Free Radical Biology & Medicine, Vol. 35, No. 12, pp. 1679 –1687, 2003 Copyright © 2003 Elsevier Inc. Printed in the USA. All rights reserved 0891-5849/03/$–see front matter
doi:10.1016/j.freeradbiomed.2003.09.016
Original Contribution INCREASED DISEASE ACTIVITY IN eNOS-DEFICIENT MICE IN EXPERIMENTAL COLITIS M. SASAKI,* S. BHARWANI,† P. JORDAN,‡ J. W. ELROD,* M. B. GRISHAM,* T. H. JACKSON,§ D. J. LEFER,* and J. STEVEN ALEXANDER* *Department of Molecular and Cellular Physiology, †Department of Pathology, ‡Department of Gastroenterology, and §Department of Microbiology and Immunology, LSU Health Sciences Center, Shreveport, LA, USA (Received 12 June 2003; Revised 14 August 2003; Accepted 19 September 2003)
Abstract—Oral dextran sodium sulfate (DSS, 3%) produces experimental colitis with many features of human inflammatory bowel disease (IBD), (leukocyte extravasation, cachexia, and histopathology). Previous studies suggest that the inducible nitric oxide synthase (iNOS) in blood cells or in the endothelium contribute to this injury. However, until now no study has been performed to directly evaluate the role of endothelial nitric oxide synthase (eNOS) in IBD. We compared disease activity in wild-type (eNOS⫹/⫹) and eNOS-deficient (eNOS⫺/⫺) mice in the DSS model of colitis. Administration of DSS induced weight loss, stool blood, and overt histopathology in both mouse strains. Disease activity was dramatically increased in eNOS⫺/⫺ mice compared to wild types. Histologically, eNOS-deficient mice had greater leukocyte infiltration, gut injury, and expressed higher levels of the mucosal addressin, MAdCAM-1. These results demonstrate that eNOS plays an important role in limiting injury to the intestine during experimental colitis and altered eNOS content and/or activity may contribute to human IBD. © 2003 Elsevier Inc. Keywords—Nitric oxide, eNOS, Inflammatory bowel disease, Free radicals
INTRODUCTION
The release of endothelium-derived nitric oxide is thought to be an essential mechanism for limiting several important indices of inflammation. In the microvasculature, NO is released both by the constitutive (eNOS, NOS3) and inducible forms of nitric oxide synthase (NOS1), and is a potent scavenger for reactive oxygen species (ROS), which trigger inflammation [5,6]. Despite these reports, the role(s) of NO in inflammation still remains controversial [7–9]. Although many reports suggest that NO derived from iNOS increases the severity of experimental colitis [10 –12], Binion et al. report that human intestinal endothelial cells (HIMEC) cultured from individuals with Crohn’s disease lack the capacity to express iNOS, which renders them hyperadhesive toward leukocytes and susceptible to leukocyte-mediated injury [13,14]. In that study, this increased leukocyte adhesivity in Crohn’s disease endothelium was relieved by the administration of exogenous NO (using NO donors), consistent with a protection from NO. With respect to eNOS, many studies indicate that the NO derived from the constitutive endothelial NOS (eNOS) decreases leukocyte-endothelial adhesion [15]. However, when eNOS protein levels were examined,
Inflammatory bowel disease (IBD) includes Crohn’s disease and ulcerative colitis (UC), which share several characteristics including edema, loss of epithelial barrier, and gut leukocyte infiltration. These effects promote overall morbidity and weight loss in individuals afflicted with IBD [1–3]. The literature suggests that all forms of IBD represent cumulative immune, genetic, and environmental influences that initiate and sustain colitis [3,4]. Although the intestinal mucosa normally maintains a higher leukocyte population than most tissues, it is not normally inflamed, or edematous. During episodes of active colitis, the colon is even more extensively infiltrated by leukocytes that release oxidants and proteases leading to tissue injury. Consequently, it is assumed that the gut has several specialized mechanisms that normally limit leukocyte activation, and that during IBD these mechanisms have been impaired. Address correspondence to: Dr. J. Steven Alexander, Department of Molecular and Cellular Physiology, LSU Health Sciences Center, 1501 Kings Highway, Shreveport, LA, 71130-3932, USA; Tel: (318) 6754151; Fax: (503) 907-7543; E-Mail:
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there was no net change in endothelial NOS expression in colitis [16], and the role of eNOS in colitis has not been studied in depth. Therefore, although therapies that modify NO bioavailability may be beneficial in the treatment of IBD and perhaps other chronic inflammatory phenomena, the dual nature of NO seems to depend on several factors, including the cell source, target tissue, the rate of NO production, and the accompanying oxidant flux. In this article we examine differences in the development of experimental DSS colitis in wild-type and eNOS-deficient mice (eNOS⫺/⫺), and further examine the effects of statins on the development of disease in these two strains of animals.
