journal

of Autoimmunity (1990) 3,307-320

Autoantibodies to Colonic Cells and Subcellular Fractions in Inflammatory Bowel Disease: Do They Exist?

M. Cantrell, T. l?rindiville* and M. E. Gershwin Division of Clinical Immunology and Gastroenterology, Medicine,

Department

of Internal

University of California at Davis, Division of Rheumatology/Allergy Clinical Immunology, TB 192, Davis, CA 95616 USA

and

Previous observations have purported to demonstrate circulating antibodies which bind to colon epithelial cells. However, the significance and reproducibility of such observations has been difficult and the data often phenomenological. To further our understanding of such autoreactivity, we studied sera and purified serum immunoglobulins from patients with ulcerative colitis, Crohn’s colitis and other inflammatory diseases, as well as normal volunteers using as a target, well-defined epithelial cell preparations from normal and diseased colon and small bowel including crude suspensions of homogenized cells, purified and characterized brush border membranes, basolateral membranes and a DEAE cellulose column purified protein fraction. Homogenates of normal liver, lung, kidney, thymus, pancreas, stomach and small and large intestine, obtained at surgery, were also included. The purified preparations were characterized by enzyme activity and were electrophoresed on SDS-polyacrylamide gels for immunoblotting. Additional studies were carried out comparing these findings with those of a previously published and described ‘positive’ colon target preparation and polyclonal antibody. There was no convincing demonstration of circulating autoantibodies in patients with ulcerative colitis. Our data, using well-defined and characterized tissue preparations, raises doubts regarding the presumptive demonstration of autoantibodies in ulcerative colitis.

Introduction

Currently available evidence on inflammatory bowel diseases suggests that immunological events occur within the inflamed mucosa with subsequent activation of *To whom correspondence should be sent. Supported in part by NIH grant CA 208 16. 307 0896-841 l/90/030307+

14 $03.00/O

0 1990 Academic Press Limited

308 M. Cantrell, T. Prindiville and M. E. Gershwin immunological effector mechanisms. Affected patients have increased production of immunoglobulin in the mucosa (IgG in ulcerative colitis, IgG and IgM in Crohn’s disease) and activation of complement [l]. Patients with ulcerative colitis (UC) or Crohn’s disease (CD) also demonstrate humoral and cellular immune responses to a wide variety of gut-associated antigens. Finally, there are also disturbances in immunoregulatory control during relapse of disease which may contribute to an enhanced immune response to gut-associated antigens [2]. Some reports have described the presence of serum antibodies in patients with ulcerative colitis that bind to colonocytes in viva [3-51 and cause premature death of colonocytes in vitro [6-111. Further, a colonic protein has been reported that is specifically recognized by serum and colon-bound antibodies in patients with idiopathic ulcerative colitis. This antigen has a molecular weight of 40 kDa and can be purified by DEAE cellulose column chromatography wth a KC1 gradient [3, 5, 12, 131. However, the data on this antigen appear inconsistent with the profile of cellular autoantigens and have raised the issue of whether antibodies are either reproducible or significant. The purpose of this study was to evaluate patients with inflammatory bowel disease for the presence of such circulating antibodies to colon and small bowel tissue. We report herein the failure to demonstrate such antibodies using welldefined and enzymatically quantitated subcellular fractions. These data have implications for the presumptive demonstration of autoantibodies in ulcerative colitis and raise basic questions regarding the pathogenic basis of disease. Materials and methods Clinical specimens

Sera were collected from patients with clinically documented and histologically diagnosed inflammatory bowel disease. These included 12 patients with ulcerative colitis and 12 with Crohn’s colitis and/or ileocolitis. All blood samples were obtained from patients in the acute phase of disease with the exception of post-colectomy sera from one UC and one CD patient. All of the UC patients were on sulfasalazine and six were also on prednisone. One CD patient was taking no medication, the other 11 were taking sulfasalazine and some were also on prednisone and flagyl. Sera from 11 patients with other active inflammatory disorders were also tested [three with rheumatoid arthritis (RA), one with RA vasculitis, one with juvenile rheumatoid arthritis (JRA), one with bypass arthritis, two with systemic lupus erythematosus (SLE), one with anti-collagen antibodies and two with primary biliary cirrhosis (PBC)]. Sera from eight healthy normal volunteers were used as controls. As a positive control, a rabbit polyclonal antibody, provided by Dr K. Das, to the described 40-kDa autoantigen of UC was used. All sera were aliquoted and stored at - 3O”C, and aliquots diluted 1:5,1: 10 and 1: 100 in phosphate buffered saline (PBS). Finally, as an additional positive quality control for the assays used herein, sera from patients with primary biliary cirrhosis were studied using purified preparations of mitochondria as antigens utilizing assays described in our laboratory [ 141. Preparation

of immunoglobulins

Serum immunoglobulins were precipitated with saturated ammonium sulfate and purified by ion exchange chromatography on DEAEsellulose [ 15,161. The purity of

