Cyclooxygenase 2 is induced in colonic epithelial cells in inflammatory bowel disease

Cyclooxygenase 2 Is Induced in Colonic Epithelial Cellsin Inflammatory Bowel Disease

IRWIN I. SINGER,* DOUGLAS W. KAWKA,* SUZANNE SCHLOEMANN,‡ TERESA TESSNER,‡

TERRENCE RIEHL,‡ and WILLIAM F. STENSON‡

*Department of Inflammation Research, Merck Research Laboratories, Rahway, New Jersey; and ‡Department of Medicine,Washington University School of Medicine, St. Louis, Missouri

Background & Aims: Prostaglandins are synthesized bycyclooxygenases (COX)-1 and -2. The expression andcellular localization of COX-1 and COX-2 in normalhuman colon and inflammatory bowel disease (IBD)surgical resections were studied. Methods: COX-1 andCOX-2 protein expression and cellular localization wereassessed by Western blotting and immunohistochemis-try. Results: COX-1 protein was expressed at equallevels in normal, Crohn’s disease, and ulcerative colitiscolonic epithelial cells. COX-2 protein was not detectedin normal epithelial cells but was detected in Crohn’sdisease and ulcerative colitis epithelial cells. Immuno-histochemistry of normal, Crohn’s colitis, and ulcer-ative colitis tissue showed equivalent COX-1 expres-sion in epithelial cells in the lower half of the coloniccrypts. COX-2 expression was absent from normalcolon, whereas in Crohn’s colitis and ulcerative colitis,COX-2 was observed in apical epithelial cells and inlamina propria mononuclear cells. In Crohn’s ileitis,COX-2 was present in the villus epithelial cells. Inulcerative colitis, colonic epithelial cells expressingCOX-2 also expressed inducible nitric oxide synthase.Conclusions: COX-1 was localized in the crypt epithe-lium of the normal ileum and colon, and its expressionwas unchanged in IBD. COX-2 was undetectable innormal ileum or colon, but it was induced in apicalepithelial cells of inflamed foci in IBD.

Cyclooxygenase (COX) is the key enzyme in thesynthesis of prostaglandins (PGs). There are two

COX isoforms: COX-1 is a constitutive enzyme ex-pressed in many tissues including the intestine and colon,whereas COX-2 is an inducible enzyme expressed inmacrophages, fibroblasts, and other cell types in inflam-mation.1 COX-2 expression is induced by phorbol estersand proinflammatory cytokines, including interleukin(IL)-1b and tumor necrosis factor (TNF)-a.2–4 Infectionof intestinal epithelial cells with invasive bacteria such asSalmonella also induces COX-2 expression.5 COX-1 isthought to be responsible for the production of the PGsassociated with the maintenance of gastrointestinal integ-

rity, whereas COX-2 is believed to be responsible for theproduction of PGs associated with the mediation ofinflammation.1,6 Inhibition of COX-1 activity by nonste-roidal anti-inflammatory drugs (NSAIDs) is the mostprobable mechanism of NSAID-induced gastrointestinalinjury.

COX-1, but not COX-2, messenger RNA (mRNA)has been identified in the normal human colon byreverse-transcription polymerase chain reaction.7 Immu-noblotting analysis of COX-1 and COX-2 proteins hasshown expression of COX-1 but not COX-2 in normalhuman colon.8 However, COX-2 mRNA and proteinhave been found in human colon adenomas and adenocar-cinomas.9,10 There have been few reports of cellularlocalization of COX-1 and COX-2 expression in thegastrointestinal tract. COX-1 is localized to the cryptepithelium in normal mouse small intestine,11 in endothe-lial cells of the rat gastrointestinal tract,8 and in mucousneck cells in normal mouse stomach,12 but there havebeen no reports of COX-1 or COX-2 cellular localizationin the human colon.

There are markedly elevated levels of PGs in inflamma-tory bowel disease (IBD) mucosa and in rectal dialysatesfrom patients with IBD.13,14 The cellular origin of thesePGs and the relative contributions of COX-1 and COX-2to their production have not been investigated. Currentlyavailable NSAIDs inhibit both COX-1 and COX-2.NSAIDs can exacerbate clinical activity in ulcerativecolitis.15 Whether the exacerbation of clinical activityrelates to inhibition of COX-1 or COX-2 and theidentity of the cell types involved are unknown.

