| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
,
* Department of Medicine, Division of Gastroenterology,
Department of Pathology, and
Greenebaum Cancer Center, School of Medicine, University of Maryland, and
Veterans Affairs Maryland Health Care System, Baltimore, MD 21201;
¶ Laboratoire de Chimie et Biochimie Pharmacologiques, Unité Mixte de Recherche 8601, Centre National de la Recherche Scientifique, Université René Descartes, Paris, France; and
|| Department of Oncology, Sidney Kimmel Comprehensive Cancer Center, School of Medicine, Johns Hopkins University, Baltimore, MD 21231
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, IL-1, and TNF-
mRNA expression in colitis tissues that was markedly attenuated with L-Arg treatment or iNOS deletion. Treatment with the arginase inhibitor S-(2-boronoethyl)-L-cysteine worsened colitis in both wild-type and iNOS–/– mice. Polyamine levels were increased in colitis tissues, and were further increased by L-Arg. In addition, in vivo inhibition of ODC with -difluoromethylornithine also exacerbated the colitis. Taken together, these data indicate that arginase is protective in C. rodentium colitis by enhancing the generation of polyamines in addition to competitive inhibition of iNOS. Modulation of the balance of iNOS and arginase, and of the arginase-ODC metabolic pathway may represent a new strategy for regulating intestinal inflammation. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, and IL-10 (2), and a proinflammatory effect of IL-12 (3). However, in the case of NO, produced by the enzyme-inducible NO synthase (iNOS),4 there have been inconsistent results concerning its role in colitis. For example, iNOS inhibition has been reported to either reduce (4) or increase (5) the severity of trinitrobenzene sulfonic acid (TNBS) colitis, while iNOS deletion has been shown to exacerbate acetic acid (6) or TNBS (7) colitis, but to improve dextran sulfate sodium (DSS) colitis (8, 9). Evidence is accumulating that the activity of the enzyme arginase, which competes with iNOS for the common substrate, L-arginine (L-Arg), has important biologic effects on host innate immune response (10, 11, 12) and epithelial cell functions (13, 14). The cytoplasmic type I arginase (15) and the mitochondrial type II arginase (16) catalyze the same conversion of L-Arg into ornithine, the latter being converted to polyamines by ornithine decarboxylase (ODC). Thus, arginase is an inhibitor of NO synthesis and can also modulate polyamine synthesis (17). Through these functions, arginase may have important biological effects in host mucosal immune response, which have been demonstrated in vitro (11, 18). However, the role of arginase has not been studied in the gastrointestinal mucosa in vivo. We hypothesized that arginase is an unrecognized regulator of mucosal inflammation and host-pathogen interactions in the gut.
Citrobacter rodentium is the rodent equivalent of enteropathogenic Escherichia coli (EPEC), which causes diarrhea in humans. C. rodentium colonizes the surface of colonic epithelial cells, resulting in signal transduction events, cytoskeletal rearrangements, formation of attaching and effacing lesions, epithelial hyperplasia, and a strong mucosal Th1 response (19, 20). Murine infection provides an ideal model, with reproducible histologic changes similar to human inflammatory bowel disease. The aim of this study was to investigate the roles of iNOS, arginase, and ODC in C. rodentium colitis. There was marked up-regulation of iNOS, arginase I, and ODC in the colon of infected mice. Both wild-type (WT) and iNOS–/– mice supplemented with L-Arg had improvement in colitis, while disease was worsened when mice were treated with S-(2-boronoethyl)-L-cysteine (BEC), a specific arginase inhibitor (21), or -difluoromethylornithine (DFMO), an ODC inhibitor. These findings suggest a protective role for arginase in the colon through the formation of polyamines.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
A WT strain of C. rodentium (DBS100), provided by D. Schauer (Massachusetts Institute of Technology, Cambridge, MA), was used. Before infection, C. rodentium were grown in Luria broth overnight, washed, and resuspended in PBS, and bacteria concentration was determined by OD and confirmed by serial dilution and culture (18).