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the gamma globulin preparation was confirmed by radial immunodiffusion against anti-IgG, anti-IgA and anti-IgM (Meloy) with only the anti-IgG reacting [17]. Finally, the protein content of each preparation was determined [ 181 and 50 l.tg and 100 pg per membrane strip used for testing.

Tissue source

Following University of California Human Subjects Committee (AR-142), fresh operative specimens of diseased large bowel and terminal ileum were obtained at the time of surgery from two patients undergoing colectomy for active refractory ulcerative colitis. Crohn’s disease large and small bowel tissues were also obtained from two patients during bowel resection. Normal bowel tissues were obtained during small bowel resection for gastric bypass and obstruction. Normal tissue obtained at the time of surgery and used for cross-reactivity testing included; thymus, lung, pancreas, stomach, liver, kidney and rat liver.

Tissue preparation

Colon tissues were treated with Type VII collagenase (Sigma, St Louis, MO) for 3 h. The mucosa was then removed by gentle scraping and the cells washed with cold 0.01 M phosphate buffer with 0.15 M NaCl (PBS) pH 7.2, containing 2 mM phenylmethylsulfonyl fluoride (PMSF) and 2 mM sodium azide, and viability was confirmed by trypan blue dye exclusion. All tissue samples were minced and washed five times with cold PBS-PMSF buffer, resuspended in 5 volumes (wt/vol) of the same buffer with 1 mM EDTA and homogenized on ice in a Polytron homogenizer (Brinkman Instruments, Westbury, N.Y.) at setting 5 for six 10-s intervals. Protein concentration of all tissue homogenates was determined and adjusted to 2.0 mg/ml with PBS. Beef heart mitochondria for the PBC controls were prepared as described [ 141.

Cell membrane preparations

Brush border membranes were prepared from small bowel and colon mucosa as described [ 191. The tissue was homogenized for 1 min on ice in a Polytron at speed 4 in 15 volumes (wt/vol) of mannitol-hepes buffer (50 mM mannitol, 2 mM hepes, pH 7.0). The homogenates were centrifuged for 10 min at 10,000 x g. The supernate (S 1) was saved and the pellet (Pl) rehomogenized for 1 min in 15 volumes of mannitolhepes buffer on ice at Polytron setting 4. Supernatant S 1 and Pl homogenate were combined and solid CaCI, added to a final concentration of 10 mM. The mixture was stirred for 20 min at 4°C to aggregate cytosol components and then centrifuged for 10 min at 4,000 x g to remove the aggregates. This supernatant (S2) was then centrifuged for 20 min at 30,000 x g. The resulting pellet containing the brush border membranes was finally resuspended in 0.5 ml mannitol-hepes buffer and frozen at -80°C until assay. The purity of the brush border membrane preparation was determined by sucrase assay [20]. Dilutions of the small bowel homogenate and the

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M. Cantrell, T. Prindiville and M. E. Gershwin

