COX-1 and 2, Intestinal Integrity, and Pathogenesis ofNonsteroidal Anti-inflammatory Drug Enteropathy in Mice

GUDMUNDUR SIGTHORSSON,* ROBERT J. SIMPSON,* MATTHEW WALLEY,* ANDREW ANTHONY,‡

RUSSELL FOSTER,* CHRISTOPH HOTZ–BEHOFTSITZ,* ABBAS PALIZBAN,* JOAQUIM POMBO,*JO WATTS,* SCOTT G. MORHAM,§ and INGVAR BJARNASON**Department of Medicine, Guy’s, King’s, St. Thomas’ Medical School, London; ‡Department of Histopathology, Royal Free and UniversityCollege Medical School, Pond Street, London, England; and §Myriad Genetics Inc., Salt Lake City, Utah

Background & Aims: The pathogenesis of nonsteroidalanti-inflammatory drug–induced enteropathy is contro-versial, but it is thought that cyclooxygenase-1 (COX-1)inhibition is of pivotal importance. We compared smallintestinal function and morphology in untreated wild-type, COX-1– and COX-2–deficient mice and the effect ofindomethacin, selective COX-1 (SC-560), and COX-2(celecoxib) inhibition. Methods: Intestinal permeability(51CrEDTA), inflammation (fecal granulocyte marker pro-tein), prostaglandin E2 (PGE2) levels, and macroscopicand microscopic appearances were assessed at base-line and after the drugs. Results: COX-1�/� animals werenormal except for a 97% decrease in intestinal PGE2levels. COX-1�/� and COX-1�/� animals reacted in asimilar way to indomethacin. However, celecoxib, havingcaused no damage in COX-1�/� animals, caused smallbowel ulcers in COX-1�/� animals. Selective inhibition ofCOX-1 decreased intestinal PGE2 levels in COX-2�/� andCOX-2�/� animals by 95%–97%, but caused only smallbowel ulcers in the latter group. Dual inhibition of COX-1and COX-2 in wild-type animals resulted in similar smallbowel damage. Between 40% and 50% of untreatedCOX-2�/� animals had increased intestinal permeabilityand inflammation. Some had ileal ulcers that were dis-tinctively different from indomethacin-induced ulcers.Furthermore, long-term celecoxib administration in wild-type animals was associated with similar damage as inthe COX-2�/� mice. Conclusions: COX-1 deficiency orinhibition and short-term COX-2 inhibition are compati-ble with normal small intestinal integrity. Dual inhibitionof the COX enzymes leads to damage similar to thatseen with indomethacin. Long-term COX-2 deficiency orinhibition is associated with significant intestinal pathol-ogy despite normal intestinal PGE2 levels, suggesting arole for COX-2 in the maintenance of small intestinalintegrity in the mouse.

Aspirin and other nonsteroidal anti-inflammatorydrugs (NSAIDs) are widely used for their analgesic

and anti-inflammatory properties, for cardiovascular pro-phylaxis, and for their antineoplastic actions on colonic

polyps and carcinomas.1 The main problem with thesedrugs is the high prevalence and severity of gastrointes-tinal damage.2 NSAID gastropathy with its attendantcomplications accounts for significant morbidity andmortality.3 Furthermore, 50%–70% of patients on long-term NSAID therapy develop NSAID enteropathy. Thisenteropathy is not usually associated with life-threaten-ing events, but may lead to management problems asso-ciated with bleeding, protein loss, ulcers, and strictures.4

The pathogenesis of NSAID-induced gastrointestinaldamage is controversial.4–6 There has been a generalconsensus that inhibition of cyclooxygenase-1 (COX-1)is the pivotal event for the gastric damage.7,8 This ideawas partially endorsed by the clinical experience withselective inhibitors of COX-2, celecoxib and rofecoxib.These drugs have equal efficacy to conventional NSAIDsand greatly improve gastric tolerability, as assessed byshort-term9 or long-term10,11 endoscopy studies and asserious outcomes.12,13 However, it is not clear whetherthis enhanced tolerability is due solely to selectivity forthe COX-2 enzyme or to the absence of the “topical”effect, which plays a pathogenic role in NSAID-induceddamage in the stomach4–6,14–20 and the small bowel.21,22

Recent work has shown that COX-1–deficient (COX-1�/�) mice do not spontaneously develop gastric dam-age.23 This lack of damage is difficult to reconcile withprevailing theories on the central importance of COX-1inhibition in the pathogenesis of NSAID-induced gas-trointestinal damage.6,7,24 These apparently contradic-tory findings have suggested to some workers that theremust be some compensatory processes that counteract theeffect of mucosal prostaglandin deficiency in the COX-

Abbreviations used in this paper: COX-1, cyclooxygenase-1; ELISA,enzyme-linked immunosorbent assay; GMP, granulocyte markerprotein; NSAID, nonsteroidal anti-inflammatory drug; PGE2, prosta-glandin E2.

© 2002 by the American Gastroenterological Association0016-5085/02/$35.00

doi:10.1053/gast.2002.33647

GASTROENTEROLOGY 2002;122:1913–1923

1�/� mice, such as up-regulation of COX-2 or nitricoxide synthase.25,26 Importantly, Wallace et al27 showedthat short-term and selective COX-1 inhibition does notlead to gastric damage in the rat; rather, dual inhibitionof both the COX-1 and COX-2 enzymes, in the absenceof the “topical” effect, is needed to cause gastric damage.

