenosine receptors (ARs) (12). The genes for these receptors have been cloned and are designated the A1, A2A, A2B, and A3 ARs. Although adenosine is constitutively present in the extracellular space at low concentrations, metabolic stress conditions dramatically increase its level (16).
The binding of adenosine to its receptors on the neutrophil surface may produce either proinflammatory or anti-inflammatory effects, depending on its concentration and the types of receptors stimulated. A1 AR engagement induces a proinflammatory response such as an increase in neutrophil adhesion, recruitment, and phagocytosis. On the other hand, the binding of adenosine to A2A ARs results in anti-inflammatory effects, including decreased neutrophil release of reactive oxygen species (10, 45). It is suggested that adenosine enhances the inflammatory response when present in low concentrations. However, at a site where there is significant tissue injury, adenosine is generated in high concentrations by damaged tissues or cells, acting as an inhibitor of neutrophil inflammatory functions (16). Therefore, the overall result of adenosine action is an anti-inflammatory effect, due to a dominant A2A response that exceeds the A1 response (10). Furthermore, studies indicate that the inhibition of the neutrophil oxidative burst is predominantly A2A mediated (45).
The aim of the present study was to determine the effect of an adenosine A2A receptor agonist on TxA-induced enteritis in mice, through the evaluation of secretion, mucosal disruption, inflammatory parameters, adenosine deaminase (ADA) activity, and cell death.
MATERIALS AND METHODS
Animals. For the present study, we used 174 male Swiss mice, 25 to 30 g in body weight, from the animal colony of the Federal University of Ceará. The animals received water and food ad libitum. All experimental protocols were approved by the local Animal Care and Use Committee.
Drugs and toxins. The following drugs were used: purified toxin A from C. difficile (strain 10463; molecular mass, 308 kDa), kindly provided through our collaboration with David Lyerly, Tech Lab, Blacksburg, VA; and 4-{3-[6-amino-9-(5-cyclopropylcarbamoyl-3,4-dihydroxytetrahydrofuran-2-yl)-9H-purin-2-yl]prop- 2-ynyl}piperidine-1-carboxylic acid methyl ester (ATL 313) (kindly provided by Adenosine Therapeutics, LLC). All of these substances were diluted in phosphate-buffered saline (PBS; pH 7.4). ZM241385 (38) was a gift from Simon Poucher, Astra-Zeneca Pharmaceuticals (Cheshire, United Kingdom).
Induction of intestinal inflammation. Mice were fasted overnight but allowed access to water and then anesthetized with ketamine and xylazine (60 and 5 mg/kg intramuscularly, respectively). Through a midline laparotomy, one 4-cm ileal loop was ligated and injected with either 0.1 ml of PBS (pH 7.4; control) or buffer containing TxA (1 to 10 μg) (8, 36). The abdomen was closed, and the animals were allowed to regain consciousness. Three hours after administration of TxA, mice were sacrificed and the intestinal loops were removed. The loop lengths, weights, and fluid volumes were recorded. A portion of the loop was frozen at –70°C for measurement of myeloperoxidase (MPO) and ADA activities, and tumor necrosis factor alpha (TNF-) concentration. The remaining tissue was fixed in 10% formalin and embedded in paraffin, and sections were stained with hematoxylin and eosin for histological grading of ileal inflammation (8, 36). Some mice were injected with ATL 313 (0.05, 0.05, or 5 nM final concentration) immediately followed by PBS or TxA (5 μg); another group was injected with ZM241385 (5 nM), the selective A2A AR antagonist, immediately followed by PBS or ATL 313 (5 nM) plus TxA (5 μg) in the ileal loop. In another experiment, TxA (5 μg) was injected into the ileal loop, the abdomen was closed and, 30 min later, the abdomen was reopened and ATL 313 (5 nM) was administered.
Histology. The severity of inflammation was scored in coded slides by a pathologist on a scale of 1 (mild) to 3 (severe) for epithelial damage, edema, and neutrophil infiltration as previously described (8, 36).
Cell death. Intestinal sections were also processed for TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) using ApopTag Plus Peroxidase In Situ Detection Kit (Serologicals Corp., Norcross, GA) for analysis of apoptosis or necrosis. Briefly, paraffin-embedded sections were hydrated and incubated with 20 μg of proteinase K (Sigma, New York)/ml for 15 min at room temperature. Endogenous peroxidase was blocked by treatment with 3% (wt/vol) hydrogen peroxide in PBS for 5 min at room temperature. After a washing step, sections were incubated in a humidified chamber at 37°C for 1 h with TdT buffer containing TdT enzyme and reaction buffer. Specimens were incubated for 10 min at room temperature with a stop/wash buffer and then incubated in a humidified chamber for 30 min with anti-digoxigenin-peroxidase conjugate at room temperature. After a series of washes in PBS, the slides were covered with peroxidase substrate to develop color and then wash in three changes of distilled H2O and counterstained in 0.5% (vol/vol) methyl green for 10 min at room temperature.