brush border preparation (1: 10,l: 100,1:500) were made in PBS and 100 ul added to 100 ~1 of substrate (0.056 M sucrose in 0.1 M phosphate buffer, pH 6.1). This mixture was incubated for 30 min at 37°C then 1 ml Tris glucose oxidase 0-dianisidine (TGO) [1.5 ml (1,500 units) glucose oxidase (aqueous solution, Sigma V), 6 mg (1,000 units) peroxidase (Sigma II), 1 ml (0.1 g/lOml 95% EtOH) 0.04~ 0-dianisidine, diluted to 200 ml with 0.5 M Tris buffer pH 7.01 added and incubated for 30 min at 37°C. Tubes were read at 475 nm and specific activity reported in mU/min/mg protein. It should be noted that the conditions of the brush border membrane preparation described for rats [19] produced similar results (i.e., yield and degree of purification) with rabbit, pig and human bowel tissue. The final centrifugation at 40,000 g for 15 min was altered to 30,000 g for 20 min, without loss of yield or purity. Basal lateral membranes were prepared from small bowel and colon tissue [2 1,221. Mucosal scrapings, kept at 4°C were suspended in 2 mM Tris-HCl, pH 7.4, 1 mM MgC12, 10 mM KC1 and 10 mM EDTA buffer (1:5, wt/vol). The tissues were homogenized at 4°C with a Polytron homogenizer at setting 5 for 2 min. The homogenate of isolated villus cells was centrifuged at 1,400 x g for 10 min to remove unbroken cells, nuclei and microvillus fragments. The supernatant fraction was centrifuged at 5,900 x g for 15 min and the resultant supernatant centrifuged at 20,000 x g for 30 min. Basal lateral membrane-rich preparations were then isolated by a sucrose gradient method starting with the 20,000 xg pellet. This fraction was diluted to 15 ml with dilute buffer (5 mM histidine-imidazole pH 7.0, 0.5 mu neutralized EDTA), homogenized briefly and centrifuged at 200,000 x g for 30 min. The pellet was homogenized gently in 2.0 ml of 50% (wt/vol) sucrose solution and placed at the bottom of 5-ml centrifuge tubes, overlayed with 1.5 ml of 40% sucrose, 1.0 ml of 30% and 0.5 ml of 20% sucrose solution (all sucrose solutions were made in histidine-imidazole buffer, pH 7.0). The tubes were then spun at 200,000 xg for 90min. The band formed at the 30-40% interface was carefully collected and resuspended in histidine-imidazole buffer and centrifuged again at 200,000 x g for 30min. The resulting pellet contained the highest concentration of basal lateral membranes and was resuspended in histidine-imidazole buffer for testing or storage at -80°C. The purification of the basal lateral membrane preparation was confirmed by Na+K+ATPase activity [23]. Inorganic phosphate released by this reaction was assayed according to Ames [24]. Extraction

of colon tissue protein

Colon tissue homogenates, as described above, were centrifuged at 2,000 x g for 20 min to remove large tissue particles. A clear supernatant was obtained bycentrifuging at 20,000 x g for 60 min. Homogenates of 8 g of tissue from each of two UC patients yielded an average of 99 mg of protein. The protein extract was further purified by the use of DEAE cellulose ion exchange chromatography [25] with discontinuous KC1 gradient. Ten milligrams of protein extract were passed through a DEAE cellulose column (6 ml bed volume) equilibrated with potassium phosphate buffer pH 7.4. Protein fractions were eluted with 2 bed volumes of KC1 concentrations 0.025 M, 0.05 M, 0.1 M, 0.2 M, 0.25 M, 0.3 M, 0.35 M and 0.5 M. Eluted proteins

Subcellular Fractions in Inflammatory

Bowel Disease

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were dialyzed against PBS pH 7.4 overnight at 4°C. The 0.35 M eluate (final concentration 0.1 mg/ml) was used for testing [8]. Additionally, a 0.35 M KC1 extract with a protein content of 1 .O mg/ml was provided by Dr Das. SDS-PAGE

and transblot assay

SDS-PAGE was performed as described by Towbin et al. [26]. Tissue homogenates (500 pg/gel), brush border or lateral membrane preparations (100 ug/gel) and tissue extracts (100 pg/gel) were diluted 1: 1 with sample buffer (63 mM Tris-HCl pH 6.8, electrophoresed in SDS0.246 SDS, 10% glycerol, 50 mM 2-mercaptoethanol), PAGE (lo-20% gradient) with running buffer (25 mM Tris base, 196 mM glycine, 0.1 O/bSDS, pH 8.3) and transblotted to nitrocellulose sheets at 30 V overnight in transfer buffer (20 mM Tris base, 150 mM glycine, 20”/0methanol) [27]. Nitrocellulose strips were blocked with 2% bovine serum albumin (BSA) in 0.01 M PBS pH 7.4 for 2 h at room temperature. The strips were incubated with serum dilutions (1:5, 1:lO or l:lOO), serum immunoglobulin (50 ltg and 100 pg/strip) or with purified IgG (50 ug and 100 pg/strip) for 2 h at room temperature, washed three times for 10 min per wash with PBS-BSA, then incubated with ‘251-Staph protein A or with ‘251-anti-human globulin (200,000 cpm/ml) for one h at room temperature. Strips were again washed three times with PBS-BSA, rinsed with PBS and subjected to autoradiography (Kodak XAR film with one intensifying screen). Bands were assessed by comparison with known molecular weight markers (Pharmacia, Upsaala, Sweden) run with each gel. ELISA