In this study, we used immunohistochemistry toinvestigate the expression and cellular distribution ofCOX-1 and COX-2 in normal ileum, normal colon,

Abbreviations used in this paper: COX, cyclooxygenase; HUVEC,human umbilical vein endothelial cell; IL, interleukin; iNOS, induciblenitric oxide synthase; LPS, lipopolysaccharide; RPA, ribonucleaseprotection assay; TNBS, trinitrobenzene sulfonic acid; TNF-a, tumornecrosis factor a.

r 1998 by the American Gastroenterological Association0016-5085/98/$3.00

GASTROENTEROLOGY 1998;115:297–306

ulcerative colitis, Crohn’s colitis, and Crohn’s ileitis. Wereport that COX-1 is expressed in epithelial cells in thelower half of the crypt in normal ileum and Crohn’s ileitisand in the lower half of colonic crypts in normal colonsand in Crohn’s colitis and ulcerative colitis. COX-2 is notexpressed in the normal ileum but is expressed in villusepithelial cells in Crohn’s ileitis. In Crohn’s colitis andulcerative colitis, COX-2 is expressed in surface epithelialcells but not in proximal crypt epithelial cells, whereas innormal colonic epithelia, COX-2 is not expressed at all.In addition to expression in epithelial cells, COX-2 is alsoexpressed in mononuclear inflammatory cells in IBD. Wepreviously reported the expression of inducible nitricoxide synthase (iNOS) in epithelial cells in ulcerativecolitis but not in the normal colon.16 Here we found that,in ulcerative colitis, the epithelial cells that expressCOX-2 also express iNOS.

Materials and Methods

PatientsAll tissue samples were collected from surgical resec-

tions. Histologically normal portions of colon (n 5 5) andileum (n 5 3) were obtained from uninvolved areas of bowelresections from 7 patients (3 men and 4 women; age range,34–84 years) with adenocarcinoma of the colon. The ulcerativecolitis tissue was obtained from total colectomies from 5patients with intractable disease (1 man and 4 women; agerange, 18–38 years). All 5 patients had pancolitis, and nopatient showed dysplasia or toxic megacolon. Clinical activityat the time of resection ranged from moderate to severe, and thedisease duration ranged from 3 to 18 years. Crohn’s diseasetissue was obtained from surgical resections for ileitis (n 5 3)and colitis (n 5 6). All resections were for intractable disease (5men and 4 women; age range, 19–62 years). Disease durationranged from 2 to 25 years. All patients with ulcerative colitisand Crohn’s disease had undergone corticosteroid therapy inthe past, and most patients were being treated with parenteralsteroids at the time of their resections.

Antibodies

Rabbit anti-mouse COX-1 antibody raised against themouse COX-1 peptide Leu-Leu-Leu-Pro-Pro-Thr-Pro-Ser-Val-Leu-Leu-Ala-Arg-Pro-Gly-Val-Pro-Ser-Pro-Val, which recog-nizes mouse COX-1 monospecifically in Western blots of crudemacrophage lysates and cross-reacts with human COX-1, was agenerous gift from J. Masferrer (G.D. Searle, St. Louis, MO).17

Affinity-purified rabbit anti-mouse COX-2 immunoglobulin(Ig) G (termed Dina IgG) raised against the peptide Ser-His-Ser-Arg-Leu-Asp-Asp-Ile-Asn-Pro-Thr-Val-Leu-Ile-Lys, which alsorecognizes human COX-2 but not COX-1, was a generous giftfrom W. Smith and A. Spencer (Michigan State University, East

Lansing, MI). Affinity-purified rabbit anti-human COX-2 IgGraised against the peptide Asp-Asp-Ile-Asn-Pro-Thr-Val-Leu-Leu-Lys-Glu-Arg, termed no. 168 IgG, was obtained from S.Kargman, J. Evans, and the Merck-Frosst COX-2 group(Merck-Frosst Laboratories, Kirkland, Quebec, Canada). Rab-bit anti-sheep seminal vesicle COX-1 serum (termed no. 241)and rabbit anti-sheep placental COX-2 IgG (termed no. 243)were additional gifts obtained from the group at Merck-Frosst.These various antibodies monospecifically recognize COX-1and COX-2, respectively, in several species including human,dog, pig, monkey, and rat as shown by Western blotting.8,10

iNOS was immunolabeled in adjacent sections of ulcerativecolitis colon with NO-53 IgG, a rabbit antipeptide antibodythat monospecifically recognizes human iNOS on Westernblots and in tissue sections as described previously.16