Mice and experimental design
All studies were approved by the University of Maryland (Baltimore, MD) Institutional Animal Care and Use Committee. WT and iNOS–/– C57BL/6 mice were obtained from The Jackson Laboratory (Bar Harbor, ME), and bred in our facility. Eight-week-old mice were gavaged with C. rodentium in 100 µl of PBS or PBS vehicle alone. Initial studies determined that an inoculum of 5 x 108 bacteria/mouse was required for consistent induction of colitis. In some studies, mice were treated with 1% L-Arg (Sigma-Aldrich, St. Louis, MO), starting day 1 postinfection; 0.1% BEC (provided by J.-L.B.), starting day 3; or 2.5% DFMO (Ilex Oncology, San Antonio, TX), starting day 3, all at pH 7 in the drinking water. Daily weights were obtained for each mouse. Water consumption was measured and found to be similar between all experimental groups. Mice were sacrificed after 14 days or when moribund. Blood samples were obtained by intracardiac puncture, and colons were collected, weighed, and divided. Tissues snap frozen in liquid nitrogen were used for RNA studies, Western blotting, enzyme activity assays, and polyamine measurements; tissues fixed in 10% buffered formalin were used for histology and immunohistochemistry; fresh tissues were homogenized in PBS; and colonization levels were determined by serial dilution and culture.
Histology
Sections (6 µm) were cut from paraffin sections and stained with H&E. Tissues were examined in a blinded manner by a pathologist (C.B.D.). Acute (neutrophilic) and chronic (lymphocytic) inflammation, and epithelial regenerative changes were each scored on a 0–4 scale, and the sum was used as an index of histologic injury.
Immunohistochemistry
Immunohistochemistry was performed, as described (22, 23). Polyclonal Ab to bovine liver arginase I (1/200; Research Diagnostics, Flanders, NJ) or to murine iNOS (1/400; BD Transduction Laboratories, Lexington, KY) were used for 1.5 h at room temperature. Sections were washed and incubated with goat biotinylated anti-rabbit secondary Ab (1/200; Vector Laboratories, Burlingame, CA) for 15 min. The Vectastain Elite ABC kit (Vector Laboratories) was used as a peroxidase system. Color was developed using 3,3'-diaminobenzidine, and hematoxylin was used as a counterstain.
Western blotting
Frozen colon samples were homogenized in lysis buffer containing protease inhibitors (11). Protein concentrations of 14,000 x g supernatants were measured (DC Protein Assay kit; Bio-Rad, Hercules, CA), and 100 µg/lane was separated by SDS-PAGE and transferred onto Immobilon-P membranes (Millipore, Bedford, MA) by electroblotting. Membranes were blocked overnight at room temperature with PBS containing 0.1% Tween and 5% nonfat dry milk. Polyclonal Ab to arginase I (1/1000) or arginase II (1/1000; provided by R. Iyer and S. Cederbaum, University of California, Los Angeles, CA (11)) were used for 1 h at room temperature. Blots were washed and incubated with donkey anti-rabbit Ab conjugated to HRP (1/2000; Amersham Biosciences, Piscataway, NJ). Chemiluminescence was detected with SuperSignal West Pico chemiluminescent substrate (Pierce, Rockford, IL) and exposure to Hyperfilm ECL (Amersham Biosciences).
Determination of arginase activity
Protein supernatants were incubated with 0.1 µM L-[guanido-14C]Arg (NEN, Boston, MA) for 24 h at 37°C, and arginase activity assay was performed, as reported (10).
Determination of ODC activity and polyamine levels
Frozen tissues were homogenized in a solution of 25 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, and 2.5 mM DTT. Supernatants of 14,000 x g centrifugations were incubated at 37°C with 10 nmol of L-[1-14C]ornithine (NEN) for 24 h, and ODC activity was assayed, as reported (11). Bacterial ODC activity was measured after sonicating C. rodentium at log phase of growth. Polyamine concentrations were measured in tissues by HPLC, as described (24), and standardized to protein concentration. Data shown are the total concentration of the biogenic polyamines putrescine, spermidine, and spermine.