protocol

Native tissue homogenates from normal and diseased (UC) colon were used as antigen and studied by enzyme-linked immunosorbent assay (ELISA) [28]. The homogenates were spun for 20 min at 15,000 x g to obtain a clear supernatant. The protein content of the homogenate was determined (Bio-Rad) and incubated overnight at 4°C with washed, hydrated protein A-agarose (Sigma): 20 mg protein/ml of swollen gel to remove colon-bound IgG. Unbound proteins were separated by centrifugation at 600 x g for 10 min, protein concentration remeasured, diluted in carbonate buffer (0.085 M Na,CO,) pH 9.6 to a concentration of 10 pg/ml and 50 pg/ml and used to coat the ELISA microtiter plates (Dynatech, Immulon TMl, Alexandria, Virginia). Controls included the 40-kDa colon protein extract (10 pg/ml) and actin (10 pg/ml) in carbonate buffer. One hundred microliters/well were incubated overnight at 4°C in microtiter plates. Plates were washed four times with washing buffer (PBS-Tween 20, pH 7.2; 0.05%). Then 5% BSA-PBS was added (100 @/well) for 1 h to saturate unbound antigen sites on the plate. Serum from patients with ulcerative colitis or Crohn’s disease and from normal controls were added to the wells in 100~~1 aliquots at dilutions of 1: 100, 1:500 and l:l,OOO. Plates were then incubated for 1 h at room temperature. After washing four times with PBS-Tween, peroxidase-labeled goat anti-human IgG (Tago, Burlingame, CA) was diluted 1:3,000 with 5 o/oBSA-PBS, and 100 ~1 was added to each well and incubated for 1 h at room temperature. The plates were again washed four times, and 100 ~1 of substrate, 2,2-azino-di-(3-ethyl-benzthiazoline sulfonic acid-6) diammonium salt

312

M. Cantrell, T. Prindiville and M. E. Gershwin Table

Brush border Basolateral

1. Protein concentration

andpurification

of membranes

Mucosal tissue (g)

Protein in homogenate (mgiml)

Membrane yield (mg/ml)

Degree of purification

3.0 3.0

2.6 7.6

2.0 2.0

20-fold B-fold

(ABTS) (Sigma) in 0.1 M citrate buffer (pH 4.2) with 0.003% H,O, was added to each well and the plates incubated for 20 min at room temperature. The plates were then read on a computerized ELISA reader (Titertek Multiskan MC, Flow Laboratories, Helsinki, Finland). Similar assays were used previously to assay for mitochondrial autoreactivity in PBC by our laboratory [ 141.

Results Purity of sample preparation

The protein concentration of serum samples ranged from 5.7 to 8.5 g/d1with a mean of 7.1 g/dl. The serum globulin concentration ranged from 2.8 to 4.7 g/d1with amean of 3.9 g/dl. The mean concentration of the IgG fraction was 7.2 mg/ml with a range of 3.3 to 10.3 mg/ml. Brush border membranes were prepared from 3 g of small bowel or colon mucosa; total protein of the initial homogenate was 2.6 mg/ml-in a total volume of 90 ml. The brush border membrane protein yield was 2.0 mg/ml; sucrase activity indicated an 1% to 20-fold purification of small bowel brush border membranes. Basolateral membranes were prepared from 3 g of mucosal tissue. The protein concentration of the partially purified homogenate was 7.6 mg/ml in a total volume of 15 ml, which yielded 2.0 mg/ml of basolateral membrane protein. An &fold purification of basolateral membranes was indicated by Na+K+ ATPase activity as shown in Table 1.