MPO assay. The extent of neutrophil accumulation in ileal tissue was estimated by measuring MPO activity assay as previously described (44). Briefly, 50 to 100 mg of ileal tissue was homogenized in 1 ml of hexadecyltrimethylammonium bromide (HTAB) buffer for each 50 mg of tissue. Then, the homogenate was centrifuged at 4,000 x g for 7 min at 4°C. MPO activity in the resuspended pellet was assayed by measuring the change in absorbance at 450 nm using o-dianisidine dihydrocloride and 1% hydrogen peroxide. The results were reported as MPO units/milligram of tissue. A unit of MPO activity was defined as that converting 1 μmol of hydrogen peroxide to water in 1 min at 22°C.
Quantification of TNF- by ELISA. Ileal tissue was harvested from animals in order to measure TNF- concentration by enzyme-linked immunosorbent assay (ELISA) as described previously (2). The results are expressed as pg of TNF-/ml.
ADA activity. Tissue samples and their contents were collected from PBS- and TxA-injected mice. The tissues were homogenized in 8 volumes of cold phosphate buffer (50 mmol/liter; pH 7.2). This preparation and the ileum contents were centrifuged at 10,000 x g in a refrigerated centrifuge at 5 to 8°C for 30 min. The sediments were discarded and supernatants assayed for ADA activity and protein content (26).
The ADA assay is based on the measurement of ammonia produced during the deamination of adenosine by the method of Giusti (14), with slight modifications. In brief, to a final volume of 220 μl, 200 μl of adenosine (21 mM) in phosphate buffer (50 mM; pH 7.2) were added to the sample (20 μl; supernatant of tissue homogenate or ileum secretion). A control tube (200 μl of adenosine 21 mM), a standard tube (200 μl of ammonium sulfate 75 μM in phosphate buffer) and a blank tube (200 μl of phosphate buffer) were also produced. One hour after incubation at 37°C the reactions were stopped with 600 μl of phenol-potassium nitroprussiate (106 mM-101.7 mM), and 20 μl of sample was added to control, standard, and blank tubes. After the addition of 600 μl of sodium hypochloride (11 mM) in 125 mM NaOH, all tubes were again incubated at 37°C for 30 min and read at 628 nm. The enzyme activity in the tissue and in the ileum contents was expressed as micromoles of ammonium formed/milligram of protein/hour and as micromoles of ammonium formed/hour, respectively.
Statistics. Results are reported as means ± the standard error of the mean (SEM) or as median values and range, where appropriate. Univariate analysis of variance (ANOVA), followed by Bonferroni's test was used to compare means, and the Kruskal-Wallis followed by Dunn's test was used to compare medians. A probability value of P < 0.05 was considered to indicate significant differences.
RESULTS
Effect of C. difficile TxA in murine ileal loops. To evaluate the inflammatory and secretory effects of TxA in murine ileal loops, we observed enteritis in response to graded amounts of TxA exposure (1, 2, 5, and 10 μg), measuring both loop weight and accumulation of fluid in the intestinal lumen as endpoints. There was a trend toward higher ileal weight/length and volume/length ratios at 1 to 2 μg of TxA that reached statistical significance (P < 0.05) at 5 and 10 μg compared to the PBS control (Fig. 1).
Effect of ATL 313 on murine ileal loops injected with C. difficile TxA. Treatment with the A2A AR agonist, ATL 313 (5 nM), significantly (P < 0.05) reduced the TxA (5 μg)-induced increase in weight/ileal loop length and secretion volume/ileal loop length ratios. ATL 313 (5 nM) alone did not alter weight/ileal loop length and secretion volume/ileal loop length ratios in the absence of TxA (Fig. 2).
At an equimolar concentration (5 nM), the A2A AR antagonist, ZM241385, significantly reversed the protective effect of ATL 313, restoring both weight/ileal loop length and secretion volume/ileal loop length ratios to levels comparable to TxA challenge alone (Fig. 2). ZM241385 alone did not increase weight/ileal loop length (ZM241385 = 19.9 ± 0.9 versus PBS = 34.5 ± 3.2 mg/cm) and secretion volume/ileal loop length (ZM241385 = 0.0 ± 0.0 versus PBS = 1.25 ± 1.25 μl/cm) ratios.
The treatment with ATL 313 30 min after injection of TxA also significantly (P < 0.05) reduced the TxA (5 μg)-induced increase in weight/ileal loop length ratio (TxA = 39.9 ± 3.5 versus ATL 313 = 28.3 ± 1.4 mg/cm).
Effect of ATL 313 on C. difficile TxA-induced histological alterations and cell death. Histological analysis demonstrated that TxA (5 μg/loop) induces intense mucosal disruption, hemorrhage, edema, and inflammatory cell infiltration, resulting in a median injury score of 3 and a range of 2 to 3 (Fig. 3C). TxA also caused a large amount of mucosal cell death in mouse ileal loops (indicated by the brown staining of the cells detected by the TUNEL method; Fig. 3D) compared to PBS that received median score 0 (0 to 0) (Fig. 3A and B). The group treated with ATL 313 was significantly (P < 0.05) protected from the disruptive effects of TxA receiving a median score of 1 (0 to 2) (Fig. 3E) and exhibited reduced number of cell death (Fig. 3F) similar to the level of PBS control. The A2A AR antagonist (ZM241385) blocked the protective effect of ATL 313, with damage (median score of 2.5 and a range of 2 to 3 similar to the group challenged with TxA alone (Fig. 3G and H). Of importance, neither ATL 313 nor ZM241385 alone induced any histological evidence of injury receiving a median score of 0 (0 to 0).