Immunohistochemistry

For immunohistochemical localization of COX-1, depa-raffinized sections of Bouin’s-fixed tissue were incubated with a1:2000–1:8000 dilution of rabbit anti-mouse COX-1 serum.After quenching of endogenous peroxidase activity with 1%hydrogen peroxide, the sections were washed, and bound rabbitanti-mouse COX-1 was detected by fluorescein-conjugatedtyramide signal amplification (TSA direct; Du Pont NewEngland Nuclear Corp. Life Science Products, Boston, MA)according to the manufacturer’s directions after incubationwith biotin-labeled donkey anti-rabbit IgG (Jackson Immunore-search Laboratory, Bar Harbor, ME) and subsequent incubationwith streptavidin–horseradish peroxidase. For immunohisto-chemical localization of COX-2, surgical resections were fixedin 10% neutral buffered formalin and embedded in paraffin,and 5-µm sections were cut and mounted on glass slides.Sections were immunolabeled with affinity-purified fractionsof the various COX-2 antibodies at concentrations from 1 to5 µg/mL, using the avidin-biotin peroxidase complex immu-noperoxidase method (Elite Kit; Vector Laboratories, Inc.,Burlingame, CA) exactly as described previously.16 To showspecificity, competition experiments were conducted in whichCOX-2 peptide antibodies were incubated with the peptidesused for immunization or with a COX-1 peptide as a control at100-fold mol/L excess. Additional positive controls for COX-1and COX-2 immunostaining included sections of dog andhuman prostate and placenta, respectively.

Western Blot Analysis

Epithelial cells isolated from normal colon, Crohn’scolitis colon, and ulcerative colitis colon were assayed forCOX-1 and COX-2 by Western blotting. Lysates of humanumbilical vein endothelial cells (HUVECs) that were stimu-lated overnight with 10 ng/mL TNF-a and 10 µg/mLlipopolysaccharide (LPS) were used as a positive control forCOX-2. Unstimulated HUVECs were used as a positivecontrol for COX-1. Sheep COX-1 (5 ng) and sheep COX-2(5 ng) were run as controls. Epithelial cells were harvested as

298 SINGER ET AL. GASTROENTEROLOGY Vol. 115, No. 2

described previously.18,19 Equal amounts of protein (40 µg)from each cell lysate were loaded onto 8% sodium dodecylsulfate–polyacrylamide gels and were electrophoresed. Afterelectrophoresis, the separated proteins were transferred to apolyvinylidene fluoride membrane (Immobilon-P; MilliporeCorp., Bedford, MA). For analysis of COX-1 expression, themembrane was stained with affinity-purified rabbit antibodyagainst sheep seminal vesicle COX-1 (no. 241) or with therabbit antibody against mouse COX-1. For detection ofCOX-2, the membrane was stained with affinity-purifiedrabbit antibody against sheep placental COX-2 (no. 243).Alternatively, a transfer membrane was stained with rabbitantibody against mouse COX-2 (Dina) that cross-reacts withhuman COX-2 to confirm the signals detected with Merck-Frosst no. 243 antibody. Primary antibodies were detected withdonkey anti-rabbit IgG linked to horseradish peroxidase andwere developed by enhanced chemiluminescence Western blotanalysis (Amersham Corp., Arlington Heights, IL) usingautoradiography.

Protection Assays

Human COX-2 and glyceraldehyde-3-phosphate dehy-drogenase (GAPDH) mRNA expression levels were measuredby ribonuclease protection assays (RPAs).5 First, in vitrotranscription was performed to produce 32P-labeled antisenseRNA probes, a 400–base pair for COX-2, and 434–base pairfor GAPDH (Ambion, Austin, TX). Next, 20 µg total RNAunderwent hybridization with an excess of the 32P-labeledantisense RNA probe (Ambion). After hybridization, the RNAprobe hybrids were digested with a ribonuclease A/T1 cocktail,and electrophoresis on a 6% polyacrylamide-urea gel wasperformed. The gel was vacuum dried and exposed to film. Toquantify COX-2 mRNA levels, the autoradiographs wereanalyzed using densitometry, GAPDH RNA levels were

compared, and sample mRNA levels were determined withCOX-2 standards.