SDS-PAGE

and transblot assays

Gels were run initially with normal and diseased small and large bowel tissue and normal kidney tissue homogenates. Transblots were tested with 1:lOO dilutions of eight ulcerative colitis sera, nine Crohn’s disease sera and one PBC serum. Table 2 summarizes the results of this study. Sera from a patient with PBC reactedstrongly with the expected mitochondrial52-kDa and 70-kDa autoantigens found in normal and diseased tissue (Figure 1). UC1 reacted with two bands of 95 to 105 kDa molecular weight against normal small bowel and with two bands of 95 to 105 kDa molecular weight and one at 35 kDa in normal large bowel. There were no reactions with the diseased tissue homogenates. Tissue homogenates of normal colon provide high background due to the crude nature of the preparations. However, as noted below, lower background values

Subcellular Fractions in Inflammatory Table

Bowel Disease

313

2. Immunoblot reactivity of sera for patients with active inflammatory bowel

disease against tissue preparations Diseased tissue Crohn’s

Normal tissue Small bowel Ulcerative colitis’ Crohn’s Disease

Colon Kidney

Ulcerative colitis

Small bowel

Small bowel

Colon Colon extract

118

118

O/S

O/S

O/S

018

o/7

019

019

019

019

019

119

O/S

IAll sera were studied at 1: 100. However, use of immunoglobulin-enriched fractions, as described in the text, were likewise non-reactive. Known positive, quality-control, sera from patients with PBC reacted as expected to the mitochondrial enzymes (see reference [14]).

ABCDEFGHI

JKLMNOPQRS

94-

6743302014-

Figure 1. Normal colon tissue homogenate separated on 10% SDS-PAGE, transferred to nitrocellulose and tested with eight UC sera (A-H), nine CD sera (I-Q), negative (R) and positive-PBC (S) control sera. All sera were diluted 1:lOO in PBS with lZSI-Staph protein A as probe. Patient UC1 (A) reacted with two bands at 95-105 kDa and one at 35 kDa. The PBC control patient had the expected autoantibodies to 52-kDa and 70-kDa mitochondrial antigens. The 40-kDa band evident in lane H appeared as an artifact on the autoradiograph.

become common with purification of subcellular fractions. CD3 (lane K, Figure 1) reacted with three bands of 25 to 35 kDa molecular weight with the UC large bowel but with no other tissue. Sera from seven UC patients and eight CD patients failed to react with the extracted tissue proteins. The serum from the patient with PBC, on the other hand, reacted quite strongly with all the tissues tested (Figure 1). Such activity is directed, as noted, at mitochondrial autoantigens.

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M. Cantrell, T. Prindiville and M. E. Gershwin

ABCDEFGH

I

94 67

30

20 14

Figure 2. Colon extract containing 40-kDa protein separated on l&20% gradient SDS-PAGE, transferred to nitrocellulose and tested with purified IgG from sera of eight UC patients with T-Staph protein A as probe. Positive control is rabbit polyclonal anti-40-kDa antibody. No reaction is seen with IgG from UC patients. However, note that, as in the text, the rabbit polyclonal antibody reacts non-specifically.

Colon protein extracts None of seven sera from patients with UC and none of eight patients with CD reacted with the colon tissue homogenates and extracts by transblot assay. Furthermore, none of the serum dilutions reacted with the 0.35 M KC1 extract. Testing was then repeated with serum immunoglobulin and purified IgG against the same colon tissue homogenates and the 0.35 M KC1 extract. In only one patient, UC5, IgG reacted with a 67-kDa protein in one UC colon homogenate and in the 0.35 M KC1 extract from that tissue but not,with the other tissue or extract. Thus we failed to demonstrate reactivity even when using the highly purified characterized target tissue. Colon target protein A 40-kDa protein extract (0.35 M KC1 eluate) provided by Dr Das was run against 10 UC sera, two CD sera, three RA sera, one JRA serum, two SLE sera, one anticollagen serum, serum from one patient with RA vasculitis, one with bypass arthritis and one normal control. All sera were diluted 1:5 in PBS-BSA. Parallel strips were probed with either 1251-anti-human immunoglobulin or lz51-Staph protein A. No bands were seen on autoradiography with these sera. The rabbit anti-40-kDa polyclonal antibody also provided by Dr Das as a control reacted with the 40-kDa band (‘251-Staph protein A was used as a probe). The transblots of the 40-kDa protein were then tested with the serum immunoglobulins and with the purified IgG from 10 UC patients, 10 CD patients, seven normal controls and the serum from two PBC patients diluted 1:5,000 and 1:2,500 respectively (Figure 2 shows the results of eight UC patients IgG). No bands were

Subcellular Fractions in Inflammatory Bowel Disease

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seen on autoradiography. The rabbit polyclonal anti-40-kDa antibody was run against tissue homogenates from UC and normal colon, CD and normal small bowel, normal kidney, lung, pancreas, thymus, liver, stomach, rat liver and against the 40-kDa protein as a positive control. Surprisingly, all of the human tissue extracts reacted with the polyclonal antibody at 40 kDa.