Each score was determined as follows, change in weight (0: ⱕ 1%, 1: 1–5%, 2: 5–10%, 3: 10 –15%, 4: ⬎ 15%), stool blood (0: negative, 2: positive) or gross bleeding (4), and stool consistency (0: normal, 2: loose stools, 4: diarrhea) as previously described [23]. Histological analysis
The animals used in this study were C57BL/6 and B6.129P2-NOS3TMLUNC (eNOS⫺/⫺) mice, which were obtained from Jackson Laboratories (Bar Harbor, ME, USA). All mice were male, and were used at 8 –10 weeks of age (at the initiation of the trial), (wgt. ⫽ 21–24 g). Mice were kept in an environmental room at 24°C with a controlled 12/12 h light-dark cycle, with free access to a standard pellet diet and water. Mice were kept in cages containing up to 5 animals, and acclimated for at least 7 d before starting experiments.
Distal colon samples were fixed in Zamboni’s fixative (overnight) and embedded in JB-4 (Polysciences). Five micrometer thickness sections were stained with hematoxylin/eosin, and scored (blinded) by a GI pathologist. Histological damage was scored using the criteria described by Cooper et al. [23]. Crypt damage was scored on a 0 – 4 grade (grade 0: intact crypt, grade 1: loss of the basal one-third of the crypt, grade 2: loss of the twothirds of the crypt, grade 3: loss of entire crypt with the surface epithelium remaining intact, grade 4: loss of the entire crypt and surface epithelium [erosion]). These changes were quantified as the percent of area involved in the disease process: (i) 1 to 25%; (ii) 26 to 50%; (iii) 51 to 75%; (iv) 76 to 100%. Crypt damage score was calculated as the sum of the grade of the crypt and percent area score. The inflammation was evaluated subjectively on a 0 –3 grade, and the extent of involvement estimated as: (i) 0 to 25%; (ii) 26 to 50%; (iii) 51 to 75%; (iv) 76 to 100% of total surface area. The inflammation score was determined as the sum of the inflammation grade and the percent extent score.
Measurement of plasma NOx species
Expression of MAdCAM-1
Plasma NOx species were measured using the standard Griess reaction after treatment with Aspergillus nitrate reductase with 50 uM NADPH and 5 uM FAD (0.1 U/ml, Boehringer-Mannheim, Indianapolis, IN, USA) [17], and read colorimetrically using sodium nitrite standards as controls. Animals were fasted for 48 h before blood sampling to avoid over-contamination from gut flora-derived nitrite.
Distal colon samples were held in Zamboni’s fixative overnight at 4°C and embedded in Tissue-Tek O.C.T Compound (Sakura Finetek, Torrance, CA, USA) and frozen at ⫺20°C. Ten micrometer thickness sections were cut by cryostat. Nonspecific staining was blocked by incubating samples in normal donkey serum (10%, Sigma Chemicals, St. Louis, MO, USA) diluted in antibody diluent (Biogenex, San Ramon, CA, USA) for 30 min at 25°C. Sections were incubated in 1° antibody (rat anti-MAdCAM-1 [clone MECA 367, Pharmingen, San Diego, CA, USA]) (1 h, 25°C), washed in PBS (3⫻, 10 min), and incubated in 2° antibody (diluted 1:200) goat anti-rat conjugated to Cy3 (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). After tissues were incubated with 2° antibody, they were washed in PBS (3⫻, 10 min) and mounted in 10 l Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA) to minimize fluorescent photo-bleaching.
