7 Clin Pathol 1992;45:784-787

Collagenous colitis: Jejunal and colorectalpathology

J Armes, D C Gee, F A Macrae,W Schroeder, P S Bhathal

AbstractAims: To determine: (1) whether there isan association between collagenous colitisand coeliac disease or lymphocytic colitis;(2) the distribution of lymphocyte subsetsand macrophages in the lamina propriaand surface epithelial layer in collagenouscolitis; and (3) the colorectal distributionof the disease and whether a mucosalbiopsy specimen, using a flexible sigmoid-oscope, is sufficient to diagnose it.Methods: The clinical data and colorectalbiopsy specimens from 38 patients withcollagenous colitis were studied. In 10,small bowel biopsy specimens were alsoavailable for review. Immunostaining ofthe mucosal lymphoid infiltrate with apanel of relevant antibodies was carriedout on formalin fixed tissue in seven cases;in three the phenotyping was performedon fresh biopsy specimens separately fro-zen or fixed in B5 solution.Results: Coeliac disease was found in fourout of the 10 patients with collagenouscolitis who had had a small bowel biopsy,in contrast to the prevalence ofthe diseasein Australia of 1 in 3000. Collagenouscolitis did not respond to gluten with-drawal. Five of 29 (17%) of the patientshad a mixed pattern of lymphocytic andcollagenous colitis. Immunostaining ofthe lymphoid infiltrate showed that thestriking increase in intraepithelial lym-phocytes in collagenous colitis was due toan influx ofCD8 positive cells. The occur-rence and severity of collagenous colitisalong the large bowel were independent ofthe anatomical site, and in more than 90%of cases biopsy specimens from the sig-moid colon or rectum were diagnostic.Conclusions: There is a very high inci-dence of coeliac disease among patientswith collagenous colitis so that jejunalbiopsy should be an essential part of theirinvestigations, especially if symptomspersist. However, only a minority showeda mixed pattern of lymphocytic and col-lagenous colitis. The intraepithelial lym-phocytes in collagenous colitis are CD8positive cells. Collagenous colitis can bediagnosed from rectal or sigmoid colonbiopsy specimens in more than 90%/o ofcases.

Collagenous colitis is characterised by chronicwatery diarrhoea, minimal or absentendoscopic findings, and the presence of

microscopic mucosal inflammation, togetherwith thickening of the subepithelial collagenplate.'

Sporadic cases of collagenous colitis inpatients with coeliac disease have been repor-ted2 and a similar aetiology for coeliacdisease and collagenous colitis has been pro-posed.6 However, in two recent studies ofpatients with coeliac disease no associationwith collagenous colitis was found, althoughlymphocytic colitis was detected in some of thepatients.8 9 Lymphocytic colitis is similar clini-cally to collagenous colitis, but differs micro-scopically in the absence of a thickenedcollagen plate.6 7The association between collagenous colitis

and both coeliac disease and lymphocyticcolitis was investigated in a histopathologicaland limited immunohistological study of 38patients with collagenous colitis. We also deter-mined the anatomical distribution of col-lagenous colitis within the large bowel, becauseawareness of its distribution should helplocalise the site for a diagnostic endoscopicbiopsy.

MethodsLarge bowel biopsy specimens from 38patients with collagenous colitis taken between1986 and 1991 were reviewed from the files ofThe Royal Melbourne Hospital and a largeprivate pathology practice. All patients hadchronic watery diarrhoea and met the histo-logical criteria of collagenous colitis compris-ing band-like thickening of the collagen plate( t 10 pm), an increase in surface intra-epithelial lymphocytes, surface epithelial dam-age with loss of mucin production and cellularcuboidalisation, and a mixed inflammatory cellinfiltrate in the lamina propria and normalcrypt architecture.6 7 10 The biopsy specimenshad been routinely fixed in formalin, embed-ded in paraffin wax, and stained with haema-toxylin and eosin and Masson's trichrome orthe picro-Mallory stain. In each case thethickness of the collagen plate was measuredwith a graticule.Duodenal or jejunal biopsy specimens were

also available from 10 of the 38 patients. Dataregarding the anatomical site of multiple largebowel biopsy specimens were available in 19 ofthe 38 cases; 12 had specimens taken from theleft and right colon and the rectum, and sevenhad them taken from the left and right colon,but not the rectum. Nineteen of the 38 patientshad specimens taken from either the rectum orthe sigmoid colon. Twenty nine of the 38