The pathogenesis of gastric and small bowel damagefrom NSAIDs differs in many respects.4,6 In particular,small bowel damage is less dependent on COX-1 inhi-bition than is gastric damage.5 Our purpose was toestablish a mouse model of indomethacin-induced smallbowel damage and to characterize and compare smallintestinal morphology and function (i.e., intestinal pros-taglandin E2 [PGE2] levels, intestinal permeability, andinflammation) in wild-type, COX-1–, and COX-2–de-ficient mice. We assessed the effects of short-term selec-tive COX-1 and COX-2 inhibition on wild-type andCOX-1– and COX-2–deficient mice and the long-termeffects of COX-2 deficiency or inhibition.

Materials And MethodsAnimals and Drugs

The generation of COX-1�/� and COX-2�/� miceused in these experiments has been described in detail previ-ously.23,28 All of the animals were bred and maintained at ourfacility. Mice from wild-type/wild-type, heterozygous/het-erozygous, and homozygous (male)/heterozygous (female) mat-ings were used for the experiments after being genotyped bypolymerase chain reaction of tail DNA. The COX-1�/� andCOX-2�/� mice were bred on pure C57BL/B6 and mixed 129and C57BL/B6 mouse strain backgrounds, respectively. Themice were studied at 8–24 weeks of age unless otherwisestated, weighing 18–25 g. Wild-type animals were studied atthe same age and weight, and where comparisons betweenwild-type and deficient mice were carried out, animals werematched for age, sex, and genetic background. Preliminarystudies showed no gender differences in the parameters stud-ied. The U.K. Home Office and the local animal ethicalcommittee approved these studies.

The drugs used in the experiments were indomethacin, anacidic dual inhibitor of the COX enzymes used widely ingastrointestinal toxicity studies29; SC-560, a selective non-acidic COX-1 inhibitor30; and celecoxib, a selective nonacidicCOX-2 inhibitor.31 All of the drugs were initially dissolved in100% dimethylsulfoxide (DMSO) and then diluted so that thefinal concentration of DMSO was less than 10%. Controlexperiments used the same DMSO concentration.

Surgery (laparotomy) was performed under anesthesia withinhalation halothane (3.5%) at constant temperature unlessotherwise stated.

Intestinal Prostanoid Levels

Samples for PGE2 assessment were collected 3 hoursafter gastric gavage with vehicle or study drugs given at 8 AM.

Tissue sampling. The abdomen was opened by amidline incision under anesthesia. A small incision was madein the duodenum, and the gut was flushed with subzero 0.9%saline after the ileum was cut at the ileocecal valve. The wholeof the small intestine was then quickly removed from theanimal and placed on a plastic film sitting on ice. Between 2and 3 cm were trimmed from both the proximal and distalends of the gut, and the remaining saline was gently squeezedout from the gut before it was snap-frozen in liquid nitrogen.The intestines were then transferred to cryogen tubes, pre-cooled in liquid nitrogen, and stored at �80°C until PGE2

extraction was done.Homogenization. The gut samples were ground in

liquid nitrogen using pestle and mortar. Roughly 100 mg ofthe pulverized sample was weighed into a microcentrifugetube, precooled in liquid nitrogen, with care taken to not allowthe samples to thaw. The samples were then put back intoliquid nitrogen and maintained at �80°C until extraction.

Extraction. In this step, 0.5 mL of ice-cold buffercontaining COX inhibitor (50 mmol/L phosphate [pH 7.4], 1mmol/L EDTA, 90 mol/L indomethacin) was added to thetubes containing the frozen samples, sitting on ice. Sampleswere vortexed briefly and left for 15 minutes, sitting on ice.

Purification of the samples was performed according to aprotocol provided with the PGE2 enzyme-linked immunosor-bent assay (ELISA) kit used for sample quantification (BiotracPGE2 enzyme immunoassay; Amersham, Buckinghamshire,England). Briefly, PGE2 was extracted with Amprep C18columns, eluted with ethyl acetate, and dried under nitrogen.Samples were reconstituted in 0.5 mL of assay buffer (suppliedwith the ELISA kit) just before the PGE2 ELISA was run.

The kit’s ELISA protocol was followed precisely, and theoptical density was read at 450 nm within 30 minutes using aMicroplate Reader Model X (MRX) ELISA plate reader fittedwith Dynex Revelation Software, version 3.2 (Dynex Technol-ogies, Chantilly, VA). This kit’s lowest reliable limit of PGE2

detection is 20 pg/mL. The lowest PGE2 measurement in thisstudy was 1.4 pg/mg of tissue, equivalent to 70 pg/mL, orapproximately 4 times above the detection limit and wellwithin the functional part of the standard curve of the assay.

Intestinal Permeability51CrEDTA was used as a marker for assessing intestinal

paracellular permeability.32,33 One hour after administration ofthe drug or solvent, mice received 1 �Ci 51CrEDTA in avolume of 100 �L of water (containing approximately 5 nmol51CrEDTA; Amersham Buckinghamshire, England) by gastricgavage followed by another 100 �L of water. The mice werehoused in individual metabolic cages. All urine passed over thenext 24 hours was collected. The urine samples were countedalong with standards (10 �L of stock solution, i.e., 10% of thedose given) in a Wallac 1284 gamma counter (Pharmacia,Sweden) for 1 minute, allowing detection of less than 0.01% ofthe dose administered. Results are presented as percentage ofthe oral dose excreted in urine.