Results

COX-1 and COX-2 Protein Expression inNormal, Crohn’s Colitis, and UlcerativeColitis Epithelial Cell Lysates

Epithelial cells were isolated from surgical resec-tions for Crohn’s colitis and ulcerative colitis and fromhistologically normal areas of resections for colon cancer.Epithelial cell purity was assessed by staining withmonoclonal antibodies against cytokeratin and commonleukocyte antigen to identify epithelial cells and mono-nuclear cells, respectively. The cell populations used were.97% epithelial cells. Viability of epithelial cell popula-tions was .90% as assessed by trypan blue exclusion.Immunoblotting was performed using polyclonal rabbitanti-sheep COX-1 antibody (no. 241). This antibodyrecognized sheep COX-1 but not sheep COX-2 (Figure1A). This antibody labeled a single band of molecularweight 72 kilodaltons in lysates of normal colonic,Crohn’s colitis, and ulcerative colitis epithelial cells. Aband with the same molecular weight was identified inboth control and stimulated HUVECs. Similar resultswere obtained with the rabbit anti-mouse COX-1 anti-body.

Immunoblotting was also performed with a rabbitpolyclonal antibody against sheep COX-2 (no. 243). Thisantibody recognized sheep COX-2 but not sheep COX-1(Figure 1B). Two bands of molecular weight 74 and 68kilodaltons were stained in the sheep COX-2 sample. The

A B

Figure 1. Detection of COX-1 and COX-2 in epithelial cells isolated from normal, Crohn’s colitis, and ulcerative colitis mucosa by Western blotting.Lysates were made of epithelial cells isolated from Crohn’s colitis, ulcerative colitis (UC), and normal colonic mucosa. Sheep COX-1, sheep COX-2,lysates of unstimulated HUVECs, and lysates of HUVECs incubated with LPS and TNF-a were included as controls. Samples were separated by 8%sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Immunoblotting was performed using (A) polyclonal rabbit anti-sheep COX-1 antibodyno. 241 and (B) polyclonal rabbit anti-sheep COX-2 antibody no. 243. Bands were detected by enhanced chemiluminescence.

August 1998 CYCLOOXYGENASE 2 IN IBD 299

range in the molecular mass of COX-2 reflects the extentof glycosylation of the protein. The fully glycosylatedprotein is glycosylated at four sites and has a molecularmass of 74 kilodaltons, and the unglycosylated proteinhas a molecular mass of 65 kilodaltons.20 This antibodylabeled a single band of molecular weight 74 kilodaltonsthat was present in lysates of HUVECs stimulated withLPS and TNF-a and in lysates of epithelial cells isolatedfrom ulcerative colitis and Crohn’s colitis resections.However, no bands of these molecular masses weredetectable in lysates of unstimulated HUVECs or inlysates of colonic epithelial cells from histologicallynormal colon. Although the staining was less intense,bands migrating at these molecular masses were alsolabeled with Dina anti-COX-2 IgG (data not shown),thus confirming the results obtained with the no. 243COX-2 antibody.

COX-2 mRNA Levels in Normaland IBD Resections

To corroborate COX-2 protein expression data, weassayed Crohn’s colitis, ulcerative colitis, and normalmucosa for COX-2 mRNAs using RPAs. A representa-tive COX-2 RPA is shown in Figure 2. Comparison of thedensities of the bands for the normal and ulcerative colitissamples with the standards allows the quantification ofCOX-2 mRNA. COX-2 mRNA levels in regions withulcerative colitis (Table 1) or Crohn’s colitis were 3–5-fold greater than in normal resections.

Immunolocalization of COX-1 and COX-2in Normal, Crohn’s Colitis, and UlcerativeColitis Colonic Mucosa and in Normaland Crohn’s Ileitis Ileal Mucosa

