SCANDINAVIAN JOURNAL OF GASTROENTEROLOGY Scandinavian Journal of Gastroenterology is the scientific periodical for Dansk Gastroenterology Selskab, Finsk Gastroenterologisk Forening, Islandsk Gastroenterology Forening. Norsk Gastro- enterologisk Forening. and Svcnsk Forening for Gastroenterologi.

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Typeset bv Gardincr-Caldvvcll Communications Limited. Macclesfield, CONCEPTS AND CONTROVERSY IN GASTROENTEROLOGY

Proceedings from a satellite symposium Säo Paulo, Brazil

10 September 1986

Edited by: James Freston, U.S.A. Roy Pounder, U.K.

Associate Editor: David Caldwell, U.K.

Contents

Preface

James Freston and Roy Pounder 5

SESSION I: Concepts

C h a i r m a n: Jam es Freston 7 Chairman's Introduction

James Freston # CASE 1: Even Simple Ulcers Can Become Tricky

Melvyn Korman 9 CASE 2: The Chronically Recurring Ulcer

Anders Walan 14 CASE 3: Bleeding Ulcer - An Unwanted Travel Companion

John Alexander-Williams 21 CASE 4: Ulcers Recurring During Maintenance Treatment - Are They Different?

Henk Festen 25 Discussion 27 Chairman's Summary 27 References 28 Appendix I: Demographic Data 29 SESSION II: Controversy- Chairman: Roy Pounder 31 Introduction to the Debate ROY Pounder 32 Proposing the Motion

Michael Clark 33 Opposing the Motion

Duncan Colin-Jones 35 Seconding the Motion

Wilfred Weinstein 37 Seconding the Opposition

Cab fiel Bianchi Porro 39 Contribution from the Floor 41 Summary Against

Duncan Colin-Jones 44 Summary For

Michael Clark 45 The Vote 46 References 46 Appendix II: Demographic Data 47 Autoimmunity to Pancreatic Juice in Crohn's Disease Results of an Autoantibody Screening in Patients with Chronic Inflammatory Bowel Disease

W. STÖCKER, M. OTTE, S. ULRICH, D. NORMANN, H. FINKBEINER. K. STÖCKER, G. JANTSCHEK & P. C. SCRIBA Dept. of Medicine and Dept. of Psychosomatic and Psychotherapy, Medical University of Lüheck, Lübeck, FRG

Stöcker W, Otte M, Ulrich S, Normann D, Finkbeiner FI. Stöcker K, Jantschek G. Scriba PC. Autoimmunity to pancreatic juice in Crohn's disease. Results of an autoantibody screening in patients with chronic inflammatory bowel disease. Scand J Gastroenterol 1987, 22 (suppl 139), 41-52

The sera of 59 patients with Crohn's disease (CD) and of 46 patients with ulcerative colitis (UC) were tested for autoantibodies (Aab) by indirect immunofluorescence with modern histochemical techniques using 19 different human tissues as antigenic substrates. Control collectives consisted of 19 patients with coeliac disease and of 100 healthy subjects. It was possible to demonstrate a specific marker for CD: Aab against exocrine (Pab) were present in 39% of the CD sera (UC 4%, coeliac disease 0%, healthy controls 3%). High Pab titres were only detectable in CD sera (29%). The CD-related autoantigen was demonstrated to be a component of normal pancreatic juice. Pab in CD were fundamentally different from those sometimes occurring in chronic and acute pancreatitis. It is suggested that CD is caused by autoimmune reactions against a component of pancreatic juice. Pab in CD correspond to Aab against intestinal goblet cells (Gab), which occurred exclusively in UC (28%). Pab and Gab, but obviously none of the other Aab investigated in this study, are of diagnostic value in chronic inflammatory bowel disease.