1914 SIGTHORSSON ET AL. GASTROENTEROLOGY Vol. 122, No. 7

Intestinal Inflammation

Granulocyte marker protein (GMP) is a 70-kilodaltonprotein isolated from rat neutrophils. GMP is stable and notsignificantly degraded for at least 1 week at room temperature.GMP isolated from feces has been shown to be a sensitive,noninvasive marker of gastrointestinal inflammation in therat.22,34

Preliminary experiments showed a cross-reactivity betweenfecal GMP of rats and mice. Complete daily fecal collectionsfor GMP assessment were made from mice housed in individ-ual metabolic cages. Collections were made for 5 days beforegavage with vehicle or drug and were continued for a few daysafterward. Stool samples were maintained at �20°C untilhomogenized in homogenizing buffer (Tris 50 mmol/L, NaCl150 mmol/L, CaCl2 10 mmol/L, and thiomersal 0.25 mmol/L,pH to 8.4; volume 4 times the weight of each sample) using anUltra Turrax homogenizer (IKE Werke, Staufen, Germany) for30 seconds at 10,000g. A sample of the homogenate wastransferred to a microcentrifuge and spun at 10,000g for 10minutes. The top half of the supernatant was then transferredto a new tube and stored at �20°C until quantitation byELISA.

Microtiter plates (Maxisorp F96; Nunc Immunoplate,Nunc, Denmark) were coated by adding 200 �L of anti-GMPimmunoglobulin G (IgG) fraction diluted (1:2000) in phos-phate-buffered saline containing 0.25 mmol/L thiomersal toeach well. The plates were covered with mylar foil and main-tained at �4°C until use. Plates were washed 4 times withwashing buffer (Tris 50 mmol/L, NaCl 150 mmol/L, MgCl2

0.5 mmol/L, KCl 2.5 mmol/L, thiomersal 0.25 mmol/L, andTween-20 0.05%, pH to 8.0) before use. GMP standards,5–320 mg/L, were prepared by diluting purified GMP (28g/mL in assay buffer (Tris 50 mmol/L, NaCl 150 mmol/L,MgCl2 0.5 mmol/L, KCl 2.5 mmol/L, thiomersal 0.25mmol/L, Tween-20 0.05%, and bovine serum albumin 1%,pH to 8.0). The frozen fecal extracts were thawed and dilutedto 1:20 and 1:200 (with further dilutions made if required).

Next, 50 �L of standards and diluted samples (1:200) wereadded to the plate (in duplicates), then covered and incubatedat room temperature for 45 minutes on a plate shaker with anagitating speed of 600/minute. Each well was then washed 4times with washing buffer using an automatic plate washer.Then 50 �L of alkaline phosphatase conjugated anti-GMPantibody diluted (1:800) in assay buffer was added to each welland incubated again at room temperature for 45 minutes at thesame agitating speed. Thereafter the wells were washed 4 timesand 100 �L of substrate (p-nitrophenyl phosphate [1 mg/mL]in substrate buffer [10% diethanolamine, 0.5 mmol/L MgCl2 ,0.25 mmol/L thiomersal, pH 9.6]) was added to each well.Using a wavelength of 405 nm on an ELISA plate reader(MRX plate reader plus Dynex Revelation software, version3.2), the optical density of the highest standard was monitoreduntil it read between 1.2 and 1.8. Then the reaction wasstopped with 100 �L of 1 mol/L NaOH solution, and the platewas read. The intra-assay and interassay coefficients of variation

were 13% and 10%, respectively. Results are expressed as fecalconcentrations of GMP (mg/L).

Morphologic Studies

For the macroscopic assessment, the animals under-went anesthesia and laparotomy. When the stomach was ex-amined, it was removed and opened by a cut along the greatercurvature. The whole of the small intestine was removed andflushed with 0.9% saline without overdistension and evertedwith the aid of a plastic tube. The intestine was then incubatedin a 16 mmol/L HEPES buffer containing 125 mmol/L NaCl,3.5 mmol/L KCl, 10 mmol/L glucose, and 1 mmol/L nitrobluetetrazolium for 20 minutes. Nitroblue tetrazolium facilitatesthe detection of intestinal mucosal damage by staining the villisurrounding erosions and ulcers. The intestine was then exam-ined under 10� magnification with a Leica GZ6 stereomicro-scope (Leica, Deerfield, IL) for ulcer counts. Preliminary stud-ies had shown that the smallest detectable lesions identifiedafter NSAID administration without staining with nitrobluetetrazolium were about 1–2 mm. By staining the intestinaltissue, we were able to reliably detect lesions of 0.1 mm andlarger, which was used to define the smallest lesion included inthe ulcer count. The histopathologic correlates of these smallerlesions are erosion, because they do not usually penetrate themuscular layer. Our ulcer count is therefore a combination oferosions and ulcers, because strictly, the latter can be verifiedonly microscopically.