Histologically normal areas of ileum and colonresected from patients with colon cancer and resectionsfor Crohn’s colitis, ulcerative colitis, and Crohn’s ileitiswere examined for expression of COX-1 using a rabbitpolyclonal antibody raised against a peptide from mouseCOX-1 recognizing human COX-1. In both Crohn’sileitis (Figure 3A) and normal ileum, there was expres-sion of COX-1 in the crypt epithelial cells but not invillus epithelial cells. There was also expression ofCOX-1 in scattered lamina propria mononuclear cells. Inthe epithelial cells, COX-1 expression was cytoplasmic.COX-1 expression in the normal ileum was identical tothat observed in Crohn’s ileitis (data not shown). Innormal colonic mucosa, (Figure 3B), COX-1 was ex-pressed in epithelial cells in the lower half of the cryptsand in scattered lamina propria mononuclear cells. Therewas no COX-1 expression in epithelial cells in the upperhalf of the crypts or in surface epithelial cells. Theimmunolocalization is cytoplasmic, the large apical holesin the immunofluorescence are mucus droplets, and thesmaller more basal holes are nuclei. The distribution ofCOX-1 immunolabeling in Crohn’s colitis and ulcerativecolitis mucosa was identical to that observed in normalcolonic mucosa. No staining was observed when normalrabbit serum was substituted for the primary antibody(data not shown).

Comparison of the histology of normal human colon(Figure 4A) with ulcerative colitis tissue (Figure 4C)shows a dense inflammatory infiltrate with macrophagesand neutrophils in the ulcerative colitis mucosa. COX-2immunohistochemistry using the rabbit anti-mouseCOX-2 IgG (Dina) in the normal colon (Figure 4B)showed no immunolabeling. In contrast, there wasintense immunolocalization of COX-2 epitopes to surfaceepithelial cells and lamina propria mononuclear cells inulcerative colitis mucosa (Figure 4D) with no detectableCOX-2 expression in the deeper crypt epithelium. Fur-ther investigation of COX-2 immunohistochemistry in

Figure 2. Detection of COX-2 mRNA in normal colonic mucosa andulcerative colitis mucosa by RPAs. Human COX-2 and GAPDH mRNAlevels were assayed by RPA. In vitro transcription was performed toproduce 32P-labeled antisense probes: 400 base pairs for COX-2 and434 base pairs for GAPDH. Total RNA (20 µg) extracted from themucosa of human colonic surgical resections was hybridized withexcess 32P probe as described in Materials and Methods. mRNA wasquantified by comparing the densitometric intensity of the protectedbands from the samples with the protected bands from knownamounts of in vitro transcribed sense-strand standards. This figure isa representative COX-2 RPA with samples from a normal colon, anulcerative colitis colon, and three different quantities of sense-strandcontrol RNA.

Table 1. COX-2 mRNA Levels in Lysates of Normal, UlcerativeColitis, and Crohn’s Disease Colons

Normal(n 5 4)

Ulcerativecolitis(n 5 3)

Crohn’sdisease(n 5 6)

COX-2 mRNA (pg/20 mgtotal RNA) 0.36 6 0.08 1.31 6 0.26a 1.77 6 0.69

NOTE. Values are expressed as means 3 6 SEM.aP , 0.05 compared with normal.


ulcerative colitis mucosa showed that staining of surfaceepithelial cells with rabbit anti-human COX-2 IgG (no.168) could be blocked with the peptide against which theantibody was raised (Figure 4F) but not with a controlCOX-1 peptide (Figure 4E).

In Crohn’s ileitis, COX-2 is expressed in epithelialcells on the villus (Figure 5A) but not in the crypt. In thenormal ileum, as in the normal colon, there is noepithelial staining for COX-2 (not shown). The pattern ofepithelial cell expression for COX-2 in Crohn’s colitis issimilar to that observed in ulcerative colitis with stainingof epithelial cells on the surface and in the upper half ofthe crypts (Figure 5B). In Crohn’s ileitis, the epithelialcell COX-2 staining localized to the perinuclear area,especially below the inferior portion of the nucleus, andin the Golgi, whereas in Crohn’s colitis, epithelial COX-2staining is only perinuclear. In both Crohn’s ileitis andCrohn’s colitis, there is COX-2 staining of some laminapropria mononuclear cells.

In active ulcerative colitis, surface epithelial cells areadjacent to inflammatory cells (Figure 6A). We previ-ously reported that iNOS was expressed by epithelial cellsin areas of inflammation in ulcerative colitis.16 Todetermine if COX-2 and iNOS were expressed in thesame population of epithelial cells in ulcerative colitis, weimmunolocalized COX-2 (Figure 6B) and iNOS (Figure6C) on adjacent sections of ulcerative colitis mucosa.COX-2 and iNOS were immunolocalized to the samepopulations of surface epithelial cells in the ulcerativecolitis colon, whereas nonimmune rabbit IgG producedno labeling (Figure 6D).