Key words: Autoantibodies; autoimmunity; chronic inflammatory bowel disease; coeliac disease; Crohn's disease, aetiology; Crohn's disease, serodiagnosis: flu• orescent antibody techniques; pancreatic autoantigens; pancreatitis; ulcerative colitis, serodiagnosis

Dr. med. W. Stöcker, Klinik für Innere Medizin, Medizinische Universität, Rat• zeburger Allee 160, D-2400 Lübeck, FRG

In chronic inflammatory bowel disease (CIBD), Crohn's disease (CD), only very few studies have a number of observations support the concept of dealt with the prevalence of Aab directed against an autoimmune aetiology. Remarkably, in the other than bowel antigens in CIBD, and the sera of some patients with ulcerative colitis (UC) number of Aab tested were limited (8, 11-13). To autc antibodies (Aab) can be observed which obtain a comprehensive overview on Aab in react with antigens of intestinal goblet cells. These CIBD, Aab profiles were established in sera from antibodies were first demonstrated by means of patients with CD and UC. Patients with coeliac hemagglutination and gel diffusion methods (1) disease, as well as healthy subjects without evi• and later by indirect immunofluorescence (2-10). dence of bowel disease, served for control. S.nce in CIBD primarily the bowel is As reported earlier (10), in these experiments concerned, studies were usually restricted to Aab the occurrence of Aab to intestinal goblet cells against intestinal tissue. Despite the frequently (Gab) in UC could be confirmed. Beyond this, occirring extraintestinal manifestations of it was possible to demonstrate CD-specific Aab 42 W. Stöcker et al. against an antigen of the exocrine pancreas (blood group O) as antigenic substrates which (Pab). In the present paper, the results of the included human adult pancreas, parotis, liver, Aab screening are summarized and further data lung, striated and smooth muscle, heart, thyroid are provided to evaluate the significance of the (unfixed sections plus methanol-fixed sections), established CD-specific autoimmunity. parathyroid, adrenal, ovary, placenta, testis, hypophysis, kidney, and sterile human fetal esophagus, stomach, , ileum, and MATERIALS AND METHODS colon. Normal adult human tissue was obtained Subjects at surgery, fetal tissue from a still-born infant at Serum samples were obtained from patients 32 weeks' gestation. Organs were snap-frozen in with Crohn's disease (n = 59), ulcerative colitis melting isopentane and stored up to 3 years at (n = 46), coeliac disease (n = 19), and healthy -196°C. subjects (n = 100). Diagnoses of the patients were The test procedure was facilitated by new rapid established by clinical examination and, in each and economic microtechniques described earlier instance, confirmed both by endoscopy and (15, 16). In brief, 4-//m tissue sections were cut histology. with a Leitz-1720 cryotome (Leitz. Wetzlar, Patients with Crohn s disease (CD). This group ERG) at -22 °C. Sections were applied to chemi• includes 22 men (average age, 26.0 years) and 37 cally activated glass cover slides, thawed, and women (average age, 28.4 years). At time of dried. The cover slides were cut with a glazier's blood drawing, 49 patients with CD received sali- diamond, and broken into pieces (fragmenting cylazosulfapyridin, 43 patients of them were technique). Chemical activation (17) was per• treated additionally with corticosteroids. Disease formed, to enable covalent coupling of the tissue activity was estimated according to Best et al. (14; sections to the glass surface, thus effectively CD AI). In 14 of the CD patients symptoms were improving the adhesion of the sections. recorded which can be regarded as extraintestinal Section fragments, carrying relevant organ manifestations of the disease. None of the patients structures, were sealed in blisters and stored in with CD had clinically apparent chronic or acute liquid nitrogen until use. They were glued on pancreatitis. reaction areas of a glass plate (sections" support), Patients with ulcerative colitis (UC). Of 46 samples or fluorescein-labelled antisera were pip• patients with UC, 23 men (average age, 40.3 etted on hydrophilic reaction areas of another, years) and 23 women (average age, 34.5 years), plane, glass plate (reagents' support). The reac• 40 had been treated with salicylazosulfapyridin, tion areas were surrounded by a hydrophobic 30 also with corticosteroids. The current activity coating, which prevented mixing of neighbouring of UC was assessed using a modified CDAI. samples. The sections' support was laid above the Control subjects. Collectives of 19 patients with reagents' support in such a way that the tissue coeliac disease (6 men, average age 52.2 years, sections were immersed in the liquid (titerplane and 13 women, average age 54.8 years) and of technique). 