Treatment with ATL 313 30 min after injection of TxA also significantly (P < 0.05) reduced the TxA (5 μg)-induced histological evidence of injury, resulting in a score of 1 (0 to 2).
Effect of ATL 313 on C. difficile TxA-induced MPO activity. MPO is an enzyme present in the azurophil granules of neutrophils, and its presence in tissues has been used as an index of neutrophil infiltration (44). TxA (5 μg/loop) caused a statistically significant increase (P < 0.05) in MPO activity in ileal tissue compared to the loops from the control group, injected with only PBS (TxA = 12.6 ± 2.6 versus PBS = 1.6 ± 0.6 U/mg).
The group treated with the A2A AR agonist, ATL 313 (5 nM), and then challenged with TxA showed markedly reduced MPO activity (P < 0.05; ATL 313 + TxA = 1.0 ± 0.6 versus TxA = 12.6 ± 2.6 U/mg). The MPO activity in ileal tissue collected from animals injected with ATL 313 plus TxA was comparable to that observed in the PBS-treated control animals. ATL 313 (5 nM) alone did not increase MPO activity compared to PBS (ATL 313 = 2.1 ± 0.2 versus PBS = 1.6 ± 0.6 U/mg).
Effect of ATL 313 on C. difficile TxA-induced TNF- production. The injection of TxA (5 μg/loop) into mouse ligated ileal loops significantly increased TNF- production within the ileal tissue (P < 0.05) compared to the control group challenged only with PBS. Treatment with ATL 313 significantly reduced (P < 0.05) TNF- production in ileal tissue compared to loops injected with only TxA. The selective A2A AR antagonist, ZM241385, blocked ATL 313 action, promoting an increase in TNF- expression, which reached levels similar to those seen in the group injected with only TxA (Fig. 4).
Effect of ATL 313 on C. difficile TxA-induced ADA activity. The injection of TxA (5 μg/loop) into mouse ligated ileal loops significantly increased ADA activity within the ileal tissue (P < 0.05) and ileal secretion compared to the control group challenged only with PBS (Fig. 5).
Treatment with ATL 313, the A2A AR agonist, significantly reduced (P < 0.05) ADA activity in ileal tissue and ileal secretion in TxA-challenged animals (Fig. 5).
DISCUSSION
In the present study we demonstrated that ATL 313, a potent and selective agonist of adenosine A2A receptors, significantly reduces C. difficile TxA-induced mouse ileal secretion, edema, hemorrhage, inflammatory cell infiltration, TNF- production, mucosal disruption, and DNA fragmentation. Even when administered 30 min after the injection of TxA, ATL 313 reduced these alterations. The protective effects of ATL 313 on secretion, edema, mucosal disruption, TNF- production and DNA fragmentation were reversed by an A2A AR antagonist, ZM241385. Together, these data suggest that the actions of ATL 313 result from its specific interaction on A2A ARs and do not result from blockage of the binding of the toxin to its receptor. Furthermore, specific binding of ATL 313 to A2A ARs and functional effects on neutrophils, platelets, monocytes, and lymphocytes has been demonstrated previously (23). We also demonstrated that TxA substantially increases the tissue expression and secretion of ADA. This is interesting since, by deaminating endogenous adenosine, that action is likely to exacerbate the inflammatory actions of TxA.
Here, we also show that ATL 313 reduces TxA-induced mouse ileal MPO activity, suggesting a potent effect of ATL 313 to inhibit neutrophil infiltration. MPO is a microbicidal enzyme found predominantly in azurophilic neutrophil granules. However, there is evidence that MPO release can contribute to tissue damage during inflammation, and thus promote disease (20). MPO activity within tissues has been used to indicate neutrophil infiltration (44). The important role of neutrophils in the pathogenesis of colitis caused by infection with C. difficile and the abundant presence of neutrophils within TxA-induced pseudomembranes are well known (6, 27). Inhibition of neutrophil recruitment by antibodies to 2-integrins or P-selectin decreases intestinal damage in TxA-exposed animals (19, 21). Thus, it is reasonable to conclude that the protective effect of ATL 313 on TxA-induced mucosal inflammation and disruption is mediated, at least in part, by an inhibition of neutrophil recruitment. In accordance with this hypothesis, it has been reported that selective activation of A2A ARs is involved in the regulation of inflammatory responses and in the reduction of neutrophil infiltration and function (10, 15). In addition, ATL 313 does not reduce the cholera toxin- induced secretion in mouse ileal loop (data not shown), suggesting that ATL 313 acts by preventing the inflammatory response rather than affecting noninflammatory secretion. Glover et al., for instance, have observed that ATL146e, another selective A2A AR agonist, markedly reduces P-selectin expression and neutrophil infiltration in a dog cardiac reperfusion model (15). In addition, the neutrophil production of oxygen radicals is also inhibited by A2A AR agonists (10, 45), which could contribute to the reduction of tissue damage in TxA-induced enteritis seen here. TxA is a potent stimulus for release of proinflammatory cytokines (11, 41). Thus, another possible explanation for ATL 313 protective effects on TxA-induced enteritis is the already reported inhibitory effect of adenosine and A2A AR agonists on resident cell proinflammatory cytokine and/or chemokine production, such as TNF-, interleukin-8 (IL-8), and IL-6 (3, 33). Consistent with this hypothesis, we showed that ATL 313 inhibits TxA-induced increase in TNF- production, and this effect is reversed by simultaneous treatment with ZM241385. In addition, adenosine has been shown to induce release of IL-10, an anti-inflammatory cytokine, from human monocytes (32). This action could also contribute to the anti-inflammatory effects of ATL 313 seen in TxA-induced enteritis.