The epithelial cell COX-2 expression in ulcerative

colitis shown in Figures 4D and E and 6B represents thelabeling patterns observed in three surgical resectionsobtained from different patients with ulcerative colitis.We examined a total of five colonic resections frompatients with ulcerative colitis and six colonic and threeileal resections from patients with Crohn’s disease; all ofthem showed epithelial COX-2 expression. We studiedhistologically normal areas in five colonic resections andthree ileal resections from patients with cancer, and noneof them showed detectable COX-2 expression.

Discussion

In this study, we have shown by immunohisto-chemistry that COX-2 is expressed in surface epithelialcells in areas of inflammation in Crohn’s colitis andulcerative colitis and in villus epithelial cells in Crohn’sileitis. The induction of COX-2 expression in epithelialcells was confirmed by Western blotting of epithelial celllysates and was corroborated by the finding of an increasein COX-2 mRNA in Crohn’s colitis and ulcerative colitismucosa compared with normal mucosa as assessed byRPA. COX-2 protein was also localized in mononuclearcells in Crohn’s colitis, ulcerative colitis, and Crohn’sileitis mucosa. Although COX-2 was expressed in bothepithelial cells and mononuclear cells, the vast majorityof the protein appears to be in epithelial cells. Thepresence of large amounts of COX-2 in epithelial cells inCrohn’s disease and ulcerative colitis raises the possibilitythat the induction of epithelial COX-2 expression mayexplain the increased levels of PGE2 in IBD mucosa.13

COX-2 expression in epithelial cells may also explain the

Figure 3. COX-1 is expressed (A) in crypt epithelial cells in Crohn’s ileitis and (B) in epithelial cells in the lower half of the crypt in normal humancolonic mucosa. Immunohistochemical localization of COX-1 colon was assessed in tissue fixed in Bouin’s solution and embedded in paraffin.Immunoreactive COX-1 was detected by incubation with rabbit anti-mouse COX-1 antibody at a 1:2000 dilution. Bound antibody was visualizedusing the fluorescein-conjugated tyramide signal amplification system (green fluorescence). (A) In Crohn’s ileitis, COX-1 is localized to the cryptepithelial cells (closed arrow) and to lamina propria mononuclear cells (arrowhead) but not to villus epithelial cells (open arrow). (B) In normalhuman colon, COX-1 is localized to crypt epithelial cells in the bottom half of the crypt (closed arrow) and to scattered lamina propria mononuclearcells (arrowhead) but not to epithelial cells in the upper half of the crypt (open arrow) (bar 5 100 µmol/L).


large amounts of PGE2 present in rectal dialysates frompatients with ulcerative colitis.14

COX-1 was expressed in epithelial cells in the lowerhalf of the crypts in histologically normal colons, in

Crohn’s colitis and ulcerative colitis, and in the crypts innormal ileum and Crohn’s ileitis. This pattern of epithe-lial COX-1 expression corresponds to the zone of prolif-eration. The absence of COX-1 expression in epithelial

Figure 4. Immunolocalization of COX-2 expression in the normal and ulcerative colitis colon. Adjacent sections of (A and B) normal or (C–F) colitichuman colon were stained (A and C) with H&E, (B and D) with Dina IgG recognizing COX-2, or (E and F ) with no. 168 IgG recognizing COX-2. (A)Histologically normal colon shows an intact epithelium and no evidence of inflammation. (B) No COX-2 expression is detected in the apical(arrowheads) or crypt (arrows) epithelium or within the lamina propria (*). (C) Ulcerative colitis colon shows crypt dilatation (arrows) andinflammation and edema within the lamina propria. (D) Intense expression of COX-2 can be seen in the apical epithelium (arrowheads) and in cellsof the lamina propria (*) but not within the crypt epithelium (arrows). (E ) High levels of COX-2 labeling are detected in the surface epithelium inulcerative colitis (arrowheads) by affinity-purified no. 168 IgG in the presence of control COX-1 peptide. (F ) Incubation with the COX-2–blockingpeptide completely prevents staining of the surface epithelium (arrowheads) with the no. 168 anti–COX-2 IgG (bar 5 100 µmol/L).