100 healthy volunteers (62 men; average age, 34.3 Aab screening was performed by a composite- years; and 38 women; average age. 28.6 years) section technique. Section fragments of 19 dif• served for control. ferent tissues were glued side by side on large Blood was drawn from all subjects for deter• reaction areas and co-incubated in the same drop mination of autoantibody profiles by indirect of sample or reagent (200//l per 19 sections). The immunofluorescence. Sera were separated and single-section technique was used to determine stored at -80 °C for up to 2 years before testing. the immunoglobulin classes of established Aab. to identify their ability to fix complement, anc to Screening for autoantibodies determine Aab concentrations by titration: Sec• Aab were determined by indirect immuno• tion fragments were glued on small reaction areas fluorescence, employing a broad panel of tissues and incubated in droplets of 10 ul, each section Autoantibodies to Pancreatic Juice in CD 43 fragment being immersed individually in a single Pab neutralization technique (18): Diluted Pab- droplet of the liquids. containing CD sera were incubated for one hour with equal volumes of different diluted organ immunoflnoreseence staining homogenates or different liquids: Composite sections were incubated for 60 min Supernatants of homogenized human liver, w ith sera which were usually diluted 1: 10 ih pho• heart, pancreas (homogenization was accom• sphate-buffered saline. Sections were stained for plished by producing 5-j.im-thick frozen sections a further 60 minutes with a polyvalent fluorescein- with the cryotome; to the slices nine volumes of conjugated rabbit antihuman immunoglobulin, buffered saline were added); directed against IgA + IgG + IgM + kappa + Human duodenal juice; lambda (DAKO, Hamburg, FRG). The sections Human pancreatic juice (collected by endo• were washed for 60 min after each stage in three scopic cannulation of the main ); changes of phosphate-buffered saline, with Fluids of pancreatic pseudocysts (obtained gentle agitation. Positive sera were titrated in from patients with chronic pancreatitis for diag• steps of 1:32, 1:100, 1:320, etc., using single nostic purposes); or sections. FPLC column effluents (fast protein liquid Heavy- and light-chain specificities of Pab and chromatography). Gab were determined using monospecific fiuor- After incubation, titres of free Pab in the mix• escein-conjugated rabbit antihuman IgA, IgD, tures were determined by the indirect fluorescent IgE, IgG, IgM, kappa and lambda antibody test using frozen sections of human adult (Behringwerke, Marburg, FRG), instead of the pancreas, as described above. The capacity of polyvalent antiserum. these substances to extinguish specific immuno• Each serum sample was tested for complement- fluorescence was recorded. fixing Pab and Gab by a three-layer indirect immunofluorescent test. The first layer was com• Ch ro m a tograp hy posed of 1:10 diluted patient's serum (incubation Human pancreatic juice and fluids of pancreatic at room temperature for 1 h), the second of undi• pseudocysts were analyzed by gel filtration luted normal human serum (blood group AB) chromatography FPLC (Superose 6, Pharmacia as complement source (1 h), the third contained Fine Chemicals, Freiburg, FRG). The antigen- fluorescein-conjugated rabbit antihuman Clq, containing fractions were identified by monitoring C3c\ or C4 (1 h; Behringwerke). Essential con• the capacity of the column effluents to neutralize trols in each serum included staining with FITC- Pab. conjugated rabbit antihuman globulin directed against albumin.