Cell membrane preparations No activity was detected by autoradiography

with either of the UC serum immunoglobulin samples against the brush border or the basal lateral membrane preparations. None of the UC, CD or normal control serum IgG extracts reacted with either the brush border or the basal lateral membranes. PBC serum diluted 1~5,000 did not react with either membrane preparation; however, the 1:2,500 PBC serum elicited a band at 52 kDa with both the brush border and the basal lateral membranes. This latter is again expected, based on other data from our laboratory on PBC.

ELISA

results

IgG from colon tissue homogenates was removed by incubation with protein Aagarose prior to its use in the ELISA. There was negligible non-specific binding in the control wells using serum blank. A total of 10 UC and 10 CD patients were tested at dilutions of l:lOO, 1:500 and l:l,OOO against two concentrations (1 yg/well and 5 l.tg/well) of normal and ulcerative colitis colon tissue homogenates. Control proteins included actin, a 43-kDa protein.( 1 yg/well), a known 40-kDa extract of colon tissue (1 l.tg/well), homogenates of liver and lung tissue (1 yg/well and 10 ug/well) and pyruvate dehydrogenase (PDH) (0.2 yg/well) as a positive control for the PBC sera. Seven sera from known normal healthy adults were included as negative controls and a known anti-40-kDa (rabbit polyclonal) antibody and 10 PBC sera (1: 100 and 1: 1,000) as positive controls. In addition, an anti-actin monoclonal antibody with activity against actin and alpha actinin (ACl) was included in the study. Enzyme and substrate controls were also included. Table 3 summarizes the results of the ELISA study. Lower dilutions of sera resulted in higher background in PAGE and ELISA testing. This was alleviated in the PAGE results by using purified immunoglobulins and IgG at two different concentrations. A positive result was considered to be twice the normal control value. There was no reactivity between any of the UC or CD patients with normal colon tissue. One UC patient reacted weakly with the UC colon homogenate but not with the normal colon tissue, while the known anti-40-kDa antibody reacted more strongly with the normal colon tissue than with the UC colon protein. The anti40-kDa antibody also reacted strongly with lung homogenate but not with actin. The PBC sera reacted weakly with liver homogenate at 1:lOO and strongly with PDH at l:l,OOO dilution [ 141, but not with the lung or colon tissues. The presence of immunoglobulin in the 40-kDa protein preparation resulted in high background values with all sera tested; however, the reactions with the anti-40-kDa antibody and the anti-a&n antibody were significantly stronger than all other sera tested. (Immunoglobulin A, albumin and kappa light chains were demonstrated in the

316 M. Cantrell, T. Prindiville and M. E. Gershwin

Subcellular Fractions in Inflammatory Bowel Disease

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40-kDa preparation by Ouchterlony radial immunodiffusion.) The anti-actin antibody reacted with normal and UC colon protein, liver and lung as well as with actin and the 40-kDa protein. Enzyme and substrate controls not shown on this table revealed no reactivity. Multiple ELISA determinations with varying concentrations of antigen and antibody (data not shown). produced similar results. Discussion