Department ofAnatomical Pathology,The Royal MelbourneHospital, Parkvi1le,Victoria 3050,AustraliaJane ArmesW SchroederP S BhathalMelbourne Pathology,Collingwood, Victoria,AustraliaD C GeeDepartment ofGastroenterology, TheRoyal MelbourneHospitalF A MacraeCorrespondence to:Professor P S BhathalAccepted for publication24 February 1992

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Collagenous colitis

patients had multiple synchronous large bowelbiopsy specimens taken, allowing the histo-logical features within one patient to becompared.Immunostaining of the mucosal lymphoid

infiltrate in formalin fixed, paraffin wax

embedded tissue was carried out in seven cases

using a panel of antibodies (table) and theperoxidase anti-peroxidase technique. In an

additional, three patients fresh biopsy speci-mens were separately frozen or fixed with 10%formalin or B5 solution. Lymphocyte subsetdistribution in cryostat sections was deter-mined by using additional antibodies (table).Non-inflamed colorectal mucosal biopsy speci-mens were available from four patients under-going colonoscopy for diarrhoea, who were

subsequently found to have the irritable bowelsyndrome. These were used as control speci-mens in the immunohistological analysis.

ResultsOf the 38 patients with collagenous colitis inthe study, 27 (71%) were female and I 1(29%) were male, giving a gender ratio of2-45:1. The age range for males was 50-86years, with a mean (SD) of 67-8 (9-92) years;

the age range for females was 25-83 years, witha mean (SD) of 65-0 (16-3) years.

ASSOCIATION BETWEEN COLLAGENOUS COLMS

AND COELIAC DISEASE

Four of the 10 patients from whom duodenaland jejunal biopsy specimens were availablehad microscopic features consistent with coe-

liac disease. In two the diagnosis of coeliacdisease preceded that of collagenous colitis andboth were already excluding gluten from theirdiet. Of the four patients with coeliac disease,two had broad collagen bands within thelamina propria, an appearance consistent withcollagenous sprue. Two of the four patients(including one with collagenous sprue) had a

small bowel biopsy repeated after adherence toa gluten free diet and both showed reversal oftheir small bowel pathology. However, thecollagenous colitis did not respond to a glutenfree diet. The remaining six patients withinthis subset had normal upper small bowelhistology.

LYMPHOCYTE SUBSET DISTRIBUTION

Compared with the control colorectal tissue,immunohistological analysis of large bowelbiopsy specimens from seven patients withcollagenous colitis showed that the increase ofintraepithelial lymphocytes was caused byhigher numbers of T lymphocytes. Further-

more, in three of these cases, immunostainingfor lymphocyte subsets on frozen sectionsshowed that the intraepithelial lymphocyteswere CD8 positive. In contrast, the laminapropria contained more CD4 positive lympho-cytes than CD8 positive lymphocytes (fig 1).Both B lymphocytes and macrophages were

present throughout the lamina propria, butmacrophages were more numerous within thesuperficial lamina propria, in close proximityto the surface epithelium.

ASSOCIATION BETWEEN LYMPHOCYTIC AND

COLLAGENOUS COLITIS

To determine whether multiple biopsy speci-mens taken from different sites from the same

patient would show a combination of bothlymphocytic and collagenous colitis (mixedpattern of disease) or whether only collagenouscolitis would be present, 29 of the 38 cases

were studied because biopsy specimens frommultiple sites were available for them. Only fiveof the 29 (17%) patients had both collagenouscolitis and lymphocytic colitis when the speci-mens were taken from different sites synchro-nously (fig 2). In four of the five patients withmixed colonic disease the thickened collagenplate was present in the distal large bowel, butthe proximal colon showed features of lympho-cytic colitis. Interestingly, in contrast to thefemale predominance of the study group as a

whole, four of the five patients with the mixedpattern of colonic disease were male.