Separate animals underwent anesthesia and laparotomy withremoval of the entire intestine (stomach and small and largeintestine) for histopathologic study. The small bowel was firstperfused with formalin through a duodenal incision and im-mersion-fixed overnight. When examined, the stomach andcolon were immersion-fixed. The whole of alternative 2-cmsegments of intestine (stomach and colon) was cut into 1- to2-mm segments and embedded in paraffin for the preparationof 3 �m H&E–stained sections. A total of about 60 smallintestinal sections from each animal were assessed for histo-logic damage.35,36

Nitric Oxide Products

Measurement of nitrate and nitrite was performed inserum, 3 24-hour mouse urines, and incubation medium usinga modified Greiss method.37 Jejunal tissue was obtained fromanesthetized animals and incubated in an oxygenated HEPESbuffer for 5 minutes. The tissue was removed and weighedwhile the medium was stored before assay for nitric oxideproducts.

Statistics

Results are presented as mean � standard deviation.Most, but not all, of the data were normally distributed.Statistical differences between groups of animals were thereforeassessed by Wilcoxon rank sum test. Sequential data wereassessed with the paired Student t test using Bonferroni’scorrection and correlations with Pearson’s correlation coeffi-cient.

June 2002 PATHOGENESIS OF NSAID-ENTEROPATHY 1915

ResultsStudies in COX-1�/� and COX-1�/� Animals

Characterization of COX-1�/� and COX-1�/�. Al-together during the studies, we examined 54 untreated(apart from solvent) COX-1�/� animals for small bowelulceration, including 12 animals 18 months of age. Noneof these were found to have any small bowel pathology,and none died unexpectedly.

Table 1 and Figure 1 show that COX-1�/� animalshave normal intestinal permeability and no increasedinflammation, with intestinal PGE2 levels only 3% ofthose of the control mice (P � 0.001) and unaffected bysolvent. The low PGE2 levels in COX-1�/� mice argueagainst intestinal up-regulation of COX-2 as a compen-satory mechanism to account for the lack of spontaneoussmall bowel damage. Another common hypothesis toexplain this lack of damage proposes up-regulation ofintestinal nitric oxide synthase.25,26 This hypothesis wastested, but Table 1 reveals no significant differencesbetween the products (nitrite and nitrate) of nitric oxidesynthase in wild-type and COX-1�/� animals.

Dose–response studies of indomethacin-induced smallintestine damage were carried out; the results are shownin Table 1. There were significantly fewer ulcers in theCOX-1�/� mice at doses of 2.0 and 3.75 mg/kg, butotherwise the damage was comparable. The ulcers had a

predilection for the proximal ileum (7–10 cm proximalto the ileocecal junction) with the smaller doses. Withthe higher doses, the ulcers were also found more prox-imally and distally within the small bowel. Histologi-cally, the ulcers resembled the acute indomethacin-in-duced small bowel ulcers in the rats,36 with a “punched-out” appearance on the mesenteric side.

Indomethacin’s effects on intestinal PGE2, ulcers,permeability, and inflammation in COX-1�/� and COX-1�/� animals. Figure 1 shows that indomethacin ad-ministrated at doses of 3.75 and 15 mg/kg reducedintestinal PGE2 levels equally in COX-1�/� (by 97%,P � 0.001) to levels not significantly different (P � 0.5)from those found in untreated COX-1�/� animals. Thesmall intestine ulcer counts were 25 � 4 and 23 � 3after the 2 doses (Figure 1 and Table 1).

Indomethacin (3.75 and 15 mg/kg) further decreasedthe already low intestinal PGE2 levels in COX-1�/�

animals by 50%–60% (P � 0.01), so that the post–indomethacin administration levels in these animals were1.4%–1.7% of those in the COX-1�/� animals. Thesmall intestine ulcer counts (Figure 1 and Table 1) 24

Figure 1. Small intestine PGE2 levels and ulcer counts. Open bars inthe upper part of the Figure represent mean PGE2 levels (as percent-age of untreated COX-1�/�) in wild-type animals; shaded bars, thelevels in COX-deficient mice (� standard error [SE]). Small intestineulcer counts (mean � SE) carried out in separate groups of animalsare shown in the lower part of the Figure. The number of animals ineach group varied from 5 to 16. *Significantly less than untreatedCOX-1�/� animals (P � 0.001). �Significantly less than untreatedCOX-1�/� animals (P � 0.001) and significantly (P � 0.05) less thanthat of untreated or DMSO-treated COX-1�/� animals. All of the ulcercounts above 0 are significantly different from control or DMSO-treated animals. °Significantly (P � 0.05) fewer ulcers than in COX-1�/� animals receiving 3.75 mg/kg indomethacin. Significantly (P �0.05) more ulcers than in COX-1�/� animals receiving 3.75 mg/kgindomethacin.