Our results indicate that TxA induces a marked increase in the activity of the adenosine catabolizing enzyme ADA in mouse ileal tissue. ADA catalyzes the irreversible deamination of adenosine and deoxyadenosine to inosine and deoxyinosine, respectively. This ubiquitous enzyme is present in all mammalian cells and plays a central role in the differentiation and maturation of the lymphoid system (9). It has been reported that ADA has an important role in acute immune-inflammatory reactions, and its serum level has been used as a biochemical marker for inflammation and disease (34, 42). Considering that adenosine has anti-inflammatory effects, it is reasonable to predict that the increase in ADA activity induced by TxA may enhance adenosine degradation, resulting in increased leukocyte recruitment and potentiation of the inflammatory response and tissue damage. In addition, we found that ATL 313 reduced ADA activity to the level of the PBS-challenged controls. This could occur because increased ADA activity is a consequence of inflammation. In agreement with our data, it has been shown that in pleurisy induced by carragenan, MPO and ADA levels peaked in parallel to neutrophil presence (13). Thus, it is possible that ATL 313 is decreasing ADA activity in the tissue by reducing neutrophil infiltration, which is consistent with the observed ATL 313-induced decreases in MPO activity in ileal tissue of TxA-challenged mice.
We also observed that the A2A AR agonist, ATL 313, reduces mucosal ileal cell death induced by TxA. It has been previously demonstrated the necrotic and apoptotic effect of TxA in human and murine cells (4, 5, 29, 30, 43). This TxA effect may contribute to the pathogenesis of C. difficile colitis. Thus, the finding that ATL 313 can prevent intestinal mucosal cell death induced by TxA may have critical implications on the prevention of mucosal disruption and inflammatory infiltration induced by TxA, although the mechanism involved in this antiapoptotic/necrotic effect of ATL 313 deserves further investigation.
In conclusion, the present study demonstrates for the first time an important effect of the A2A AR agonist, ATL 313, on toxin A-induced mucosal disruption, cell death, ADA activity, and inflammatory cell infiltration. In addition, our results also suggest a possible role for ADA in the pathogenesis of toxin A-induced inflammation.
ACKNOWLEDGMENTS
This study was supported by grants from Conselho Nacional de Pesquisa and Fundao Cearense de Apoio ao Desenvolvimento Científico e Tecnologico. This research was supported in part by the NIH Fogarty International Center Action for Building Capacity training grant #D43-TW01136 at the University of Virginia.
We gratefully acknowledge the technical assistance of Maria Silvandira Frana Pinheiro and Jose Ivan Rodrigues de Sousa.
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University of Virginia Adenosine Therapeutics, LLC, Charlottesville, Virginia
Faculty of Medicine of Ribeiro Preto, So Paulo University, So Paulo, Brazil
ABSTRACT
Clostridium difficile is a spore-forming, anaerobic, gram-positive bacillus that releases two main virulence factors: toxins A and B. Toxin A plays an important pathogenic role in antibiotic-induced diarrhea and pseudomembranous colitis, a condition characterized by intense mucosal inflammation and secretion. Agonist activity at A2A adenosine receptors attenuates inflammation and damage in many tissues. This study evaluated the effects of a new selective A2A adenosine receptor agonist (ATL 313) on toxin A-induced injury in murine ileal loops. ATL 313 (0.5 to 5 nM) and/or the A2A adenosine receptor antagonist (ZM241385; 5 nM) or phosphate-buffered saline (PBS) were injected into ileal loops immediately prior to challenge with toxin A (1 to 10 μg/loop) or PBS. Intestinal fluid volume/length and weight/length ratios were calculated 3 h later. Ileal tissues were collected for the measurement of myeloperoxidase, adenosine deaminase activity, tumor necrosis factor alpha (TNF-) production, histopathology, and detection of cell death by the TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) method. Toxin A significantly increased volume/length and weight/length ratios in a dose-dependent fashion. ATL 313 treatment significantly (P < 0.05) reduced toxin A-induced secretion and edema, prevented mucosal disruption, and neutrophil infiltration as measured by myeloperoxidase activity. ATL 313 also reduced the toxin A-induced TNF- production and adenosine deaminase activity and prevented toxin A-induced cell death. These protective effects of ATL 313 were reversed by ZM241385. In conclusion, the A2A adenosine receptor agonist, ATL 313, reduces tissue injury and inflammation in mice with toxin A-induced enteritis. The finding of increased ileal adenosine deaminase activity following the administration of toxin A is new and might contribute to the pathogenesis of the toxin A-induced enteritis by deaminating endogenous adenosine.