RESULTS Mieroscop:c examination Results were read subjectively with a Leitz Autoantibody profiles Dialux-20 microscope by two individuals, inde• The prevalence of 22 different autoantibodies pendently of each other, without knowledge of (Aab) was determined in Crohn's disease (CD) the patients1 diagnoses. The microscope was fitted and in ulcerative colitis (UC), as well as in the with epi-illumination, 50-W HB-50 mercury lamp, control collectives, which consisted of patients filter combination J 2, mirror RKP510, mag• with coeliac disease and of healthy persons. nification x250 (Leitz, Wetzlar, FRG). All results In CD, Aab against exocrine pancreas (Pab) were read double-blind with positive and negative turned out to be very frequent, and they oc• control sera in each batch. curred in high concentrations (Figs. 1 and 2). The Aab screening confirmed the prevalence of Neutralization tests Aab against intestinal goblet cells (Gab) in UC The CD-related autoantigen was detected by a (Fig. 3). 44 W. Stöcker et al.

Figs. 1 and 2. Autoantibodies against exocrine pancreas in Crohn's disease. Deter• mination by indirect immunofluorescence. Unfixed frozen sections of human adult pancreas were incubated in the first step with 1:10 diluted sera of patients with CD, in the second step with a polyvalent fluorescein-conjugated rabbit antihuman immunoglobulin. Fig. 1 {top). Serum of CD patient 22; reticulogranular pattern. Fig. 2 (bottom). Serum of CD patient 27; droplet-like pattern.

Results of the Aab screening are summarized Aab to and nerve tissue (elevated in in Table I. In addition to Pab and Gab, in patients both). None of these Aab correlated with Pab with CD and UC the following Aab occurred with and Gab. Their bearing on the pathogenesis of a higher frequency than in coeliac disease and in CIBD is not yet understood. healthy subjects: Aab to gastric mucosa (pref• Aab to the other tissues employed in this study erence to CD), Aab to 'single cells1 of connective have not been more frequent in CIBD than in tissue and to cell nuclei (preference to UC), and healthy control subjects. Autoantibodies to Pancreatic Juice in CD 45

Fig. 3. Autoantibodies against intestinal goblet cells in ulcerative colitis. Deter• mination by indirect immunofluorescence. An unfixed frozen section of human fetal duodenum was incubated in the first step with the 1: 10 diluted serum of UC patient 38, in the second step with a polyvalent fluorescein-conjugated rabbit anti-human immunoglobulin.

Autoantibodies to exocrine pancreas (Pab) was 50%, in patients without extraintestinal mani• Aab to the exocrine pancreas (Figs. 1 and 2) festations only 33%. were discernible by a striking reticulogranular The prevalence of Pab was dependent neither fluorescence in the cytoplasm of the pancreatic on sex, old age or blood group (A, B, AB, O) of acinar cells, with an increasing intensity in the the patients, nor on the intestinal localization of direction of the acinar centre. With the most the disease or on a therapy with salicyla• positive sera fluorescent droplets became visible zosulfapyridin. Corticosteroids seemed to reduce in the centre of the acini. Pancreatic islets were the prevalence of Pab (15 of 43 patients = 35%), not stained by Pab. patients without corticosteroids showed Pab in 8 Pab could be observed in 23 out of 59 patients of 16 cases (50%). with Crohn's disease (39%). In 17 patients (29%) Pab were also found in 2 out of 46 patients with the litre of Pab was 1 :100 or higher. Such high the diagnosis of UC (4%; titres 1: 10 and 1:32). concentrations have been found neither in UC, Three sera of 100 control subjects exhibited Pab nor in coeliac disease and healthy controls. in titres of 1:10. Six of the 23 Pab fixed complement. In these cases, the CDAI was twice as high (225.3 ± 139.4) Autoantibodies to intestinal goblet cells (Gab) than in 17 patients with non-complement-fixing Autoantibodies to intestinal goblet cells (Gab) Pab (99.7 ± 85.2). (Fig. 3) were a distinguishing mark of UC. Gab In patients whose CD had been present for were discernible in the epithelium of the mucosa less than 2.5 years the prevalence of Pab was as hazy confined, cloudy spots. They could be determined as being only 25%. If CD existed detected with each segment of the bowel used as longer than 2.5 years, patients exhibited Pab in antigenic substrate from duodenum to the rectum. 46%. In CD patients having extraintestinal mani• But positive and negative staining could be distin• festations of the disease, the prevalence of Pab guished better with than with 46 W. Stöcker et al.

Table I. Autoantibody profiles in Crohn's disease, ulcerative colitis, coeliac disease, and healthv subjects Prevalence of clinically significant Aab titres

Prevalence (%) of Crohn's disease Ulcerative colitis Coeliac disease Healthv control autoantibodies to (/i = 59) (// = 46) (n - 19) (ti = 100)

Exocrine pancreas (Pab), titre 1:100-1:1000 29 Exocrine pancreas (Pab), titre 1:10-1 : 1000 39 Intestinal goblet cells (Gab), titre 1 : HM : 1000 0 Gastric mucosa, (granular pattern) 41 'Single cells' of connective tissue 2 17 Cell nuclei 8 26 6 Enterocytes 39 33 10 14 Nerve tissue 26 5 Mitochondria 0 10 Smooth muscle 2 0 Thyroid microsomes 7 5 Parietal cells 7 0 Sinusoids of the liver 0 20