Table 1. Comparison of Intestinal PGE2 Levels, IntestinalPermeability, Intestinal Inflammation, and Productsof Nitric Oxide Synthase in COX-1�/� and COX-1�/�

Animals

Experiment COX-1�/� COX-1�/�

Intestinal prostaglandinsPGE2 (pg/mg tissue) 340 � 25 9 � 2a

Intestinal permeabilityUrinary excretion of 51CrEDTA(% oral dose) 4.1 � 0.4 4.0 � 0.5

Intestinal inflammationGMP fecal excretion (mg/L) 7.1 � 0.9 7.9 � 1.1

Metabolites of nitrite and nitrateSerum concentration (�mol/L) 36.3 � 3.5 35.8 � 3.0Urinary excretion (�mol/units) 3.10 � 0.86 2.99 � 0.84Jejunal nitric oxide production(�mol � L�1 � mg�1) 37.5 � 0.5 38.3 � 0.4

Effect of indomethacinSolvent (ulcer counts) 0 01.0 mg/kg 0 02.0 mg/kg 4 � 0.6 1 � 0.4a

3.75 mg/kg 25 � 3 14 � 3a

7.5 mg/kg 28 � 4 26 � 315 mg/kg 23 � 3 22 � 4

NOTE. The number of animals was 8–16 per group, except for theintestinal permeability test, in which there were 28 animals in eachgroup.aDiffers significantly from control (P � 0.001)

1916 SIGTHORSSON ET AL. GASTROENTEROLOGY Vol. 122, No. 7

hours after indomethacin administration (3.75 and 15mg/kg) were 14 �3 and 22 �4, respectively, the formerbeing significantly (P � 0.001) lower than that foundafter the same dose in COX-1�/� animals.

Figure 2 shows that 3.75 mg of indomethacin in-creased intestinal permeability equally in COX-1�/� andCOX-1�/� animals 1–25 hours after gastric gavage. Thisincreased intestinal permeability normalized 72–96hours after administration. The same pattern is seen afterthe 15-mg/kg dose, but the permeability values at 1–25hours are significantly (P � 0.01) higher than after the3.75-mg/kg dose in both the COX-1�/� and COX-1�/�

animals.Figure 3 shows that intestinal inflammation increases

significantly (P � 0.001) for 2 days after the 3.75-mgdose in both COX-1�/� and COX-1�/� animals, andthere is no significant (P � 0.5) difference between thegenotypes. The increase in inflammation was signifi-cantly greater (P � 0.01) and more prolonged (4 days)after the 15-mg/kg dose, but again there was no signif-icant (P � 0.5) difference between the COX-1�/� andCOX-1�/� animals.

Comparison of the effect of selective COX-2 inhi-bition in COX-1�/� and COX-1�/� animals. Figure 1shows that indomethacin significantly decreased the al-ready very low PGE2 levels in COX-1�/� animals. Thissuggests that the effect may be COX-2 mediated. Weassessed the effect of selective COX-2 inhibition onintestinal PGE2 levels and ulcers in COX-1�/� and

COX-1�/� mice. Figure 1 shows that whereas celecoxibdid not affect intestinal PGE2 levels significantly or causeulcers in COX-1�/� animals (n 6), it caused dose-dependent ulcerative damage in COX-1�/� animals. Atthe 30- and 300-mg/kg doses, celecoxib reduced intes-tinal PGE2 levels significantly (by 50%–60%, which isnot significantly [P � 0.5] different from the effect ofindomethacin in these animals) from the baseline levels

Figure 2. Effect of COX-1 deletion on intestinal permeability afterindomethacin administration. Baseline intestinal permeability did notdiffer significantly between the COX-1�/� (n 16/group) and COX-1�/� animals (n 8/group). Indomethacin at both doses (n 8/group) increased intestinal permeability significantly in the 1- to25-hour period, with restoration of normal intestinal permeability at72–96 hours. The 15-mg/kg dose increased intestinal permeability toa significantly greater extent than the 3.75-mg/kg dose. The boxesrepresent mean, and the bars represent �SE; the open boxes areCOX-1�/� animals, and the shaded boxes are COX-1�/� animals.*Differs significantly (P � 0.01) from baseline. �Differs significantly(P � 0.01) from baseline and the 3.75 mg/kg indomethacin dose.

Figure 3. Effect of COX-1 deletion on intestinal inflammation afterindomethacin administration. Baseline fecal excretion of GMP did notdiffer significantly (P � 0.8) between any of the groups. Solvent hadno significant effect on fecal GMP concentrations, whereas bothdoses of indomethacin increased GMP concentrations significantly inboth genotypes. The effect of a 15-mg/kg dose of indomethacin wassignificantly greater than that of the 3.75-mg/kg dose, but there wasno significant difference between the COX-1�/� and COX-1�/� ani-mals at any time point. Bars represent the mean (�SE) daily fecalconcentration of GMP. The open boxes are COX-1�/� animals, and theshaded boxes are COX-1�/� animals. �Differs significantly (P � 0.01)from baseline. *Differs significantly (P � 0.001) from baseline.

June 2002 PATHOGENESIS OF NSAID-ENTEROPATHY 1917

found in COX-1�/� animals. This strongly suggests thatthe residual PGE2 levels in COX1�/� mice are COX-2dependent.

Studies in COX-2�/� and COX-2�/� Animals

Characterization of COX-2�/� and COX-2�/�mice.Untreated COX-2�/� animals did not differ significantly(P � 0.6) from COX-1�/� animals in respect of intes-tinal morphology (all normal; n 14–32), PGE2 levels(103 � 16% of control levels; n 9), intestinal perme-ability (24-hour urinary excretion of 51CrEDTA 5.8� 0.8%; n 11) or inflammation (GMP concentra-tions 6.5 � 0.9 mg/L; n 17).