mRNA analysis
Colon samples were homogenized in 1 ml of TRIzol reagent (Invitrogen Life Technologies, Grand Island, NY), and total RNA was isolated. Reverse transcription, PCR cycle conditions, and primer sequences for iNOS, arginase I, arginase II, ODC, and -actin were as described (11). PCR products were run on 2% agarose gels with 0.4 µg/ml ethidium bromide. Bands were visualized under UV light and photographed with a digital gel documentation system (EDAS 290; Kodak Digital Science, Rochester, NY). Real-time PCR was performed with a Bio-Rad iCycler iQ detection system for iNOS, arginase I, arginase II, and ODC, and with an MJ Research DNA Engine Opticon 2 (Waltham, MA) for IFN-, TNF-, and IL-1. Fluorescein dye and SYBR green (Molecular Probes, Eugene, OR) were included in the PCR mix. For ODC, 3 pmol, and for iNOS, arginase I, arginase II, IFN-, TNF-, IL-1, and -actin, 7 pmol each of sense and antisense primers was used in each reaction. Sense and antisense primer sequences for the cytokines were: IFN-, 5'-GCCACGGCACAGTCATTGAA-3' and 5'-CGCCTTGCTGTTGCTGAAGA-3'; TNF-, 5'-CTGTGAAGGGAATGGGTGTT-3' and 5'-GGTCACTGTCCCAGCATCTT3'; and IL-1, 5'-GCTGAAGGAGTTGCCAGAAA-3' and 5'-GTGCAAGTGACTCAGGGTGA-3'. One PCR cycle consisted of the following: 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s. After standardization to the housekeeping gene -actin, relative expression of each gene compared with that in control animals was calculated according to the manufacturer’s instructions.
Serum analysis
Serum was deproteinized with an equal volume of 6% 5-sulfosalicylic acid. Concentrations of L-Arg and total reactive nitrogen metabolites were determined in the serum, by HPLC analysis and chemiluminescence with an NO analyzer (NOA 280; Sievers, Boulder, CO), respectively, as previously described (18).
Statistical analysis
Data are expressed as the mean ± SEM. When comparisons between multiple groups were made, the Student-Newman-Keuls test was used, and for comparisons between two groups, Student’s t test was used. For survival data, 
2 or Fisher’s exact test was used as appropriate at each time point, and Cox hazard regression analysis was used to determine the effect of L-Arg throughout. SAS (SAS Institute, Cary, NC) was used for the Cox regression, and Statview v. 5.01 for the Macintosh (SAS Institute) was used for all other analyses.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
As shown in Fig. 1A, expression of both arginase I and arginase II was very low in control tissues. Arginase I was increased in the colon of C. rodentium-infected WT mice, while arginase II mRNA was not induced. By real-time PCR, arginase I mRNA levels of infected mice were increased by 9.7 ± 2.5-fold compared with uninfected mice (n = 6 for each; p < 0.01), while there was no significant change in arginase II mRNA levels from the low control levels (data not shown). In addition, iNOS mRNA levels were also found by RT-PCR (Fig. 1A) and by real-time PCR (data not shown) to be markedly increased in the colon of mice infected with C. rodentium, and absent in the colon of uninfected mice. As shown in Fig. 1B, arginase I protein was present in the tissues of infected mice and absent from control, while arginase II levels were very low in both control and infected mice.
|
Focally intense staining for arginase I in colonic tissues of infected mice is shown in Fig. 2, B, C, and H. Staining was present in epithelial cells (Fig. 2, B and C), but was also found throughout the mucosa in severe colitis (Fig. 2H), with staining of infiltrating inflammatory cells. In serial sections from the same tissues, iNOS localized to the epithelium, and the lamina propria and submucosal inflammatory cells (Fig. 2, E, F, and I). Staining was absent in uninfected tissues with Ab to arginase I (Fig. 2A) or iNOS (Fig. 2D) or in infected tissues incubated with an isotype Ig control (Fig. 2G). iNOS staining was completely absent in tissues from iNOS–/– mice (data not shown).
|
In the colon of infected animals, arginase activity was increased by 
2.4-fold vs control mice (Fig. 3A). A significant increase in NO concentration was observed in the serum of infected mice compared with control mice (Fig. 3B). A concomitant marked decrease of L-Arg concentration was observed in the serum of infected mice (Fig. 3C).