The Aab screening was carried out employing the following human tissues as antigenic substrates: Adult pancreas, parotis, liver, lung, striated and smooth muscle, heart, thyroid, parathyroid, adrenal, ovary, placenta, testis, hypophysis, kidney, and sterile human fetal esophagus, stomach, duodenum, ileum, and colon. In addition to the Aab listed in the table, the control group exhibited Aab to thyroglobulin (5%), striated muscle (1%), and pancreatic islets (1%). Further Aab were not detectable in any of the four groups.

colon, since in the small intestine goblet cells are (13%). Additionally, the distribution of Gab in separated from each other and in the colon goblet patients with different blood groups was not even: cells appear as a coalescing cloudy mass. In blood group A the prevalence was 38% (6 of Gab were present in 13 of 46 patients with UC 16), in group B 40% (2 of 5), in group AB 66% (28%), but in no case of CD and coeliac disease (2 of 3), in group O only 16% (3 of 19). and in none of the healthy control persons. Serum concentrations varied from patient to patient, Immunoglobulin classes of Pab and Gab titres were determined between 1:10 and 1:1000. Pab mainly belonged to IgG and Ig A, one The (UC-adapted) activity index in patients patient exhibited IgD and one patient IgM. Pab with Gab was 136 (±93). In patients without Gab, of class IgE were not observed (Table II). disease activity was 85 (±75). In Gab, only immunoglobulin classes IgA and None of the established Gab fixed complement. IgG could be observed (Table II). IgM were not The occurrence of Gab correlated neither with detected. the patients' old age, nor with the duration of the disease or the therapy. Light chains of Pab and Gab A preponderance of Gab was observed in To find out whether Pab and Gab were of males: Gab occurred in 10 of 23 male UC patients oligoclonal or of polyclonal origin, their light (43%), but only in 3 out of 23 female patients chains were analyzed by indirect immuno- Autoantibodies to Pancreatic Juice in CD 47

Table II. Immunoglobulin classes of Pab and Gab, and their ability to bind complement factor C3c. Pab contained in two cases additionally IgD, but never IgE. Gab consisted exclusively of IgA and/or IgG

Reaction dotal IgA alone IgG alone IgA + IgG IgM alone with C3c

Pab in CD 23 2 8 12 1 6 Gab in UC 13 1 3 9 0 0

fluorescence: kappa and lambda were evenly of nearly every organ including pancreas, liver, distributed in 11 of 23 Pab; in 7 cases kappa over• kidney, and thyroid. The target cells have not yet balanced lambda, and in 5 cases lambda over• been identified. Serum titres between 1:10 and balanced kappa (Table III). Similar data were 1:320 were observed (UC, 17% ; CD, 2% ; coeliac obtained in Gab: kappa was equal to lambda in disease, 0%; controls, 0%). Clinical symptoms ^ of 13 cases, kappa predominated in 3 cases, and could not be detected, which would explain the lambda in 1 case. implication of these Aab. Significant concentrations of Aab to cell nuclei Other au to antib odies (titre 1:100 or higher) have been stated with Aab to gastric mucosa gave a granular staining a significantly higher frequency than in control in single, up to now not identified cells of the subjects in UC and with a lower extent in CD gastric glands. They were present in only very low (UC 26%; CD, 8% ; coeliac disease, 5% ; healthy concentrations (to detect these Aab, the patients' controls, 6% ). sera had to be used undiluted in the first step of Aab to enterocytes were common in both CD the indirect fluorescent antibody test). Of the CD (39% ) and UC (33% ), but also in coeliac disease patients 41% exhibited this antibody (UC. 11%; (10% ) and in healthy controls (14%; Table I). coeliac disease. 5% ; healthy controls, 6%), which They reacted with the cytoplasm of the entire correlated neither with Pab nor with population of intestinal epithelial cells including antibodies. the goblet cells. These Aab exhibited the same Aab to 'single cells' of connective tissue: In the affinity to each of the different bowel segments. sera of some patients with UC an Aab was found Aab to enterocytes were different from Pab and which reacted with the cytoplasm of large, spor• Gab, they occurred independently from these adically distributed cells in the connective tissue Aab in CD and UC. Aab to nerve tissue reacted with nerves and with the cytoplasm of plexus cells in many Table III. Reactions of Pab and Gab with fluorescein- organs. They were especially well discernible with labelled anti-kappa or anti-lambda. Titre 1:3.2 and human fetal tissue of the 1:10 - +; 1 :32 and 1 : 100 = + + ;!: 320 and 1 : 1000 = (plexus Auerbachi et Meissneri). Their preva• + + + lence in CD was 22% and in UC 26% (coeliac Lambda disease, 5%; healthy controls, 2%).