ANATOMICAL DISTRIBUTION OF COLLAGENOUS

COLITIS

In 19 of the 38 patients multiple biopsyspecimens taken from different named siteswithin the large bowel were available. In 12 ofthese patients it was possible to assess whetherthe right or left colon or rectum were more

severely affected (determined by the thicknessof the collagen plate), and in a further seven

patients the severity of the disease between theleft and right colon could be compared. Nosignificant difference emerged. Of the 19patients who had biopsy specimens taken fromeither the rectum or sigmoid colon, 18 showedthat a biopsy specimen from one of these twosites was diagnostic for collagenous colitis.

DiscussionCollagenous colitis causes chronic watery diar-rhoea that is associated with well definedhistopathological changes in the large bowelmucosa. However, there are uncertaintiesregarding its pathogenesis, its association with

Panel of antibodies used in immunohistological study

Formalin or B5-fixed, paraffin wax embedded Fresh frozen tissue, cryostat sections

Antibody (source) Moleculelcell detected Antibody (source) Moleculelcell detected

L26 (Dako) B cell BI (Coulter) B-cell4KB5 (Dako) B cell B2 (Coulter) B-cellLeu22 (B-D*) T cell Leu4 (B-D) CD3/T-cellUCHL1 (Dako) T cell Leu3/3a (B-D) CD4/TH/I-cellMac387 (Dako) Macrophage, monocyte, OKT8 (Ortho) CD8/Ts/c-cell

granulocyte LeuM5 (B-D) Macrophage, monocyte

*B-D Becton Dickinson

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Figure I Frozen sections of large bowel mucosa in collagenous colitis immunostainedthe peroxidase-antiperoxidase method. (A) The infiltrate in the lamina propria (7p) iCD4 + T cells. The lymphocytes in the surface epithelium are CD4 - (arrows). (B) A

contrast, CD8+ T cells populate both the lamina propria and epithelium.

both coeliac disease and lymphocytic coland the anatomical distribution of the dis4within the large bowel.Of the 10 patients from whom small bc

biopsy specimens were available, four shoimicroscopic features consistent with coedisease, indicating a significant associatioiview of the prevalence of coeliac disease inAustralian population of 1 in 3000. Thusstudy complements earlier case reports wlindicate that patients with coeliac disease 1

also develop collagenous colitis2'- and diffrom the studies by Du Bois et al andWolbal which did not detect collagenous colitipatients with coeliac disease, but rather folymphocytic colitis in some of them.8" Inestingly, two of our four cases also hadincrease of collagen within the small bclamina propria, consistent with collagensprue, an association that has been ncbefore by other workers.'2 13 Our findisuggest that jejunal biopsy should beessential part of the investigative worku;patients with collagenous colitis, especial]symptoms persist.The pathogenesis of collagenous cc

remains undetermined. Attention has beendrawn to similarities between the pathogenesisof collagenous colitis and coeliac disease.6Coeliac disease is thought to be an immuno-pathological disease caused by sensitivity to thewheat protein gluten, perhaps triggered byadenovirus type 12 infection in certain suscep-tible individuals.14 In our four patients withcoeliac disease collagenous colitis persisteddespite a gluten free diet. Indeed, in two ofthese patients the discovery of collagenouscolitis followed the diagnosis of coeliac diseaseand the start of a gluten free diet. Theresistance of collagenous colitis to a gluten freediet has been reported before.24 Thus itwould seem that collagenous colitis is not theresult of sensitivity to gluten present within thelarge bowel. However, as the increased numberof intraepithelial lymphocytes is a strikingfeature of both coeliac disease and collagenouscolitis, it is still possible that, in collagenouscolitis, sensitivity to a luminal agent other thangluten may stimulate an immunopathologicalresponse.Immunostaining of the biopsy specimens in