|
Because arginase I and iNOS were both abundantly expressed in the colon of C. rodentium-infected mice, and L-Arg was completely metabolized, we investigated the effect of L-Arg supplementation. In WT mice, C. rodentium infection was associated with a high level of mortality that began on day 9 postinfection (Fig. 4A). When WT animals were treated with L-Arg, mortality was inhibited by 42 and 62% compared with mice receiving water alone, after 12 and 14 days of infection, respectively (Fig. 4A). To further assess the impact of L-Arg on survival, Cox regression analysis was performed. WT mice treated with L-Arg had only a 31% hazard of death compared with mice receiving water alone (p < 0.0009). In iNOS–/– mice, no deaths were observed, with or without L-Arg (Fig. 4A).
|
Improvement in histopathology with iNOS deletion and additional beneficial effect of L-Arg supplementation
When compared with control mice (Fig. 5A), WT mice infected with C. rodentium (Fig. 5B) developed changes that included transmural inflammation, ulceration, mucosal bacterial aggregates, and epithelial hyperplasia with mucin depletion. There was reduction of colitis in iNOS-deficient mice (Fig. 5C), with less colonic thickening and inflammatory cell infiltration, and L-Arg further ameliorated the inflammation, epithelial reactivity, and mucin depletion (Fig. 5D). Because individual scores for acute inflammation, chronic inflammation, and epithelial reactivity had similar patterns, a composite score is shown in Fig. 5E. iNOS deletion and iNOS deletion + L-Arg inhibited the mean histologic injury score by 48 and 72%, respectively, compared with WT mice. L-Arg treatment of iNOS–/– mice resulted in a 46% improvement compared with water-treated iNOS–/– mice. It should be noted that the mean histologic injury score in the WT mice is likely to be underestimated, due to the early mortality that precluded obtaining histologic samples in some of the mice that would be expected to have had high injury scores. In uninfected mice, no differences were observed between the colons of WT and iNOS–/– mice, with or without L-Arg treatment (data not shown).
|
Consistent with the decrease in histopathology, there was a significant reduction in C. rodentium colonization levels in the iNOS–/– mice (WT mice, 8.3 ± 3.0 x 109 CFU/g colon, n = 12; iNOS–/– mice, 5.6 ± 3.3 x 106 CFU/g colon, n = 18; p < 0.01 vs WT). However, L-Arg supplementation did not alter colonization levels in WT mice (WT L-Arg, 3.2 ± 1.7 x 1010 CFU/g colon, n = 12), and actually increased levels in iNOS–/– mice compared with mutant mice receiving water alone (iNOS–/– L-Arg, 2.8 ± 1.8 x 108 CFU/g colon, n = 13; p < 0.05 vs iNOS–/–), indicating that the clinical and histologic improvement with L-Arg is not simply due to inhibition of bacterial growth by this amino acid.
Induction of proinflammatory cytokines in colitis tissues is attenuated by L-Arg treatment or iNOS deletion
Because C. rodentium colitis has been strongly associated with activation of the Th1 cytokine IFN-, and the associated proinflammatory cytokines TNF- and IL-1 (19, 20), we sought to determine the relationship between the clinical and histologic effects that we observed with these immunologic parameters. Real-time PCR analysis demonstrated a marked increase in IFN- (Fig. 6A), TNF- (Fig. 6B), and IL-1 (Fig. 6C) in C. rodentium colitis tissues compared with normal tissues, and a significant attenuation of these increases with either iNOS deletion or L-Arg treatment.
|
Because ornithine, the product of arginase, is metabolized by ODC to form polyamines, we investigated ODC expression in the colon of C. rodentium-infected WT mice. By real-time PCR, we found a 2.5 ± 0.5-fold increase of mRNA level in infected mice (n = 13) compared with control mice (n = 4; data not shown). However, a 40-fold increase of ODC activity was measured in the colon of either infected WT or WT L-Arg mice (Fig. 7A). This increase was not likely to be due to ODC activity from C. rodentium itself, because we measured bacterial ODC activity, and determined that it represented no more than 1% of the total ODC activity in the tissue. There was a 1.8 ± 0.1-fold increase in colonic polyamines in C. rodentium-infected WT mice, and a significant, further increase of 2.7 ± 0.2-fold with L-Arg treatment (Fig. 7B), indicating that arginase activity was an important determinant of polyamine synthesis.