Kappa 0 + + + + + + Detection of the CD-related autoantigen in pan• Antibodies to exocrine 0 0 0 creatic juice pancreas in CD + 4 7 -) 0 Neutralization experiments have been per• (23 positive sera) + + 1 1 1 + + + 0 0 0 3 formed with different pancreas-derived sub• Antibodies to intestinal 0 0 1 0 stances to evaluate the possible role of the goblet cells in UC -r ~> 9 0 0 exocrine pancreas in the pathogenesis of CD. (13 positive sera) + -f 0 0 0 0 The neutralizing efficacy of crude duodenal + + + 1 0 0 0 juice or homogenized pancreas could not be 48 W. Stöcker et al.

Table IV. Neutralization of autoantibodies with different pancreas-derived substances, whose capacity to extinguish specific immunofluorescence was estimated by titration. Liquids were used undiluted; supernatants of organ homogenates were diluted 1 : 10 in phosphate-buffered saline. In the table, the reciprocal titres are listed

Neutralization of autoantibodies with

Phosphate- Cystic Cystic Column (Titre)-1 of buffered fluid fluid Pancreatic effluent Parotic Homog. Homog. autoantibodies to saline 1 2 juice fract. 14 liver heart

Exocrine pancreas (serum 11) 100 3.2 100 3.2 10 100 100 100 Exocrine pancreas (serum 13) 1000 32 1000 10 32 1000 1000 1000 Pancreatic islet cells 100 100 100 100 100 100 100 100 Intestinal goblet cells 320 320 320 320 320 320 320 320 Cell nuclei 1000 1000 1000 1000 1000 1000 10 100 Mitochondria 1000 1000 1000 1000 1000 1000 100 3.2