cells in the upper half of the colonic crypts and in ilealvilli suggests that epithelial cells stop expressing COX-1as they become more differentiated. COX-1 expression inintestinal and colonic epithelial cells is cytoplasmic asopposed to COX-2 expression, which is perinuclear. Thedistribution of COX-1 expression in the human colonand ileum corresponds closely to that in the mouse smallintestine where COX-1 is expressed in the crypt epithe-lial cells but not in the villus epithelial cells.11 In themouse small intestine, as in the human ileum and colon,COX-1 expression is confined to the area of epithelialproliferation and is lost as the epithelial cells becomemore differentiated. The colocalization of COX-1 expres-sion and the proliferation zone are also observed in themouse stomach where epithelial COX-1 expression isconfined to the mucous neck cells.12 The similarity ofCOX-1 distribution in IBD and normal mucosa isconsistent with previous studies that showed that COX-1expression is not induced by inflammatory cytokines. Incontrast to COX-1, COX-2 was expressed in epithelialcells in the upper portions of the crypt and on the surfacein Crohn’s colitis and ulcerative colitis and in villusepithelial cells in Crohn’s ileitis and was not detectable inthe epithelium of the normal ileum or colon. Thesefindings suggest that COX-2 expression is induced byinflammatory mediators in the differentiated portion ofthe epithelium in Crohn’s colitis and ulcerative colitisand in Crohn’s ileitis.

What is the functional role of PGs produced throughepithelial cell COX-2 in ulcerative colitis and Crohn’sdisease? PGE2 induces epithelial cell chloride secretion21;it is possible that PGs produced through epithelialCOX-2 may contribute to the diarrhea observed in IBD.Salmonella infection induces epithelial cell COX-2 expres-sion and PGE2 production. It has been suggested that

PGE2 produced by epithelial COX-2 may contribute tothe secretory diarrhea observed in salmonellosis.5 Epithe-lial proliferation is increased in IBD, and PGE2 increasesepithelial proliferation in the face of injury.22,23 IBD ischaracterized by mucosal hyperemia, and PGE2 is avasodilator.24 PGE2 produced by epithelial cells maycontribute to the erythema observed in IBD. PGs alsoparticipate in the regulation of cytokine synthesis bymononuclear cells,25 and it is possible that PGs producedin epithelial cells may act to regulate cytokine productionby lymphocytes and macrophages.

There have been reports suggesting that NSAIDsincrease the level of clinical activity and worsen thesymptoms in IBD.15 The mechanism of the clinicalexacerbation of IBD by NSAIDs is not known. Whichcell types and which of the isoforms of COX (COX-1 orCOX-2) are involved in NSAID-induced exacerbationshave not been studied. Currently available NSAIDsinhibit both COX-1 and COX-2. There have beensuggestions that the anti-inflammatory effects of NSAIDsrelate to COX-2 inhibition and that the gastrointestinalinjury, particularly the gastropathy, induced by NSAIDsrelates to COX-1 inhibition.1 The association of NSAID-induced injury with COX-1 is reasonable in that, innormal gastrointestinal mucosa, COX-1 is expressed insome abundance, whereas little or no COX-2 is expressedin the normal human gastrointestinal tract.26,27

COX-2 expression has also been reported in thetrinitrobenzene sulfonic acid (TNBS)-induced rat modelof colitis. There was a sixfold increase in COX-2 mRNA72 hours after administration of TNBS.28 Immunolocal-ization of COX-2 expression in the TNBS model showedexpression in smooth muscle, infiltrating cells of thesubmucosa, and submucosal blood vessels but not inepithelial cells. Treatment with a selective COX-2 inhibi-

Figure 5. Localization of COX-2 in (A) Crohn’s ileitis and (B) Crohn’s colitis. (A) In Crohn’s ileitis, COX-2 is expressed in villus epithelial cells (arrow)and lamina propria mononuclear cells (arrowhead ) but not in crypt epithelial cells. In the villus epithelial cells, COX-2 is expressed in theperinuclear area, especially at the base of the nucleus, and in the Golgi. (B) In Crohn’s colitis, COX-2 is expressed in surface epithelial cells (arrow)and scattered lamina propria mononuclear cells (arrowhead ) but not in crypt epithelial cells. In the surface epithelial cells, COX-2 is expressed inthe perinuclear area, especially at the base of the nucleus.


tor resulted in worsening of the colitis and perforation ofthe colon. Thus, this study suggests that PGs producedthrough COX-2 promote wound healing in gastrointesti-nal mucosal injury. The TNBS study did not address themechanism by which PGs may affect wound healing.