Untreated COX-2�/� animals did not differ signifi-cantly from COX-2�/� animals with respect to PGE2

levels (96 � 23% of control levels; n 6) or intestinalpermeability (24-hour urinary excretion of 51CrEDTA 8.7 �1.1%; n 12; P 0.086), although 6 (50%) ofthe animals were above the normal range. Fecal GMPlevels in COX-2�/� animals (14.8 � 3.9 mg/L; n 28)were significantly (P � 0.05) higher than those in thewild-type animals, with 11 (40%) of these animals hav-ing values above the normal range. There were no sig-nificant correlations (P � 0.05) between age and intes-tinal permeability (r 0.19) or inflammation (r 0.29)in the COX-2�/� animals.

During the studies, 8 of 56 (14%) untreated COX-2�/� animals died suddenly or were killed because ofdistress. All had peritoneal sepsis with small bowel ad-hesions. Macroscopically, there was evidence of smallbowel perforation, and microscopy showed areas ofchronic multifocal mucosal necrosis and ulceration in thelast few centimeters of the ileum (stomach and colonwere normal).

More groups of untreated COX-2�/� animals werestudied to assess the relationship between intestinal

PGE2 level, inflammation, and intestinal morphology.Six animals with high fecal GMP values (15–68 mg/L)had intestinal PGE2 levels not significantly different thanthose in wild-type animals (94 � 13% of control levels).Further groups were selected on the basis of normal (n 8) and high fecal GMP (�15 mg/L; n 12) at age 8–52weeks. None of the animals with a normal GMP showedany evidence of morphologic damage, whereas 2 of the 3animals with fecal GMP concentrations above 50 mg/Lhad microscopically detected small ulcers in the terminalileum that were identical to those found in the distressedanimals and the animals that died unexpectedly.

Finally, COX-2�/� mice (n 13) were given 100mg � kg�1 � d�1 of celecoxib orally for 3 months mixedwith their food. During the studies, 3 animals diedsuddenly or were killed due to distress (after 6–10 weeksof treatment). All 3, plus 2 of those killed at the end ofthis study, had lesions identical to those described in theCOX-2�/� animals. Collectively, all of the lesions in theuntreated COX-2�/� animals and the COX-2�/� ani-mals receiving celecoxib were located on the mesentericside of the intestine, and they differ from the acuteindomethacin-induced lesions (representative histologyshown in Figure 4) in 4 respects.

First, the lesions in the COX-2�/� and COX-2�/�

animals treated with celecoxib long-term are confined tothe terminal ileum, whereas the lesions seen with low-dose indomethacin (2.0 mg/kg) are located mainly in themid to small intestine, spreading proximally and distallyat higher doses. Second, the terminal ileal ulcers associ-ated with COX-2 deficiency and inhibition do not stainwith nitroblue tetrazolium and are difficult to detectmacroscopically. Third, there is a lower preponderance(n 1–3) of ulcers in the celecoxib-treated and COX-2�/� animals than in the indomethacin-treated ones

Figure 4. Microscopic features of damage associated with indomethacin and COX-2 inhibition or absence. (A) Normal small bowel (magnification20�). (B) Acute damage 24 hours after indomethacin (3.75 mg/kg) administration demonstrating a punched-out ulcer (magnification 30�). (C)Representative damage in a COX-2�/� animal demonstrating a chronic-looking ileal ulcer (magnification 30�), distinctively different from theacute indomethacin-induced ulcer. Long-term COX-2 inhibition with celecoxib was associated with the same pathology as in the COX-2�/�

animals.

1918 SIGTHORSSON ET AL. GASTROENTEROLOGY Vol. 122, No. 7

(apart from at the 2.0-mg/kg dose). Finally, there is adominance of chronic (rather than acute) inflammatorycells seen microscopically in the spontaneous lesionsfound in the COX-2�/� and wild-type animals treatedwith celecoxib, whereas the indomethacin-induced ulcershave an acute appearance (Figure 4).

Effect of indomethacin and SC-560 on intestinalPGE2 and ulcers in COX-2�/� and COX-2�/� animals.Figure 1 shows that the solvent had no significant effecton intestinal PGE2 levels, whereas single doses of indo-methacin (3.75 mg/kg) decreased intestinal PGE2 levelsin the COX-2�/� and COX-2�/� mice significantly (P �0.001) and equally. These PGE2 levels were not signifi-cantly (P � 0.9) different than those seen in the indo-methacin (3.75 mg/kg) treated COX-1�/� and untreatedCOX-1�/� mice. However, at this dose of indomethacin,the COX-2�/� mice (n 16) developed significantly(P � 0.05) fewer small intestinal ulcers (14 � 3) thanthe COX-1�/� mice (26 � 4). The COX-2�/� mice (n 16) developed significantly (P � 0.01) more ulcers withindomethacin than their COX-2�/� controls, the con-verse of what was seen in the COX-1�/� and COX-1�/�

animals.Figure 1 shows that single doses of the selective,

nonacidic COX-1 inhibitor SC-560 (30 mg/kg; n 4–6/group) decreased intestinal PGE2 significantly (by96%–97%; P � 0.001) from control levels in the COX-2�/� and COX-2�/� animals. However, although nomacroscopic or microscopic damage was seen with SC-560 administration in the COX-2�/� animals, the drugcaused significant ulcerative (P � 0.001) damage to thesmall bowel in the COX-2�/� animals. The location andhistopathologic appearance of these ulcers were identicalto those seen with indomethacin.