|
To further demonstrate the beneficial effect of arginase and polyamine formation, we conducted experiments with BEC and DFMO, inhibitors of arginase and ODC, respectively. In uninfected control mice, BEC or DFMO treatment had no effect (Table I). However, there was a significant loss of survival in C. rodentium-infected WT mice treated with BEC or with DFMO (Table I). In fact, the experiments needed to be terminated at 10 days after infection, because of the deaths and severe disease at this point. The colons of C. rodentium-BEC and C. rodentium-DFMO groups had a greater increase in weight and histologic injury than those of the C. rodentium-water group (Table I). When compared with the infected WT mice treated with water (Fig. 8A), the colons of both BEC (Fig. 8B)- and DFMO (Fig. 8C)-treated mice showed marked transmural inflammation and mucin depletion. The BEC-treated mice had substantial submucosal abscess formation, and the DFMO-treated mice exhibited mucosal and submucosal hemorrhage, both indicative of severe acute inflammation.
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The fact that L-Arg treatment resulted in improvement most likely derives from the systemic depletion of L-Arg that occurred in the C. rodentium-infected mice. We measured serum L-Arg rather than tissue levels, because L-Arg is in constant flux between the intracellular and extracellular space (25), making serum levels a more reliable means to measure L-Arg availability. As an example of this, in mice with targeted deletion of arginase II, the metabolic effect was measured in the serum in which significant accumulation of L-Arg was demonstrated in these mice (26).
It should be noted that there was a marked (40-fold) increase in ODC activity in C. rodentium colitis tissues, but only a 2-fold increase in polyamine levels. This is due in part to the fact that polyamines are rapidly acetylated, which results in their efflux out of cells and excretion (27). Additionally, polyamine synthesis is dependent on availability of L-ornithine substrate for ODC, which derives from L-Arg by the activity of arginase; our current data show that arginase activity is increased only 2-fold in the colitis tissues, so this can be rate limiting in polyamine synthesis. Another important point is that DFMO is an irreversible inhibitor of ODC (28), and ODC is absolutely required for polyamine synthesis. Therefore, at an effective dose of DFMO, supplementation of L-Arg or iNOS deletion would not be expected to reverse the effect of DFMO on polyamine synthesis or the exacerbation of colitis.
The potent inhibition of ODC by DFMO is also illustrated by the more severe exacerbation of colitis by DFMO than by BEC. It is likely that C. rodentium infection has a direct effect on ODC induction. Because we observed a 2.5-fold increase in ODC mRNA, there is activation of mRNA expression, but because of the 40-fold increase in enzyme activity, it is likely that there may be posttranslational effects on ODC activity as well. Direct activation of ODC by C. rodentium is also supported by our findings of an 87-fold increase in ODC activity in intestinal epithelial-6 cells stimulated in vitro with C. rodentium for 4 h (our unpublished data).
To our knowledge, this is also the first report of the activation of arginase I in the gastrointestinal tract under pathophysiologic conditions. We have reported that arginase II mRNA and protein levels were up-regulated in the stomach of Helicobacter pylori-infected mice and humans, but arginase I was not induced in these tissues (11). Murine macrophages and dendritic cells express arginase I when stimulated by Th2 cytokines (29), LPS (30), or cAMP (15). Even though C. rodentium infection is a Th1-driven disease, with no increase in Th2 cytokines (20) (our own results), arginase I expression was induced in this model. We have also found that in vitro, arginase I is induced in C. rodentium-stimulated murine macrophages (our unpublished data), consistent with expression in lamina propria cells in our immunohistochemistry data. It is possible that the selective expression of arginase I may be due to: 1) a specific activation by C. rodentium and/or its released factors; 2) a colon-specific expression; or 3) selective induction in an acute form of inflammation vs chronic inflammation. In studies in mouse DSS (31) and rat TNBS colitis tissues (our unpublished data), we also found that arginase I, and not arginase II, is up-regulated; therefore, the expression of arginase I in colitis is not specific to C. rodentium. Because it has been shown that arginase I is up-regulated in a mouse model of acute immune complex-induced inflammation of the nephron (32) and in macrophages from an acute wounded rat model (30), the expression of arginase I is not specific to the colon. It is also unlikely that arginase I occurs only in acute inflammation, because we have observed increased expression of arginase I in human inflammatory bowel disease tissues (discussed below), in which there is a chronic component to the disease and the histologic injury pattern.