INTRODUCTION
Clostridium difficile is the most common cause of nosocomial bacterial diarrhea and accounts for 10 to 20% of the cases of antibiotic-associated diarrhea (1, 19). C. difficile infection can result in asymptomatic carriage, mild diarrhea, or fulminant pseudomembranous colitis (22). This anaerobic bacterium causes intestinal damage through the actions of two large exotoxins, toxin A and toxin B. A third toxin, designated CDT, with actin-specific ADP-ribosyltransferase activity was described from C. difficile strain CD196 in 1988 (35). Strains carrying CDT genes may be associated with the severity of C. difficile disease (31). Purified toxin A (TxA) causes intestinal secretion, destruction of the intestinal epithelium, and hemorrhagic colitis when introduced in vivo to the intestinal lumen (17, 24, 25, 27, 28, 40). The mechanism of TxA-induced enteritis involves toxin binding to enterocyte receptors, leading to activation of sensory and enteric nerves that results in enhanced intestinal secretion and motility, degranulation of mast cells, and infiltration of the mucosa by neutrophils (6, 18, 37). In addition to its proinflammatory and prosecretory activities, TxA induces apoptosis and nonapoptotic cell death in human and murine cells, which could contribute to intestinal mucosal disruption (4, 5, 7, 29, 43).
Adenosine is an endogenous purine nucleoside which, after its release from cells or after being formed from the breakdown of nucleotides, diffuses to the plasma membranes of surrounding cells, where it binds to specific cell surface receptors (12, 39). There are four types of G protein-coupled adenosine receptors (ARs) (12). The genes for these receptors have been cloned and are designated the A1, A2A, A2B, and A3 ARs. Although adenosine is constitutively present in the extracellular space at low concentrations, metabolic stress conditions dramatically increase its level (16).
The binding of adenosine to its receptors on the neutrophil surface may produce either proinflammatory or anti-inflammatory effects, depending on its concentration and the types of receptors stimulated. A1 AR engagement induces a proinflammatory response such as an increase in neutrophil adhesion, recruitment, and phagocytosis. On the other hand, the binding of adenosine to A2A ARs results in anti-inflammatory effects, including decreased neutrophil release of reactive oxygen species (10, 45). It is suggested that adenosine enhances the inflammatory response when present in low concentrations. However, at a site where there is significant tissue injury, adenosine is generated in high concentrations by damaged tissues or cells, acting as an inhibitor of neutrophil inflammatory functions (16). Therefore, the overall result of adenosine action is an anti-inflammatory effect, due to a dominant A2A response that exceeds the A1 response (10). Furthermore, studies indicate that the inhibition of the neutrophil oxidative burst is predominantly A2A mediated (45).
The aim of the present study was to determine the effect of an adenosine A2A receptor agonist on TxA-induced enteritis in mice, through the evaluation of secretion, mucosal disruption, inflammatory parameters, adenosine deaminase (ADA) activity, and cell death.
MATERIALS AND METHODS
Animals. For the present study, we used 174 male Swiss mice, 25 to 30 g in body weight, from the animal colony of the Federal University of Ceará. The animals received water and food ad libitum. All experimental protocols were approved by the local Animal Care and Use Committee.
Drugs and toxins. The following drugs were used: purified toxin A from C. difficile (strain 10463; molecular mass, 308 kDa), kindly provided through our collaboration with David Lyerly, Tech Lab, Blacksburg, VA; and 4-{3-[6-amino-9-(5-cyclopropylcarbamoyl-3,4-dihydroxytetrahydrofuran-2-yl)-9H-purin-2-yl]prop- 2-ynyl}piperidine-1-carboxylic acid methyl ester (ATL 313) (kindly provided by Adenosine Therapeutics, LLC). All of these substances were diluted in phosphate-buffered saline (PBS; pH 7.4). ZM241385 (38) was a gift from Simon Poucher, Astra-Zeneca Pharmaceuticals (Cheshire, United Kingdom).
Induction of intestinal inflammation. Mice were fasted overnight but allowed access to water and then anesthetized with ketamine and xylazine (60 and 5 mg/kg intramuscularly, respectively). Through a midline laparotomy, one 4-cm ileal loop was ligated and injected with either 0.1 ml of PBS (pH 7.4; control) or buffer containing TxA (1 to 10 μg) (8, 36). The abdomen was closed, and the animals were allowed to regain consciousness. Three hours after administration of TxA, mice were sacrificed and the intestinal loops were removed. The loop lengths, weights, and fluid volumes were recorded. A portion of the loop was frozen at –70°C for measurement of myeloperoxidase (MPO) and ADA activities, and tumor necrosis factor alpha (TNF-) concentration. The remaining tissue was fixed in 10% formalin and embedded in paraffin, and sections were stained with hematoxylin and eosin for histological grading of ileal inflammation (8, 36). Some mice were injected with ATL 313 (0.05, 0.05, or 5 nM final concentration) immediately followed by PBS or TxA (5 μg); another group was injected with ZM241385 (5 nM), the selective A2A AR antagonist, immediately followed by PBS or ATL 313 (5 nM) plus TxA (5 μg) in the ileal loop. In another experiment, TxA (5 μg) was injected into the ileal loop, the abdomen was closed and, 30 min later, the abdomen was reopened and ATL 313 (5 nM) was administered.