Dual inhibition of COX-1 and COX-2 in wild-typeanimals. The foregoing suggests, but does not provideconclusive proof, that inhibition of both the COX-1 andCOX-2 enzymes in the short term leads to small intes-tinal ulcers resembling those seen with indomethacin.Thus COX-2�/� mice (n 4) were given single doses of5 mg/kg SC-560 and 30 mg/kg celecoxib, neither ofwhich causes damage in wild-type animals when admin-istered alone at these doses. Small intestinal ulcer counts24 hours later showed 23�2 ulcers with the same mac-roscopic and microscopic appearance and location as in-domethacin-induced ulcers.

DiscussionNSAID-induced gastrointestinal damage is a

complex, multistep pathogenic event. In theory, theinitial damaging event is dependent on one or more

biochemical action(s) shared by all conventionalNSAIDs, namely inhibition of COX-1 and COX-2 andthe “topical” effects. It has been suggested that becauseof these biochemical actions, a number of interactingpathophysiologic events follow, including recruitment ofneutrophils and other cells involved in the inflammatorycascade, ischemia, villus contraction, and so on. Theseevents constitute the host response to the initial damageand eventually lead to the characteristic gastrointestinalinflammation, ulcers.

The relative importance and contribution of the initialbiochemical actions of conventional NSAIDs in thestomach and small bowel may differ. In particular, it hasbeen suggested that the small bowel damage is lessdependent on COX-1 inhibition than the stomach.5 Fur-thermore, the pathophysiology of the damage differssignificantly at the 2 sites because of the participation ofluminal factors in the tissue damage. The importance ofelucidating the pathogenesis of NSAID-induced gastro-intestinal damage is that such knowledge might lead tomeasures to reduce the frequency and severity of thesedrugs’ gastrointestinal side effects.2,4,5 Although the sec-ondary host and luminal factors can be modified todecrease the damage, optimally a therapeutic measure tominimize NSAID toxicity would be directed against adrug action that initiates the damage.

The gastric damage caused by NSAIDs has been stud-ied in greater detail than the small bowel damage be-cause the clinical consequences of gastric ulcer bleeds andperforations in humans are common and may be life-threatening.38,39 It is possible to cause gastric damage inthe rat through dual inhibition of COX-1 and COX-2,27

and there are pervasive data to suggest that the “topical”effect may also play a role.15,16,18,19,40

In this study, we used genetically modified mice toassess the long-term effect of COX-1 and COX-2 defi-ciency on the small intestine and the role of the 2enzymes in acute damage, using SC-560 and celecoxib,neither of which has a “topical” effect.41 Our resultsexpand on previous studies that concentrated largely ongastric integrity23 to show that long-term deficiency ofCOX-1 deficiency is not associated with a disruptedsmall intestinal barrier function, intestinal inflammation,or ulcers. Moreover, we have shown that short-termselective COX-1 inhibition with SC-560 in wild-typeanimals does not cause small intestinal damage despitedecreasing intestinal PGE2 to levels seen at ulcerogenicdoses of indomethacin. These results argue against theidea that COX-1 inhibition is the sole mechanism un-derlying the small intestinal toxicity of NSAIDs and arein keeping with similar studies in the stomach.27 Fur-

June 2002 PATHOGENESIS OF NSAID-ENTEROPATHY 1919

thermore, our results show that up-regulation of intes-tinal nitric oxide synthase, a commonly postulated mech-anism,25,26 does not explain the lack of damage in theface of the low PGE2 levels.

The alterations in small intestine permeability andinflammation and the location and histopathologic fea-tures of the damage caused by indomethacin in micewere almost identical to those seen in rats.22,36 Theeffects of indomethacin on these parameters did not differsignificantly between the COX-1�/� and COX-1�/� an-imals, except the latter were less susceptible to theulcerative damage at the lower dosage range (2.0 and3.75 mg/kg) of indomethacin. In the stomach, a similarreduced susceptibility to the damaging effect of indo-methacin was seen at the 10-mg/kg dose, with the20-mg/kg dose associated with equal damage in wild-type and COX-1�/� animals.23

Untreated COX-1�/� animals and wild-type animalsreceiving indomethacin (3.75 and 15 mg/kg) and SC-560 all had similarly low intestinal PGE2 levels. How-ever, the degree of small bowel damage differed, sug-gesting that concomitant inhibition of COX-2 and/orthe topical effect is important in the pathogenesis ofsmall bowel damage. The former possibility was studiedfurther. Single doses of celecoxib did not affect intestinalPGE2 levels or cause damage in wild-type animals, inkeeping with similar studies in rats.42,43 However, cele-coxib caused significant small bowel damage with afurther 50% decrease (30 and 300 mg/kg) in the alreadylow intestinal PGE2 levels of the COX-1�/� animals.The mechanism by which short-term COX-2 inhibitionleads to small bowel damage in COX-1�/� mice isuncertain, because no significant COX-2 mRNA signalwas detected in the intestine of COX-1�/� mice.23,44

Having demonstrated that COX-2 inhibition is det-rimental in COX-1�/� animals, we proceeded to showthat short-term COX-1 inhibition, associated with de-creased intestinal PGE2 levels, caused small bowel ulcersin COX-2�/� animals, as did short-term dual selectiveinhibition of both enzymes in the wild-type animals. Themacroscopic and microscopic appearances of these ulcerswere similar to the indomethacin-induced ones. Thisprovides strong support for the idea that both enzymesare involved in the pathogenesis of the small boweldamage induced by NSAIDs in mice.