evaluated. Their inherent proteinases became DISCUSSION activated and digested the tissue sections, thus it was not possible to interpret the results of the Autoantibody profiles were established in chronic fluorescent antibody test. inflammatory bowel disease (CIBD) by indirect Well-interpretable results were obtained, how• immunofluorescence with modern histochemical ever, when pancreatic juice or fluids of pancreatic techniques. Results of this study confirm that pseudocysts were used for neutralization. In both significant autoimmune reactions do exist in cases positive Pab reactions were abolished or CIBD. In Crohn's disease (CD) as well as in titres were reduced by at least 4 double dilution ulcerative colitis (UC), disease-specific Aab could steps in each of the 23 Pab-positive sera studied. be demonstrated, which were directed against In contrast, pancreatic juice and fluids of pan• exocrine pancreas in CD (10, 19) and against creatic pseudocysts did not neutralize auto• intestinal goblet cells in UC. With regard to serum antibodies against cell nuclei, mitochondria, concentration and disease specificity, Pab and smooth muscle, gastric parietal cells, intestinal Gab were as conspicuous as other autoantibodies goblet cells, thyroid microsomes, and pancreatic in proven autoimmune diseases. islets (Table IV). A number of further Aab were found which Separation of the CD-related autoantigen: The occurred in CIBD with a higher frequency than hypothetical CD-related autoantigen was con• in control collectives, but were of less significance tained in one single peak and behaved chro- than Pab and Gab: Aab to enterocytes and to cell matographically as a macromolecule of about 106 nuclei (neither specific for CD nor for UC), Aab dalton. It was different from functionally active to gastric mucosa (occurred in very low con• , lipase, trypsin, and , which centrations), and Aab to 'single cells1 of con• were identified by biochemical standard pro• nective tissue (low prevalence). The other Aab cedures in separate peaks (18). determined in this study were as rare in CIBD as With the pancreatic autoantigen containing in healthy persons and in patients with coeliac fraction, each of the positive Pab reactions in the disease. 23 CD sera studied could be abolished in the The results made evident that Crohn's disease neutralization experiments. Thus, the antigenic is associated with potent autoimmune reactions specificity of Pab seems to be uniform. against pancreatic juice: In CD, autoantibodies to Autoantibodies to Pancreatic Juice in CD 49 exocrine pancreas were frequent and they usually autoimmunity to pancreatic antigens is essential occurred in considerably high concentrations in in CD. thie patients' sera. High titres of Pab could be The CD-related autoantigens were distributed recorded neither in patients with UC or coeliac like secretory proteins in the exocrine pancreas, di:sease nor in healthy subjects. as could be observed in the indirect fluorescent In rare cases of chronic and acute pancreatitis antibody test. Are these antigens secreted into Aab can be found, which also react with acinar the bowel lumen? In this study it was documented cedls of the pancreas (20-22). One may ask by neutralization experiments that the CD-related whether there exist immunological differences autoantigens really are contained in normal pan• between the two conditions. Hellwig et al. (22) creatic juice. Therefore, it is imaginable that the have characterized serum Aab against exocrine exocrine pancreas is implicated in the patho• pancreas in pancreatitis: The prevalence of genesis of CD: significant concentrations was as low as 6.5% in Crohn's disease may be caused by autoimmune chronic and acute pancreatitis (5 of 77 cases) and, reactions against the exocrine pancreas. The if these antibodies were present, titres did not bowel has developed a state of hypersensitivity exceed 1:32. Pab in pancreatitis consisted only of against a component of pancreatic juice. The IgA, did not fix complement and could not be autoantigens are synthesized in the pancreas and neutralized by the CD-related autoantigen (Table secreted into the bowel. In the bowel wall they V). On the other hand, Pab were frequent in react with locally present autoantibodies and form CD, they usually occurred in high concentrations, immunocomplexes which induce activation of contained IgG in most cases, sometimes fixed complement and inflammation. It is also con• complement and could be neutralized by the CD- ceivable that the autoantigens bind unspecifically related autoantigen in each case. Thus, Pab in to tissue structures and transform them to targets pancreatitis differed fundamentally from those in of immunoattacks. CD. The in vitro observed autoimmunity to pan• In patients with pancreatitis it is conceivable creatic juice in CD corresponds to a number of that antigens released from the injured pancreas histological, clinical, and epidemiological induce a secondary immunization without impor• phenomena: tant pathogenetical implications. If pancreatic A striking augmentation of plasma cells in the immunity were also an epiphenomenon in CD, intestinal CD lesions (23,24) reflects a response immunoreactions should be of comparable to hitherto unknown antigens. dimensions. But in CD, humoral immunity Diversion of the fecal stream by an ileo- or against exocrine pancreas exceeds by far that colostoma usually results in an immediate clinical found in pancreatitis, thus it is suggested that improvement of acute Crohn colitis. In UC, this

Table V. Characteristics of autoantibodies to exocrine pancreas in CD and in pancreatitis