Histology. The severity of inflammation was scored in coded slides by a pathologist on a scale of 1 (mild) to 3 (severe) for epithelial damage, edema, and neutrophil infiltration as previously described (8, 36).
Cell death. Intestinal sections were also processed for TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) using ApopTag Plus Peroxidase In Situ Detection Kit (Serologicals Corp., Norcross, GA) for analysis of apoptosis or necrosis. Briefly, paraffin-embedded sections were hydrated and incubated with 20 μg of proteinase K (Sigma, New York)/ml for 15 min at room temperature. Endogenous peroxidase was blocked by treatment with 3% (wt/vol) hydrogen peroxide in PBS for 5 min at room temperature. After a washing step, sections were incubated in a humidified chamber at 37°C for 1 h with TdT buffer containing TdT enzyme and reaction buffer. Specimens were incubated for 10 min at room temperature with a stop/wash buffer and then incubated in a humidified chamber for 30 min with anti-digoxigenin-peroxidase conjugate at room temperature. After a series of washes in PBS, the slides were covered with peroxidase substrate to develop color and then wash in three changes of distilled H2O and counterstained in 0.5% (vol/vol) methyl green for 10 min at room temperature.
MPO assay. The extent of neutrophil accumulation in ileal tissue was estimated by measuring MPO activity assay as previously described (44). Briefly, 50 to 100 mg of ileal tissue was homogenized in 1 ml of hexadecyltrimethylammonium bromide (HTAB) buffer for each 50 mg of tissue. Then, the homogenate was centrifuged at 4,000 x g for 7 min at 4°C. MPO activity in the resuspended pellet was assayed by measuring the change in absorbance at 450 nm using o-dianisidine dihydrocloride and 1% hydrogen peroxide. The results were reported as MPO units/milligram of tissue. A unit of MPO activity was defined as that converting 1 μmol of hydrogen peroxide to water in 1 min at 22°C.
Quantification of TNF- by ELISA. Ileal tissue was harvested from animals in order to measure TNF- concentration by enzyme-linked immunosorbent assay (ELISA) as described previously (2). The results are expressed as pg of TNF-/ml.
ADA activity. Tissue samples and their contents were collected from PBS- and TxA-injected mice. The tissues were homogenized in 8 volumes of cold phosphate buffer (50 mmol/liter; pH 7.2). This preparation and the ileum contents were centrifuged at 10,000 x g in a refrigerated centrifuge at 5 to 8°C for 30 min. The sediments were discarded and supernatants assayed for ADA activity and protein content (26).
The ADA assay is based on the measurement of ammonia produced during the deamination of adenosine by the method of Giusti (14), with slight modifications. In brief, to a final volume of 220 μl, 200 μl of adenosine (21 mM) in phosphate buffer (50 mM; pH 7.2) were added to the sample (20 μl; supernatant of tissue homogenate or ileum secretion). A control tube (200 μl of adenosine 21 mM), a standard tube (200 μl of ammonium sulfate 75 μM in phosphate buffer) and a blank tube (200 μl of phosphate buffer) were also produced. One hour after incubation at 37°C the reactions were stopped with 600 μl of phenol-potassium nitroprussiate (106 mM-101.7 mM), and 20 μl of sample was added to control, standard, and blank tubes. After the addition of 600 μl of sodium hypochloride (11 mM) in 125 mM NaOH, all tubes were again incubated at 37°C for 30 min and read at 628 nm. The enzyme activity in the tissue and in the ileum contents was expressed as micromoles of ammonium formed/milligram of protein/hour and as micromoles of ammonium formed/hour, respectively.
Statistics. Results are reported as means ± the standard error of the mean (SEM) or as median values and range, where appropriate. Univariate analysis of variance (ANOVA), followed by Bonferroni's test was used to compare means, and the Kruskal-Wallis followed by Dunn's test was used to compare medians. A probability value of P < 0.05 was considered to indicate significant differences.
RESULTS
Effect of C. difficile TxA in murine ileal loops. To evaluate the inflammatory and secretory effects of TxA in murine ileal loops, we observed enteritis in response to graded amounts of TxA exposure (1, 2, 5, and 10 μg), measuring both loop weight and accumulation of fluid in the intestinal lumen as endpoints. There was a trend toward higher ileal weight/length and volume/length ratios at 1 to 2 μg of TxA that reached statistical significance (P < 0.05) at 5 and 10 μg compared to the PBS control (Fig. 1).
Effect of ATL 313 on murine ileal loops injected with C. difficile TxA. Treatment with the A2A AR agonist, ATL 313 (5 nM), significantly (P < 0.05) reduced the TxA (5 μg)-induced increase in weight/ileal loop length and secretion volume/ileal loop length ratios. ATL 313 (5 nM) alone did not alter weight/ileal loop length and secretion volume/ileal loop length ratios in the absence of TxA (Fig. 2).