MATERIALS AND METHODS
Animals
Induction of colitis Dextran sulfate sodium (DSS) colitis was induced by mixing 3% (wt/vol) DSS (mol wt, 44 kilo Daltons; source: TdB Consultancy AB, Uppsala, Sweden) into the drinking water (distilled) provided ad libitum as described [18 –22]. Control groups received distilled water without DSS. Evaluation of clinical colitis In all animals, weight, stool blood, presence of gross blood, and daily stool consistency were determined daily as previously described. Disease activity index (DAI) was determined by combining scores of (i) weight loss, (ii) stool consistency, and (iii) bleeding (divided by 3).
Statistical analysis Results are expressed as mean ⫾ SE. Significant differences were assessed by one-way ANOVA plus
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knockout mice had significantly less plasma nitrite than controls, (eNOS knockout ⫾ vs. wild types) (Fig. 1). Severity of DSS-induced colonic injury
Fig. 1. Plasma NOx species in wild-type and eNOS⫺/⫺ mice. NOx (nitrite/nitrate) species in the plasma of wild-type control and eNOS⫺/⫺ was evaluated using the Griess reaction after treatment with nitrate reductase. eNOS⫺/⫺ mice exhibited ⬃ 50% lower levels of plasma NOx species compared to wild-type controls. *p ⬍ .05 wild-type mice vs. eNOS⫺/⫺ mice. Student’s t-test.
Fisher’s PLSD test. p Values ⱕ .05 were accepted as statistically significant. RESULTS
Levels of plasma NOx in eNOS knockout and wildtype mice were compared using the Griess assay. eNOS
The initial weight of the mice in this study was 22.5 ⫾ 0.4 gms (day 0); weight was monitored for 10 d. Histological parameters were evaluated in day 10 tissue samples. Clinically, a progressive loss of body weight was noted after the 6th day of 3.0% DSS administration. After induction of colitis, weight was significantly decreased in both the wild-type and eNOS knockout mouse groups treated with DSS. A decrease in weight of 11.6 ⫾ 2.2% and 18.6 ⫾ 1.1% in the wild-type DSS group and the eNOS⫺/⫺ DSS group was significant compared to the wild-type control group and eNOS⫺/⫺ control group, respectively. However, eNOS⫺/⫺ mice receiving DSS lost significantly more weight than wild-type mice receiving DSS (Fig. 2). Several other indices of colitis were seen to appear at different times after DSS. Occult blood was common after the 3rd day of DSS in both strains; diarrhea appeared at day 9, and at day 8 in DSS-treated wildtype mice and eNOS⫺/⫺ mice, respectively. Hematochezia was observed only in DSS-treated eNOS⫺/⫺ mice, and appeared on day 8. A progressive increase in the overall disease activity index (DAI) was observed in the DSS-treated mice. The increase of the DAI to 3.8 ⫾ 0.2 in the eNOS⫺/⫺ DSS group was markedly
Fig. 2. Body weight change of wild-type and eNOS⫺/⫺ mice in DSS model. Both wild-type control and eNOS⫺/⫺ control show no apparent change in body weight. Dextran sulfate significantly reduced body weight (*p ⬍ .05) in both wild-type and eNOS⫺/⫺ mice; this decrease in body weight was significantly greater in eNOS⫺/⫺ mice than wild-type mice. (*p ⬍ .05 vs. control respectively, #p ⬍ .05 wild-type mice with DSS vs. eNOS⫺/⫺ mice with DSS. One-way ANOVA plus Fisher’s PLSD test).