During these studies, we also found that COX-1�/�

and COX-2�/� animals differed in their susceptibility tothe ulcerative damage of indomethacin, which is in keep-ing with known interspecies and intraspecies varia-tions.29 However, whereas the COX-1�/� animals were,if anything, less susceptible to the small bowel damage of

indomethacin, the COX-2�/� animals were significantlymore susceptible to this damage than their COX-2�/�

counterparts. Moreover, we demonstrated that long-termabsence or inhibition of COX-2, which does not lead tosignificant decreases in intestinal PGE2 levels, is associ-ated with small intestine damage. This damage is dis-tinctively different from the acute damage seen in theshort-term experiments in terms of location and his-topathologic appearance and occurred without concomi-tant gastric damage. Collectively, this suggests thatCOX-2 is important for maintenance of small bowelintegrity. This might be an extraintestinal effect, butthere is increasing (albeit controversial45) evidence thatCOX-2 is constitutively expressed in the gastrointestinaltract.46,47 If so, then the COX-2 products may regulateneutrophil adherence in the microvasculature in a similarway as described in the stomach.27 However, the COX-2products may be important immunomodulators in theintestinal mucosal reaction to luminal antigens.48 In thiscontext, it may be relevant that the terminal ileal loca-tion of the ulcers is in the transition area from a low toa high concentration of luminal bacteria. Loss of COX-2products might impair oral tolerance at this site therebyrendering the luminal bacteria pathogenic and henceleading to ulcers. Alternatively, and not mutually exclu-sively, ileal ulcers may occur spontaneously throughoutthe life of these animals, and COX-2 inhibition or ab-sence may delay healing of these lesions, similar to theeffects demonstrated in experimental gastric ulceration.49

Extrapolation of the aforementioned data to humans isproblematic because there is significant intraspecies andinterspecies variation in the intestinal expression ofCOX-1 and COX-2.45 A detailed localization study ofthe enzymes in mice has not been performed, butwhereas no COX-2 protein was found in the humanintestine, some rats appeared to express COX-2 in thececum.45 However, there are many similarities in theshort-term gastric9,42 and small bowel43,50,51 tolerabilityto COX-2–selective agents in humans and animals.Long-term selective COX-2 inhibition is also associatedwith good gastric tolerability in humans,10,11 but thepossible small bowel consequences have been assessedonly in short-term studies.50,51 From our studies, itseems possible that long-term administration of COX-2inhibitors may lead to ileal inflammation. If so, then theclinical and theoretical implications of such inflamma-tion are of considerable importance. The serious outcomestudies with rofecoxib12 and celecoxib13 show that smallintestine perforations are at least exceedingly rare withCOX-2–selective agents, as is the case with conventionalNSAIDs.52 However, NSAID enteropathy is not rare,

1920 SIGTHORSSON ET AL. GASTROENTEROLOGY Vol. 122, No. 7

affecting 10%–60% of patients taking conventionalNSAIDs long-term, depending on the diagnosticmethod used,4,53–56 and is associated with low-gradebleeding and protein loss.4,57 The precise location of thesmall bowel inflammation associated with NSAID inges-tion is unknown, but enteroscopy shows macroscopicchanges in the mid-small bowel consistent with acutedamage. The possibility that long-term NSAID use isassociated with ileal damage has not been assessed di-rectly. Nevertheless, ileocolonoscopy with biopsy showsthat 20%–70% of patients with spondylarthropathy,58

most of whom were receiving conventional NSAIDs,have ileal inflammation, which has many histopathologicsimilarities to Crohn’s disease.59,60 However, 1 studyshowed an equally high prevalence of ileitis in patientswithout spondylitis taking NSAIDs,61 which, in con-junction with the present results, raises the possibilitythat this pathology is iatrogenically induced.

In summary, these studies have shown that short- andlong-term COX-1 deficiency or inhibition and short-term COX-2 inhibition are not detrimental to smallbowel integrity in mice. Dual inhibition of the COXenzymes, in the absence of the topical effect, leads to thecharacteristic small bowel damage seen with indometh-acin, in the absence of the topical effect. Long-termCOX-2 deficiency or inhibition is frequently associatedwith a disrupted small intestinal barrier function, intes-tinal inflammation, and ileal ulcers, despite normal in-testinal PGE2 levels. COX-2 products, therefore, seem toplay an important role in the maintenance of small bowelintegrity in mice.

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Received December 4, 2001. Accepted January 21, 2002.Address requests for reprints to: Ingvar Bjarnason, M.D., Professor of

Digestive Diseases, Department of Medicine, Guy’s, King’s, St.Thomas’ School of Medicine, Bessemer Road, London SE5 9PJ, En-gland. e-mail: ingvar.bjarnason@kcl.ac.uk; fax: (44) 20-7346-3313.

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