Crohn's disease Acute and chronic pancreatitis

Prevalence of Pab 39% 5% Concentration of Pab Usually high (titre 1:100 or higher Low (titre max. 1:10) in 3 of 4 positive cases) Immunoglobulin class Usually IgG IgA only of Pab or IgG plus IgA Complement fixation Sometimes observed Not observed of Pab Pab neutralize the In all cases observed Not observed CD-related autoantigen 50 W. Stöcker et al. treatment is without positive effects (25). Fur• in UC. Aab against intestinal goblet cells in U'C thermore, an intestinal lavage with physiologic are well established and may have a patho saline reduces the activity of the disease. This genetical implication since Gab are specific for indicates that toxic (antigenic?) substances are UC and goblet cells show macroscopically anid contained in the bowel lumen (26). microscopically the same localization as thie The beneficial effect in CD of parenteral disease: They are rare in the small intestine, buit nutrition (27) or a low-fat elemental diet (28, 29), arranged closely side by side in the colon, increas• which reduce pancreatic secretion to levels near ing in the direction to the rectum. Furthermore, those observed during fasting (30-33), might also the number of goblet cells is the highest in tine indicate an impaired tolerance to pancreatic juice crypts. Accordingly, in UC, the small intestine is in CD. not affected, but the rectum nearly in each cas»e, Patients suffering from CD develop a pre• and cryptitis is a typical feature of UC. dilection for refined sugar. They consume sig• Instead of adult tissue, fetal intestine was nificantly more carbohydrates than UC patients employed as antigenic substrate for determination and healthy control persons (34—37). This has of Gab by indirect immunofluorescence, to avoid been considered to be a causative factor in CD, unspecific reactions caused by bacterial or food but another interpretation seems also to be con• antigens. Furthermore, it is important to u:se ceivable: sugar and starch do not stimulate protein human tissue: antibodies, determined with rat synthesis in the exocrine pancreas, and glucose colon (12, 40-42), do not correlate with Gab even inhibits the /pancreocymin-stimu- (9) and exhibit positive reactions also in CD lated pancreatic secretion (38, 39). Patients with and in healthy subjects. These 'heterophile' anti• CD unconsciously realize the reduced tolerance bodies often consist of IgM (42), in contrary to to pancreatic juice, and they try to minimize the Aab against human goblet cells, which belonged pancreatic-juice-mediated injury to the bowel. only to the immunoglobulin classes IgC and The increasing incidence of CD in wealthy IgA. countries might be attributed to the nutrition, The occurrence of Gab in UC has been dis• which is rich in protein and fat. The over-con• cussed to be a corollary of an immunization sumption of food leads to a strong and long- against antigens shedded from dama• lasting stimulation of the exocrine pancreas. The ged colonic mucosa and thus to be a secondary gut-associated immune system comes perma• phenomenon (e.g. Ref. 6). But the strict absence nently into contact with large amounts of the of Gab in patients with CD of the colon is incon• (hypothetical) autoantigen, and predestinated sistent with this explanation. On the other hand, subjects develop autoimmune reactions to pan• one might also speculate that Pab result from a creatic juice and CD. secondary immunization in the gut. This should Extraintestinal manifestations in many patients take place in the small intestine, since high con• with CD might be caused by deposition of cir• centrated Pab essentially could not be detected in culating immunocomplexes (consisting of Pab and the colon-restricted UC. But significant Pab-titres the corresponding autoantigen) or by immuno- also occurred in a number of patients with CD reactions against the CD-related autoantigen confined to the colon. Thus also the pancreas- which are contained in the affected tissues and specific autoimmunity in CD seems to be a presented to the immunosystem more effectively phenomenon of primary significance. than in the pancreas. In spite of the established Furthermore, a secondary immunization pancreatic immunity, pancreatitis is not pre• against pancreatic and goblet cell antigens would dominant in CD since the bulk of autoantigens probably induce a polyclonal antibody response, comes into contact with the immune system only and kappa and lambda light chains would be outside of the pancreas. evenly distributed in each Pab or Gab. But the The autoimmunity against exocrine pancreas in high frequency of antibodies with only one type CD is a counterpart to autoimmune phenomena of light-chain detectable gives evidence of an oli- Autoantibodies to Pancreatic Juice in CD 51 goclonal response: Both Pab and Gab are pro• aetiology, the corresponding disease-specific Aab duced by a very small number of plasma cell do not reach a prevalence of 100% either. clones. This result was confirmed with a higher n umber of sera (43) and might correspond to the finding of monoclonal lymphocyte populations in ACKNOWLEDGEMENTS the peripheral blood of 12 out of 20 patients with CD or UC (44). The established oligoclonality of The authors wish to thank Prof. Dr. A. Gropp Pab and Gab speaks in favour of autoimmunity (deceased), Prof. Dr. D. Sellin (Lübeck), and as being causatively involved in the aetiology of Prof. Dr. P. Schmitz-Moormann (Marburg) for CD and UC. the histological examination of the patients' Pab and Gab seem to be disease-specific, and biopsies, and technical assistants D. Grage, A. they do not yet indicate a disposition for CD or Hagedorn, LI. Reddig, A. Stolzenberg, and D. UC. This was shown by Döscher et al. (45) and Struve for their skillful help. Kosegarten et al. (46), who determined the preva• lence of Pab and Gab in an up to now limited number of healthy appearing first-degree relatives of patients with CIBD. 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