At an equimolar concentration (5 nM), the A2A AR antagonist, ZM241385, significantly reversed the protective effect of ATL 313, restoring both weight/ileal loop length and secretion volume/ileal loop length ratios to levels comparable to TxA challenge alone (Fig. 2). ZM241385 alone did not increase weight/ileal loop length (ZM241385 = 19.9 ± 0.9 versus PBS = 34.5 ± 3.2 mg/cm) and secretion volume/ileal loop length (ZM241385 = 0.0 ± 0.0 versus PBS = 1.25 ± 1.25 μl/cm) ratios.
The treatment with ATL 313 30 min after injection of TxA also significantly (P < 0.05) reduced the TxA (5 μg)-induced increase in weight/ileal loop length ratio (TxA = 39.9 ± 3.5 versus ATL 313 = 28.3 ± 1.4 mg/cm).
Effect of ATL 313 on C. difficile TxA-induced histological alterations and cell death. Histological analysis demonstrated that TxA (5 μg/loop) induces intense mucosal disruption, hemorrhage, edema, and inflammatory cell infiltration, resulting in a median injury score of 3 and a range of 2 to 3 (Fig. 3C). TxA also caused a large amount of mucosal cell death in mouse ileal loops (indicated by the brown staining of the cells detected by the TUNEL method; Fig. 3D) compared to PBS that received median score 0 (0 to 0) (Fig. 3A and B). The group treated with ATL 313 was significantly (P < 0.05) protected from the disruptive effects of TxA receiving a median score of 1 (0 to 2) (Fig. 3E) and exhibited reduced number of cell death (Fig. 3F) similar to the level of PBS control. The A2A AR antagonist (ZM241385) blocked the protective effect of ATL 313, with damage (median score of 2.5 and a range of 2 to 3 similar to the group challenged with TxA alone (Fig. 3G and H). Of importance, neither ATL 313 nor ZM241385 alone induced any histological evidence of injury receiving a median score of 0 (0 to 0).
Treatment with ATL 313 30 min after injection of TxA also significantly (P < 0.05) reduced the TxA (5 μg)-induced histological evidence of injury, resulting in a score of 1 (0 to 2).
Effect of ATL 313 on C. difficile TxA-induced MPO activity. MPO is an enzyme present in the azurophil granules of neutrophils, and its presence in tissues has been used as an index of neutrophil infiltration (44). TxA (5 μg/loop) caused a statistically significant increase (P < 0.05) in MPO activity in ileal tissue compared to the loops from the control group, injected with only PBS (TxA = 12.6 ± 2.6 versus PBS = 1.6 ± 0.6 U/mg).
The group treated with the A2A AR agonist, ATL 313 (5 nM), and then challenged with TxA showed markedly reduced MPO activity (P < 0.05; ATL 313 + TxA = 1.0 ± 0.6 versus TxA = 12.6 ± 2.6 U/mg). The MPO activity in ileal tissue collected from animals injected with ATL 313 plus TxA was comparable to that observed in the PBS-treated control animals. ATL 313 (5 nM) alone did not increase MPO activity compared to PBS (ATL 313 = 2.1 ± 0.2 versus PBS = 1.6 ± 0.6 U/mg).
Effect of ATL 313 on C. difficile TxA-induced TNF- production. The injection of TxA (5 μg/loop) into mouse ligated ileal loops significantly increased TNF- production within the ileal tissue (P < 0.05) compared to the control group challenged only with PBS. Treatment with ATL 313 significantly reduced (P < 0.05) TNF- production in ileal tissue compared to loops injected with only TxA. The selective A2A AR antagonist, ZM241385, blocked ATL 313 action, promoting an increase in TNF- expression, which reached levels similar to those seen in the group injected with only TxA (Fig. 4).
Effect of ATL 313 on C. difficile TxA-induced ADA activity. The injection of TxA (5 μg/loop) into mouse ligated ileal loops significantly increased ADA activity within the ileal tissue (P < 0.05) and ileal secretion compared to the control group challenged only with PBS (Fig. 5).
Treatment with ATL 313, the A2A AR agonist, significantly reduced (P < 0.05) ADA activity in ileal tissue and ileal secretion in TxA-challenged animals (Fig. 5).
DISCUSSION
In the present study we demonstrated that ATL 313, a potent and selective agonist of adenosine A2A receptors, significantly reduces C. difficile TxA-induced mouse ileal secretion, edema, hemorrhage, inflammatory cell infiltration, TNF- production, mucosal disruption, and DNA fragmentation. Even when administered 30 min after the injection of TxA, ATL 313 reduced these alterations. The protective effects of ATL 313 on secretion, edema, mucosal disruption, TNF- production and DNA fragmentation were reversed by an A2A AR antagonist, ZM241385. Together, these data suggest that the actions of ATL 313 result from its specific interaction on A2A ARs and do not result from blockage of the binding of the toxin to its receptor. Furthermore, specific binding of ATL 313 to A2A ARs and functional effects on neutrophils, platelets, monocytes, and lymphocytes has been demonstrated previously (23). We also demonstrated that TxA substantially increases the tissue expression and secretion of ADA. This is interesting since, by deaminating endogenous adenosine, that action is likely to exacerbate the inflammatory actions of TxA.