It is likely that the main effect of L-Arg treatment is enhanced substrate availability for both arginase and iNOS. It should be noted that we demonstrated increased arginase activity in the colitis tissues, while showing increased iNOS activity by measuring serum NO production. There is abundant published evidence that iNOS enzymatic activity is increased in rodent models of colitis and in human inflammatory bowel disease and that this correlates with iNOS expression; iNOS activity is regulated in vitro and in vivo by enzyme abundance (see Ref. 33 for our review on this subject). We measured serum NO production to yield a more sensitive means to determine the relationship of L-Arg consumption to iNOS activity. The fact that the histologic scores of infected WT L-Arg-treated mice are not significantly different from those of water-treated mice could be due to an overproduction of NO, counteracting the beneficial effect of arginase metabolism and polyamine synthesis. Because NO can inhibit ODC by S-nitrosylation (34), this could interfere with colonic ODC activity.
Polyamines have been shown to have numerous biological functions that may be relevant to amelioration of colitis, including inhibition of monocyte activation and Th1 cytokine production (35), stimulation of epithelial restitution by enhancing cell migration (36) and proliferation (37), and regulation of apoptosis (38). It has been demonstrated that polyamine production is essential for the repair of rat duodenal mucosa after stress (39), and that ODC activity is increased in the acetic acid colitis model (40). Arginase activity can also result in increased collagen production through proline synthesis from L-ornithine by the enzyme ornithine aminotransferase (25). Collagen production and polyamine synthesis have both been proposed as major events leading to wound healing by favoring fibroblast replication (41).
We have addressed the potential effect of modulation of polyamine levels on immune response by measuring Th1 and proinflammatory cytokines and demonstrating that the large increases in IFN-, TNF-, and IL-1 in C. rodentium colitis are ameliorated by either L-Arg treatment or iNOS deletion. These findings may be attributable to enhanced substrate availability for polyamine synthesis in these groups; however, it should be recognized that these cytokines are markers of inflammation and our results may simply represent a correlation with the improvement of colitis. We have also found that there is induction of epithelial apoptosis in C. rodentium colitis tissues by TUNEL assay (data not shown) and a significant increase in apoptosis in vitro in intestinal epithelial-6 cells stimulated with C. rodentium (our unpublished data). The interaction of epithelial apoptosis, proliferation, and cell migration in response to C. rodentium with acute vs chronic infection is likely to be important and is a complex area that requires additional investigation.
Similar to our findings, iNOS expression was shown to be up-regulated in epithelial and lamina propria cells of mice infected with C. rodentium in a recent report (42). However, there was no reduction in bacterial colonization or of colonic weight or histologic injury in iNOS–/– mice in this study, although there was an improvement in survival that was not statistically significant (42). We attribute these differences from our results in the current study of significant improvement of each of the above parameters in iNOS–/– mice to several possibilities, including their use of a lower inoculum of C. rodentium, younger age of infected mice (3–4 wk vs 8 wk in our study), and specific pathogen-free housing vs conventional housing in our study. A second report showed no differences between WT and iNOS–/– mice (43), but in that study a different strain was used (E. coli attaching and effacing (eae)-deficient mutant complemented with eae from EPEC) and colonization studies revealed >2 log orders less C. rodentium/g of tissue than we recovered at day 14 in WT mice, indicating important differences in their model. Our data show a correlation of decreased colonization levels of iNOS–/– mice with decreased histologic injury, proinflammatory cytokine levels, and clinical course. However, there is a more complex relationship between colonization and indicators of disease, because L-Arg led to improvement of both WT and iNOS–/– mice without decreasing colonization. Our present results are strengthened by the fact that we have obtained similar preliminary results in the DSS model of colitis (31). L-Arg improved, while BEC worsened clinical and histologic features of colitis in both WT and iNOS–/– mice (31), indicating that the findings of a beneficial role of arginase are not limited to one model of colitis.