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Fig. 3. Disease activity index (DAI) wild-type and eNOS⫺/⫺ mice treated with DSS. The cumulative disease activity index (body weight, stool characteristic and fecal blood) was expressed on a scale of 0 – 4. Compared to control (untreated) mice, mice receiving DSS showed a significant increase in disease activity respectively (*p ⬍ .05), which was significantly exaggerated in eNOS⫺/⫺ mice (#p ⬍ .05). There are no apparent changes in DAI between wild-type and eNOS⫺/⫺ mice. One-way ANOVA plus Fisher’s PLSD test.
greater than that observed in the wild-type DSS group (2.8 ⫾ 0.3 point) measured on the10th day of DSS administration (Fig. 3). Histological analysis Figure 4A and B shows similar, normal colon histological architecture in both wild-type controls and in eNOS⫺/⫺ controls. After DSS, colon structure was characterized by severe disruption of tissue architecture, edema, and a massive mixed immune cell infiltrate (mononuclear cells, neutrophils, and eosinophils) with loss of crypts (Fig. 4C, D). In addition to these changes, ulcerations and large areas of complete epithelial denudation were observed in DSS-treated eNOS⫺/⫺ mice (Fig. 4D). The inflammation score (Fig. 5) and crypt damage score (Fig. 6) was significantly increased in DSS-treated mice. Inflammation was significantly increased by DSS in wild-type mouse (4.6 ⫾ 1.3, *p ⬍ .05 vs. wild-type control) and also significantly in eNOS⫺/⫺ mice (7.5 ⫾ 0.86, *p ⬍ .05 vs. eNOS⫺/⫺ control). Importantly, inflammation was significantly increased in eNOS⫺/⫺ mice compared to wild-type mouse (#p ⬍ .05) (Fig. 6). DSS also induced significant crypt damage in both wild-type and eNOS⫺/⫺ mice (*p ⬍ .05 vs. control, respectively, Fig. 6), which was also significantly exaggerated in eNOS⫺/⫺ mice (2.8 ⫾ 0.9 vs. 4.0 ⫾ 0) (*p ⬍ .05 vs. control, respectively, #p ⬍ .05 vs. DSS).
Immunohistochemistry localization of MAdCAM-1 A likely mechanism responsible for the increased colitis in eNOS⫺/⫺ mice could be the induction of the mucosal addressin cell adhesion molecule ‘MAdCAM-1’ on gut microvessels. MAdCAM-1 has been shown to be a key endothelial adhesion molecule that promotes infiltration of leukocytes into the colon in IBD. Consequently, we assessed MAdCAM-1 expression in the gut by immunofluorescent staining. Figure 7 shows the immunostaining for MAdCAM-1 expression in frozen sections of the distal colon sections (collected on day 10). DSS treated wild-type mouse colons (Fig. 7c) demonstrate some staining on vessels in the mucosa, whereas eNOS⫺/⫺ mice (Fig. 7d) demonstrate a dramatic increase in staining on relatively larger vessels in the gut that may be distended venules, or possibly lymphatics. By comparison, relatively low levels of MAdCAM-1 staining are observed in wild-type and in eNOS⫺/⫺ control mice (Fig. 7a and b).
DISCUSSION
Nitric oxide can both hinder and drive the progress of experimental [24] and human forms of colitis [13,14] and other types of chronic inflammation. NO modulates leukocyte-endothelial adhesion [25] through the ability of NO to restrict the cytokine-induced expression and pre-
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Fig. 4. eNOS⫺/⫺ mice show exaggerated colon histopathology in response to DSS. Control colon histology in wild-type mice (A) is altered by DSS (C) and causes mucosal thickening, leukocyte infiltration, crypt damage, and loss of goblet cells. Colon histology in eNOS⫺/⫺ knockout mice (C) is similar to that found in normal mice (A), but is much more severe when challenged with DSS (eNOS⫺/⫺/DSS) (D).