a proportion of our patients with collagenouscolitis showed that, as in coeliac disease, theintraepithelial lymphocytes were of T cellorigin. Furthermore, lymphocyte subset typingin three cases showed that the intraepitheliallymphocytes were CD8 positive, while mostlymphocytes within the lamina propria were ofCD4 subtype. This finding is similar to thatseen in coeliac disease, where an increase ofintraepithelial CD8 positive lymphocytes hasbeen described."5The distribution of lymphocyte subsets

within the inflamed large bowel mucosa ofpatients with collagenous colitis sheds addi-tional light on the pathogenesis of the disease.CD8 positive T cells show MHC class Irestriction, and in this context the antigenpresented is usually a processed peptide of anendogenous antigen selectively bound to class Imolecules, unlike antigen presented in associa-

e tion with MHC class II to CD4 positivelymphocytes, which is processed exogenousantigen.16-18 In collagenous colitis, therefore,GCD8 positive intraepithelial lymphocytes may

c be stimulated to react by a luminal agent whicha leads to immuno-crossreactivity with ane endogenous antigen produced by the surfaces enterocytes.h Collagenous colitis shows clinical andy microscopic features similar to those of

lymphocytic colitis. Indeed, the histologicalt features differ only in terms of collagen platea thickening. It has therefore been suggested thatI lymphocytic colitis is an earlier stage of col-

lagenous colitis, before the onset of collagena deposition,' " or that collagen deposition may

be absent or borderline from certain parts ofs the large bowel, particularly the distal coloni and rectum.t020 Some ical aspects of thes two diseases, however, indicate that they area separate entities: the equal gender ratio presentof in lymphocytic colitis, as opposed to the femaleif excess in collagenous colitis; and the differing

HLA predominance between the two dis-s eases-namely, HLA-A1 and HLA-DRW53 in

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Figure 2 Biopsy specimens taken simultaneously from the caecum and sigmoid colon inone patient showing: (A) features of lymphocytic colitis in the caecum comprising acollagen table of normal thickness and intraepithelial lymphocytic infiltration (arrows);but (B) features of collagenous colitis in the sigmoid colon with band-like thickening ofthe collagen plate (arrowheads) (Masson trichrome.)

lymphocytic colitis-but no distinct HIA link-age in collagenous colitis.2' A mixed pattern oflymphocytic and collagenous colitis was foundin five of the 29 patients in whom multiplelarge bowel biopsy specimens were taken at thesame time. In these patients with mixedcolonic disease absence of a thickened collagenplate did not seem to depend on the site of thebiopsy. In each case multiple biopsy specimenswere only taken on one occasion and thereforethe possibility of subsequent development ofcomplete collagenous colitis could not beassessed. Unlike the female preponderanceseen in the main study group (71% comparedwith 29%) and previously reported in patientswith collagenous colitis,6 four of the fivepatients with the mixed colonic disease in thisstudy were male. Hence these findings wouldadd support to the hypothesis that collagenouscolitis and lymphocytic colitis are separatediseases with similar, but not identical, histo-logical appearances.

Because macroscopic evidence of collage-nous colitis is not seen during endoscopicexamination, it is important to establish the

distribution of disease within the large bowel todetermine the site most likely to yield adiagnostic biopsy specimen. It has been repor-ted that in collagenous colitis the collagen plateis thicker in the proximal colon compared withthe sigmoid colon and rectum,'0 and Giar-diello et al found that the thickened collagenplate may be absent in the rectum.20 However,our study confirms that of Lazenby et al, whofound that there was little difference betweenthe degree of collagen thickening in proximaland distal large bowel in patients with col-lagenous colitis.6 Hence, on the basis of thepresent study, it seems that biopsy specimenstaken with a flexible sigmoidoscope are suffi-cient to diagnose collagenous colitis in over90% of cases.This study was presented in part at the 36th Annual ScientificMeeting of The Royal College of Pathologists of Australasia,Melbourne, 30 September-4 October 1991.We thank Mr F Fellepa and Ms L Turnham for their excellentlaboratory assistance and Ms T Dentry for typing the manu-script. We also thank the many gastroenterologists and surgeonswho allowed us to study their patients' biopsy specimens.