In human inflammatory bowel disease, an increase in ODC activity has been described in ulcerative colitis and Crohn’s disease in children (44), while a decrease in ODC activity has been reported in severe human colitis (45). Interestingly, a decrease in ODC activity, but an increase in mucosal spermidine concentration has been reported in patients with ulcerative colitis (46). We have found that expression of arginase I, arginase II, and ODC mRNA is up-regulated in both human ulcerative colitis and Crohn’s disease tissues (our unpublished data), providing relevance for the murine colitis studies presented in this work. Additionally, induction of arginase II by cAMP has been described in human colonic epithelial cells (47); we have observed that cAMP can also induce arginase I expression in colonic cell lines (our unpublished data).
In summary, our data define an important in vivo role for arginase as a regulator of mucosal inflammation. In addition to synthesis of polyamines in the colonic mucosa, alterations in luminal uptake, as well as polyamine metabolism and efflux may be important events in colitis. Chronic overproduction of polyamines could contribute to the risk for colorectal cancer associated with colitis due to the growth-promoting effects of polyamines and the oxidative stress that occurs with polyamine metabolism by polyamine oxidase. Nonetheless, enhancement of arginase and ODC activities may represent important new strategies for ameliorating inflammatory bowel disease.
|
Footnotes |
|---|
1 This work was supported by grants from the National Institutes of Health (DK 02469, DK56938, DK53620, and DK63626 to K.T.W., and CA51085 and CA98454 to R.A.C.), the Office of Medical Research, Department of Veterans of Affairs (to K.T.W.), and the Crohn’s and Colitis Foundation of America (to K.T.W.). 
2 Current address: Unité de Microbiologie, Institut National de la Recherche Agronomique de Clermont-Ferrand-Theix, 63122 Saint-Genès-Champanelle, France.
3 Address correspondence and reprint requests to Dr. Keith T. Wilson, Division of Gastroenterology, School of Medicine, University of Maryland, 22 South Greene Street, Room N3W62, Baltimore, MD 21201. E-mail address: kwilson{at}umaryland.edu
4 Abbreviations used in this paper: iNOS, inducible NO synthase; BEC, S-(2-boronoethyl)-L-cysteine; DFMO, -difluoromethylornithine; DSS, dextran sulfate sodium; L-Arg, L-arginase; ODC, ornithine decarboxylase; TNBS, trinitrobenzene sulfonic acid; WT, wild type; EPEC, enteropathogenic Escherichia coli.
Received for publication February 11, 2004. Accepted for publication May 21, 2004.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and IL-10 in the regulation of experimental colitis. J. Immunol. 168:900.-difluoromethylornithine: characterization of sequences at the inhibitor and coenzyme binding sites. J. Biol. Chem. 267:150.. J. Immunol. 168:1804.This article has been cited by other articles:
|
|
 |
|
  K. Miki, A. Kumar, R. Yang, M. E. Killeen, and R. L. Delude Extracellular activation of arginase-1 decreases enterocyte inducible nitric oxide synthase activity during systemic inflammation Am J Physiol Gastrointest Liver Physiol, October 1, 2009; 297(4): G840 - G848. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. R. Chaluvadi, R. D. Kinloch, B. A. Nyagode, T. A. Richardson, M. J. Raynor, M. Sherman, L. Antonovic, H. W. Strobel, D. L. Dillehay, and E. T. Morgan Regulation of Hepatic Cytochrome P450 Expression in Mice with Intestinal or Systemic Infections of Citrobacter rodentium Drug Metab. Dispos., February 1, 2009; 37(2): 366 - 374. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. W. Gerner and F. L. Meyskens Jr. Combination Chemoprevention for Colon Cancer Targeting Polyamine Synthesis and Inflammation Clin. Cancer Res., February 1, 2009; 15(3): 758 - 761. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Singh, R. Chaturvedi, M. Asim, D. P. Barry, N. D. Lewis, M. P. Vitek, and K. T. Wilson The Apolipoprotein E-mimetic Peptide COG112 Inhibits the Inflammatory Response to Citrobacter rodentium in Colonic Epithelial Cells by Preventing NF-{kappa}B Activation J. Biol. Chem., June 13, 2008; 283(24): 16752 - 16761. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Vareille, F. Rannou, N. Thelier, A.-L. Glasser, T. de Sablet, C. Martin, and A. P. Gobert Heme Oxygenase-1 Is a Critical Regulator of Nitric Oxide Production in Enterohemorrhagic Escherichia coli-Infected Human Enterocytes J. Immunol., April 15, 2008; 180(8): 5720 - 5726. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Chaturvedi, M. Asim, N. D. Lewis, H. M. S. Algood, T. L. Cover, P. Y. Kim, and K. T. Wilson L-Arginine Availability Regulates Inducible Nitric Oxide Synthase-Dependent Host Defense against Helicobacter pylori Infect. Immun., September 1, 2007; 75(9): 4305 - 4315. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Borenshtein, P. R. Nambiar, E. B. Groff, J. G. Fox, and D. B. Schauer Development of Fatal Colitis in FVB Mice Infected with Citrobacter rodentium Infect. Immun., July 1, 2007; 75(7): 3271 - 3281. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Vareille, T. de Sablet, T. Hindre, C. Martin, and A. P. Gobert Nitric oxide inhibits Shiga-toxin synthesis by enterohemorrhagic Escherichia coli PNAS, June 12, 2007; 104(24): 10199 - 10204. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Gao, X. Xu, S. Belmadani, Y. Park, Z. Tang, A. M. Feldman, W. M. Chilian, and C. Zhang TNF-{alpha} Contributes to Endothelial Dysfunction by Upregulating Arginase in Ischemia/Reperfusion Injury Arterioscler Thromb Vasc Biol, June 1, 2007; 27(6): 1269 - 1275. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Horowitz, D. G. Binion, V. M. Nelson, Y. Kanaa, P. Javadi, Z. Lazarova, C. Andrekopoulos, B. Kalyanaraman, M. F. Otterson, and P. Rafiee Increased arginase activity and endothelial dysfunction in human inflammatory bowel disease Am J Physiol Gastrointest Liver Physiol, May 1, 2007; 292(5): G1323 - G1336. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. I. Bussiere, R. Chaturvedi, M. Asim, K. L. Hoek, Y. Cheng, J. Gainor, A. Scholz, W. N. Khan, and K. T. Wilson Low Multiplicity of Infection of Helicobacter pylori Suppresses Apoptosis of B Lymphocytes. Cancer Res., July 1, 2006; 66(13): 6834 - 6842. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. van Erp, K. Dach, I. Koch, J. Heesemann, and R. Hoffmann Role of strain differences on host resistance and the transcriptional response of macrophages to infection with Yersinia enterocolitica Physiol Genomics, March 13, 2006; 25(1): 75 - 84. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. A. Richardson, M. Sherman, L. Antonovic, S. S. Kardar, H. W. Strobel, D. Kalman, and E. T. Morgan HEPATIC AND RENAL CYTOCHROME P450 GENE REGULATION DURING CITROBACTER RODENTIUM INFECTION IN WILD-TYPE AND TOLL-LIKE RECEPTOR 4 MUTANT MICE Drug Metab. Dispos., March 1, 2006; 34(3): 354 - 360. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. R. Martin and J. L. Wallace Gastrointestinal Inflammation: A Central Component of Mucosal Defense and Repair Experimental Biology and Medicine, February 1, 2006; 231(2): 130 - 137. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Cheng, R. Chaturvedi, M. Asim, F. I. Bussiere, H. Xu, R. A. Casero Jr., and K. T. Wilson Helicobacter pylori-induced Macrophage Apoptosis Requires Activation of Ornithine Decarboxylase by c-Myc J. Biol. Chem., June 10, 2005; 280(23): 22492 - 22496. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), n = 32 for L-Arg-treated WT-infected mice (
), n = 23 for infected iNOS–/– mice (
), and n = 20 for infected iNOS–/– mice treated with L-Arg (
). A–C, *, p < 0.05; ***, p < 0.001 vs day 0; 