The expression of COX-2 in other forms of gastrointes-tinal injury, the inhibition of gastrointestinal woundhealing by selective COX-2 inhibitors, and the therapeu-tic efficacy of exogenous PGs in gastrointestinal injury allraise the possibility that the expression of COX-2 inepithelial cells in IBD is a protective response of thewound healing process. These studies also raise thepossibility that the exacerbation of clinical activity inulcerative colitis by NSAIDs could result from inhibitionof epithelial cell COX-2 and the resultant impairment ofwound healing. Selective COX-2 inhibitors are being

developed in the hope that they will have the anti-inflammatory effects of currently available NSAIDs with-out the gastrointestinal toxicity. If epithelial COX-2activity is important in wound healing, then selectiveCOX-2 inhibitors may be as likely to exacerbate ulcer-ative colitis as conventional NSAIDs. However, the use ofselective COX-2 inhibitors as analgesics and anti-inflammatory agents would not be expected to damagethe normal gut because it does not express COX-2.

Gastrointestinal injury and inflammation result inchanges in epithelial cell gene expression. IL-8 is ex-pressed in epithelial cells in IBD29 and is induced byinfection of epithelial cells by Salmonella.30 Manganesesuperoxide dismutase is expressed in epithelial cells incolitis.31 We previously reported that iNOS is expressedin epithelial cells in ulcerative colitis17 and, in the present

Figure 6. Expression of COX-2 and iNOS are colocalized in the surface epithelium of the ulcerative colitis colon. (A) H&E-stained section showingcrypt dilatation and inflammation of the lamina propria. (B) Adjacent section stained with Dina anti–COX-2 IgG shows intense COX-2 localization inthe surface epithelium (arrowheads) that abruptly diminishes at the zone of transition into the proximal portion of the crypt (arrows). (C)Neighboring section shows intense iNOS expression localized in the same regions of the colonic epithelium that showed high COX-2 labeling(corresponding arrowheads and arrows in B). (D) Another section stained with nonimmune rabbit IgG is completely unlabeled (bar 5 100 µmol/L).


study, found that the apical epithelial cells that expressCOX-2 also express iNOS. COX-2, iNOS, and IL-8 arethree of a group of inducible proteins whose promotershave nuclear factor kB response elements.32 Nuclearfactor kB is activated in cells responding to cytokines,including IL-1b and TNF-a, and to oxidants such ashydrogen peroxide. Elevated levels of IL-1b and TNF-aare formed in IBD mucosa,33,34 and the intense infiltra-tion with activated neutrophils provides a source ofsuperoxide, hydrogen peroxide, and other oxidants.35 Thecoexpression of COX-2 and iNOS in IBD epithelial cellsraises the possibility that other nuclear factor kB–relatedproteins, including intercellular adhesion molecule 1,may also be expressed in epithelial cells in IBD. This localexpression of iNOS might also enhance the activation ofCOX-2.36

Ulcerative colitis and, to a lesser extent, Crohn’s colitisare associated with an increased risk of colon cancer.COX-2 is expressed in colonic adenomas and in adenocar-cinomas in humans.9,10 Human familial adenomatouspolyposis is associated with numerous colonic polyps andthe early onset of colon cancer. The APC knockout mouseis a model of human familial adenomatous polyposis.Mice heterozygous for the APC gene knockout have alarge number of COX-2–expressing polyps. In a recentstudy, COX-2–deficient mice were crossed with miceheterozygous for the APC gene knockout.37 Mice hetero-zygous for the APC gene knockout that also have adisrupted COX-2 gene have far fewer polyps, suggestingthat PGs produced through COX-2 may play a role in theincreased risk of colon cancer in ulcerative colitis. Thisraises the possibility that inhibition of COX-2 mayreduce the risk of malignancy.

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Received November 14, 1997. Accepted April 23, 1998.Address requests for reprints to: William F. Stenson, M.D., Wash-

ington University School of Medicine, Campus Box 8124, St. Louis,Missouri 63110. Fax: (314) 362-8959.

Supported by grant DK33165 from the National Institutes ofHealth (to W.F.S.).


Cyclooxygenase 2 is induced in colonic epithelial cells in inflammatory bowel disease

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