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2 Hamilton I, Sanders S, Hopwood D, Bouchier IAD.Collagenous colitis associated with small intestinal villousatrophy. Gut 1986;27:1394-8.

3 Breen EG, Coughlan G, Connolly CE, Stevens FM,McCarthy CF. Coeliac proctitis. Scand J Gastroenterol1987;22:471-7.

4 Cadiot G, Flourie B, Galian A, Lavergne A, Modigliani R.Coeliac disease and collagenous colitis. A fortuitousassociation. Presse Med 1990;19:1621-2.

5 O'Mahony S, Nawroz IM, Ferguson A. Coeliac disease andcollagenous colitis. Postgrad Med J 1990;66:238-4 1.

6 Lazenby AJ, Yardley JH, Giardiello FM, Jessurun J, BaylessTM. Lymphocytic ("microscopic") colitis: A compar-ative histopathologic study with particular reference tocollagenous colitis. Hum Pathol 1989;20: 18-28.

7 Yardley JH, Lazenby AJ, Giardiello FM, Bayless TM.Collagenous, "microscopic", lymphocytic, and othergentler and more subtle forms of colitis. Hum Pathol1990;21:1089-91.

8 DuBois RN, Lazenby AJ, Yardley JH, Hendrix TR, BaylessTM, Giardiello FM. Lymphocytic enterocolitis inpatients with 'refractory sprue'. JAMA 1989;262:935-7.

9 Wolber R, Owen D, Freeman H. Colonic lymphocytosisin patients with coeliac sprue. Hum Pathol 1990;21:1092-6.

10 Jessurun J, Yardley JH, Giardiello FM, Hamilton SR,Bayless TM. Chronic colitis with thickening of thesubepithelial collagen layer (collagenous colitis): Histo-pathologic findings in 15 patients. Hum Pathol 1987;-18:839-48.

11 Porter NJ, Duggan JM. Epidemiology of coeliac disease inan Australian community [abstract]. Aust NZJMed 1990;20:363.

12 Eckstein RP, Dowsett JF, Riley JW. Collagenous entero-colitis: A case of coilagenous colitis with involvement ofthe small intestine. Am Jf Gastroenterol 1988;83:767-7 1.

13 Stolte M, Ritter M, Borchard F, Koch-Scherrer G. Col-lagenous gastroduodenitis on collagenous colitis. Endo-scopy 1990;22:186-7.

14 Kagnoff MF, PatersonYJ, Kumar PJ, et al. Evidence for therole of a human intestinal adenovirus in the pathogenesisof coeliac disease. Gut 1987;28:995-1001.

15 Selby WS, Janossy G, Bofill M, Jewell DP. Lymphocytesubpopulations in the human small intestine. The findingsin normal mucosa and in the mucosa of patients withadult coeliac disease. Clin Exp Immunol 1983;52:219-28.

16 Germain RN. The ins and outs of antigen processing andpresentation. Nature 1986;322:687-9.

17 Townsend A, Bodmer H. Antigen recognition by class Irestricted T lymphocytes. Ann Rev Immunol 1989;7:601-24.

18 Guagliardi LE, Koppelman B, Blum JS, Marks MS,Cresswell P, Brodsky FM. Co-localization of moleculesinvolved in antigen processing and presentation in anearly endocytic compartment. Nature 1990;343:133-9.

19 Sylwestrowicz T, Kelly JK, Hwang WS, Shaffer EA. Col-lagenous colitis and microscopic colitis: The waterydiarrhoea-colitis syndrome. Am J Gastroenterol 1989;84:763-8.

20 Giardiello FM, Bayless TM, Jessurun J, Hamilton SR,Yardley JH. Collagenous colitis: Physiologic and histo-pathologic studies in seven patients. Ann Intern Med1987;106:46-9.

21 Giardiello FM, LazenbyAJ, BaylessTM, et al. Lymphocytic(microscopic) colitis. Clinicopathologic study of 18patients and comparison to collagenous colitis. Dig Dis Sci1989;34: 1730-8.

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