sentation of endothelial cell adhesion molecules, including MAdCAM-1 [26], VCAM-1, ICAM-1, E-selectin [27], and P-selectin [6,28], and also to regulate adhesive determinants on leukocytes like the 2 integrins [29 – 31]. The expression of all of these adhesion molecules is dramatically amplified in colitis (most likely in response to the elevated levels of Th1 cytokines released during active IBD and in models of colitis). In support of this, NO donors have been reported to reduce two events in inflammation, ECAM expression, and binding between leukocytes and the endothelium [24,32–37]. Conversely, pharmacological inhibition of NO seems to enhance leukocyte endothelial binding [25]. NO is produced from L-arginine by at least three forms of nitric oxide synthases (NOSs): the constitutive or endothelial NOS (eNOS, cNOS, NOS3), the neuronal NOS (NOS1 or nNOS) and the inducible NOS (NOS2 or iNOS). Multiple forms of NOS are present in the gut in health and disease, and consequently, nitric oxide’s role in IBD is still controversial. Of these, iNOS is capable of generating a high flux of NO, especially during inflammation, whereas eNOS only intermittently generates low levels of NO. Consequently, NO has been shown both to enhance and to reduce different manifestations of
chronic inflammation [6,13,14,24,38,39], and these divergent roles may reflect the NOS isoform generating the NO, the cell source of NO, the target tissue, and also the presence of reactive oxygen species [17]. We found that the eNOS knockout animals did in fact have lower levels of NO generation (⬃50%) in plasma, confirming that at least 50% of the NO found in the blood space seems to be derived from eNOS. Based on our data, in this experimental colitis, eNOS seems to protect against injury. Using the dextran sulfate-sodium (DSS) model of IBD, Yoshida et al. [12] showed that a general inhibition of NOS isoforms (with L-NAME) dramatically increased disease. However, it has been shown that the NO produced by any form of iNOS (either in formed blood cells or in tissues) does not protect, but dramatically worsens the course of disease [24]. In vivo, in Crohn’s colitis, Binion et al. have demonstrated that human intestinal microvascular endothelial cells of afflicted individuals may lack the capacity to express the inducible form of NO synthase, iNOS [13,14]. It was reasoned in that report that repeated rounds of injury during Crohns’ might persistently irritate the endothelium to yield a sustained “nondifferenti-
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Fig. 5. eNOS⫺/⫺ mice show an exaggerated inflammation score in response to DSS. Although eNOS⫺/⫺ mice control show no apparent change in their inflammation score compared with wild-type controls, eNOS deficiency significantly exaggerates the inflammation produced by DSS treatment compared with DSS-treated wild type. (*p ⬍ .05 vs. control, respectively, #p ⬍ .05 vs. DSS-treated wild-type mouse. One-way ANOVA plus Fisher’s PLSD test).
Fig. 6. eNOS⫺/⫺ mice exaggerate crypt damage score. Although eNOS⫺/⫺ mice control show no apparent change in their crypt damage score compared to wild-type controls, eNOS deficiency significantly exaggerates crypt damage produced by DSS treatment compared with DSS treated wild type. (*p ⬍ .05 vs. control, respectively, #p ⬍ .05 vs. DSS treated wild-type mouse. One-way ANOVA plus Fisher’s PLSD test).
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Fig. 7. eNOS⫺/⫺ mice markedly upregulate MAdCAM-1 in response to DSS. No obvious increase in MAdCAM-1 staining was seen in either control treated wild-type or eNOS⫺/⫺ mice (a and b, respectively). MAdCAM-1 staining was more common in wild-type DSS-treated colons, but intense MAdCAM-1 staining was universally observed in the mucosae of DSS treated eNOS⫺/⫺ mice (d) than in wild-type mouse (c).