Here, we also show that ATL 313 reduces TxA-induced mouse ileal MPO activity, suggesting a potent effect of ATL 313 to inhibit neutrophil infiltration. MPO is a microbicidal enzyme found predominantly in azurophilic neutrophil granules. However, there is evidence that MPO release can contribute to tissue damage during inflammation, and thus promote disease (20). MPO activity within tissues has been used to indicate neutrophil infiltration (44). The important role of neutrophils in the pathogenesis of colitis caused by infection with C. difficile and the abundant presence of neutrophils within TxA-induced pseudomembranes are well known (6, 27). Inhibition of neutrophil recruitment by antibodies to 2-integrins or P-selectin decreases intestinal damage in TxA-exposed animals (19, 21). Thus, it is reasonable to conclude that the protective effect of ATL 313 on TxA-induced mucosal inflammation and disruption is mediated, at least in part, by an inhibition of neutrophil recruitment. In accordance with this hypothesis, it has been reported that selective activation of A2A ARs is involved in the regulation of inflammatory responses and in the reduction of neutrophil infiltration and function (10, 15). In addition, ATL 313 does not reduce the cholera toxin- induced secretion in mouse ileal loop (data not shown), suggesting that ATL 313 acts by preventing the inflammatory response rather than affecting noninflammatory secretion. Glover et al., for instance, have observed that ATL146e, another selective A2A AR agonist, markedly reduces P-selectin expression and neutrophil infiltration in a dog cardiac reperfusion model (15). In addition, the neutrophil production of oxygen radicals is also inhibited by A2A AR agonists (10, 45), which could contribute to the reduction of tissue damage in TxA-induced enteritis seen here. TxA is a potent stimulus for release of proinflammatory cytokines (11, 41). Thus, another possible explanation for ATL 313 protective effects on TxA-induced enteritis is the already reported inhibitory effect of adenosine and A2A AR agonists on resident cell proinflammatory cytokine and/or chemokine production, such as TNF-, interleukin-8 (IL-8), and IL-6 (3, 33). Consistent with this hypothesis, we showed that ATL 313 inhibits TxA-induced increase in TNF- production, and this effect is reversed by simultaneous treatment with ZM241385. In addition, adenosine has been shown to induce release of IL-10, an anti-inflammatory cytokine, from human monocytes (32). This action could also contribute to the anti-inflammatory effects of ATL 313 seen in TxA-induced enteritis.
Our results indicate that TxA induces a marked increase in the activity of the adenosine catabolizing enzyme ADA in mouse ileal tissue. ADA catalyzes the irreversible deamination of adenosine and deoxyadenosine to inosine and deoxyinosine, respectively. This ubiquitous enzyme is present in all mammalian cells and plays a central role in the differentiation and maturation of the lymphoid system (9). It has been reported that ADA has an important role in acute immune-inflammatory reactions, and its serum level has been used as a biochemical marker for inflammation and disease (34, 42). Considering that adenosine has anti-inflammatory effects, it is reasonable to predict that the increase in ADA activity induced by TxA may enhance adenosine degradation, resulting in increased leukocyte recruitment and potentiation of the inflammatory response and tissue damage. In addition, we found that ATL 313 reduced ADA activity to the level of the PBS-challenged controls. This could occur because increased ADA activity is a consequence of inflammation. In agreement with our data, it has been shown that in pleurisy induced by carragenan, MPO and ADA levels peaked in parallel to neutrophil presence (13). Thus, it is possible that ATL 313 is decreasing ADA activity in the tissue by reducing neutrophil infiltration, which is consistent with the observed ATL 313-induced decreases in MPO activity in ileal tissue of TxA-challenged mice.
We also observed that the A2A AR agonist, ATL 313, reduces mucosal ileal cell death induced by TxA. It has been previously demonstrated the necrotic and apoptotic effect of TxA in human and murine cells (4, 5, 29, 30, 43). This TxA effect may contribute to the pathogenesis of C. difficile colitis. Thus, the finding that ATL 313 can prevent intestinal mucosal cell death induced by TxA may have critical implications on the prevention of mucosal disruption and inflammatory infiltration induced by TxA, although the mechanism involved in this antiapoptotic/necrotic effect of ATL 313 deserves further investigation.
In conclusion, the present study demonstrates for the first time an important effect of the A2A AR agonist, ATL 313, on toxin A-induced mucosal disruption, cell death, ADA activity, and inflammatory cell infiltration. In addition, our results also suggest a possible role for ADA in the pathogenesis of toxin A-induced inflammation.
ACKNOWLEDGMENTS
This study was supported by grants from Conselho Nacional de Pesquisa and Fundao Cearense de Apoio ao Desenvolvimento Científico e Tecnologico. This research was supported in part by the NIH Fogarty International Center Action for Building Capacity training grant #D43-TW01136 at the University of Virginia.
We gratefully acknowledge the technical assistance of Maria Silvandira Frana Pinheiro and Jose Ivan Rodrigues de Sousa.
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