ated” phenotype that lacks normal expression of iNOS, and thus exhibit increased adhesivity. However, Vento et al. have shown that in ulcerative colitis, there is an elevation in iNOS that closely correlates with disease severity [40]. Therefore, the roles played by NO in these two different forms of IBD may indicate very different routes of etiology. Although the role of iNOS in colitis and normal gut physiology is contentious, eNOS is for the most part thought to generally provide protection against inflammation [41] in animal models of inflammation and in human disease [42– 44]. In IBD, Vento et al. have reported that the relative distribution of eNOS may be altered in UC [40], whereas Djikstra et al. [16] did not find any apparent differences in eNOS between normal and either form of IBD (Crohn’s or UC). However, besides these, few if any reports have directly evaluated the role of eNOS in IBD. Although fairly selective inhibitors for iNOS and nNOS have been produced, and successfully exploited to study these NOS isoforms, no equivalent eNOS selective inhibitor has been developed. Consequently, the most straightforward approach to directly modulate eNOS function in this type of model should be to use eNOSgene-deficient (‘knockout’) mice. eNOS-deficient animals have higher basal levels of leukocyte binding, and also seem to be more susceptible to forms of injury
dependent on leukocyte extravasation and activation [25,29]. Both endothelial cells and animals that are either deficient in eNOS (through gene-‘knockouts’, or through senescence) show an exacerbation of leukocyte-endothelial binding [45,46]. Similarly, eNOS-deficient mice show increased injury during ischemia-reperfusion [46], suggesting that eNOS-derived NO plays a similar role in experimental colitis and in human disease [45,46]. In the setting of IBD, the expression of ECAMs like ICAM-1, VCAM-1, and MAdCAM-1, is observed in both experimental colitis models [26,32,37,47], and also in the human colon during Crohn’s disease and UC [48,49]. Although ICAM-1, VCAM-1, and E-selectin are upregulated in IBD [32], and clearly contribute to its etiology, MAdCAM-1 is thought to play a preeminent role in the development of chronic gut inflammation. MAdCAM-1, most likely with the other adhesion molecules, directs leukocytes, particularly ␣47 lymphocytes, to arrest within the gut, where they may amplify inflammatory responses. Under nonpathological conditions, the gut is the only organ to express significant levels of MAdCAM-1, and during inflammation the expression of MAdCAM-1 in the gut is even more dramatically increased [32,37]. The functional significance of this MAdCAM-1-directed, lymphocyte-dependent injury in IBD is well supported by several reports that demonstrate that immunoneutral-
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ization of either MAdCAM-1 or its lymphocyte ligand, the ␣47 integrin, significantly attenuate gut inflammation and intestinal mucosal damage in models of colitis [50 –53]. The entry of these lymphocytes into the gut may also support the subsequent entry of other leukocytes classes (e.g., granulocytes) mediating the severe injury seen in this model. We found that eNOS-deficient mice did not exhibit evidence for gut injury (i.e., no difference in disease activity, histology or MAdCAM-1 induction) without a prior DSS challenge. However, these animals show a marked increase in the severity of colon injury when fed DSS. eNOS-deficient animals had higher disease activity (DAI), weight loss, hematochezia, leukocyte infiltration into the colon, colon thickening, and edema in the submucosa, consistent with the loss of protection that might normally be maintained by eNOS-derived NO. Although levels of eNOS protein do not seem to be different between normal individuals and those with Crohn’s disease or UC [16], it is unclear whether the bioavailability of NO is altered in IBD, and whether net iNOS or eNOS activity is modified. The results of our study clearly show that eNOS plays a significant protective role at least against experimental colitis, and indicates that treatments like statins, which increase NO bioavailability [54], might boost eNOS capacity to reach a higher, protected level and provide relief in IBD. Based on the wide use and relative safety of statins, they might constitute a novel treatment for IBD. However, there are several potential adverse effects associated with statins, e.g., liver and kidney toxicity, myopathy, and death. Therefore, many additional studies will be needed to evaluate statin safety in IBD before they should be introduced into therapy. Acknowledgements — This study was supported in part by grants HL47615 and P02-DK43785 from the National Institutes of Health.
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