Alterations in the reactivity of the cellular and humoral elements of patients with inflammatory bowel disease (IBD) suggest that the immune system is involved in the pathogenesis of IBD [29]. However, the evidence implicating immunological mechanisms in the etiology of IBD is indirect, e.g. frequency of personal and family history of autoimmunity, occasional association with other disorders often linked with immunological mechanisms, frequency in young people and response to anti-inflammatory or immunosuppressive agents [30]. Circulating anti-colon antibodies have been detected in UC patients’ sera using sterile human fetal colon [31], adult normal or diseased colon [6], germ-free rat colon [32] and Escherichia coli 014 [8] as antigens. The titers are, however, often low and controls have not always been adequate. Of particular importance to the observations herein, autoantibodies have been demonstrated by antibody-dependent cellular cytotoxicity (ADCC) and by immunoblot to epithelial cell-associated components (ECAC) of the colon and small bowel in 70% of patients with Crohn’s disease and ulcerative colitis. These antigens were highly purified macromolecules of 160 kDa and 137 kDa derived from solubilized gut epithelium and were also reactive with the serum from 56% of healthy relatives of IBD patients [33]. In another study, peripheral blood mononuclear cells (PBMC) from most healthy donors showed low but significant levels of cytotoxicity for normal epithelial target cells with no increase in activity in patients with ulcerative colitis or Crohn’s disease. These results were irrespective of their drug therapy or disease activity [34]. A human colon-specific antigen reactive with 90% of ulcerative colitis colon tissue-bound IgG (CCA-IgG) and with serum IgG from 83% of patients with ulcerative colitis, but not from Crohn’s disease patients or normal subjects, has been described [3, 5, 121. Studies with colon tissue extracts and CCA-IgG have allowed further characterization of this colonic ‘auto-antigen’. CCA-IgG from UC patients consistently recognized a colon protein of 40 kDa on SDS-PAGE transblots. IgG preparations from colon specimens of patients with Crohn’s colitis and other colonic inflammatory diseases did not react with the 40-kDa protein. Similarly, serum IgG from symptomatic UC patients reacted with the 40-kDa protein while control serum IgG did not. The 40-kDa protein was present in all colon specimens, normal or diseased, and was reported to be organ specific since it was not found in human normal stomach, duodenum, ileum or liver. It was absent in mouse and rat tissues, including colon [8]. Further studies using a monoclonal antibody to the 40-kDa protein showed a difference in expression of the antigen between the right and left colon, more antigen being present on the distal portions of the bowel [35]. A monoclonal antibody was also used to determine cellular localization of the 40-kDa protein on tissue sections. Immunoreactivity was present primarily on the basolateral areas of the plasma membrane of epithelial cells in the crypt and luminal surface [4].

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M. Cantrell, T. Prindiville and M. E. Gershwin

In this study circulating IgG antibodies to a specific colon protein were not found in UC patients. Indeed, we were unable to demonstrate any consistent ‘autoantibody’ activity to UC colon tissue extract by these methods even in patient UC1 who was also a donor of UC small and large bowel sections. All but two of the sera used in this study were from patients in the acute phase of their disease, although presence of anticolon antibodies in the circulation has not been correlated with the extent, severity, state of activity and course of disease or with corticosteroid treatment [31]. Furthermore, there was no evidence of antibody activity with purified membrane preparations, either brush border or the basal lateral membrane, as would be expected in the study by Vecchi et al. [4]. Indeed, studies with the rabbit polyclonal anti-40-kDa antibody demonstrated a 40-kDa band with all the human tissue extracts including UC and normal colon, CD and normal small bowel, normal kidney, lung, pancreas, thymus, liver and stomach with only rat liver showing no reactivity. By increasing the sensitivity of immunoassays, autoantibodies can be detected in healthy individuals and are expected to increase in titer in autoimmune disease [36]. Anti-thyroglobulin IgG has been demonstrated in 38% of healthy subjects by a sensitive enzyme assay [37]. Autoantibodies usually react with normal antigens from humans and a variety of other animals, the antigenic sites ,generally highly conserved through evolution [38-411. The 40-kDa protein described above does not fit this normal profile for an autoantigen, and the failure, at least..in this group, to demonstrate such reactivity would be consistent with the latter observations. Other incansistencies with the so-called 40-kDa autoantigen are based, on these latter observations as well as the absence of high antibody titers evident in most autoimmune conditions and the fact that the 40-kDa antigen in question is not highly conserved. The ability to demonstrate autoantibodies in healthy individuals and the failure to demonstrate them reproducibly in patients with inflammatory bowel disease leads us to question the significance of such autoantibodies.

References 1. Baklien, K. and P. Brandtzaeg. 1978. Comparative mapping of the local distribution of immunoglobulin-containing cells in ulcerative colitis and Crohn’s disease of the colon. Clin. Exp. Immunol. 22: 197-228 2. Jewell, D. P and A. Patel. 1985. Immunology of inflammatory bowel disease. Stand. J. Gastroenterol. (suppl). 14: 119-126 3. Nagai, T. and K. M. Das. 198 1. Detection of colonic antigen(s) in tissues from ulcerative colitis using purified colitis colon-tissue-bound IgG (CCA-IgG). Gastroenterology 81: 463-470 4. Vecchi, M., S. Sakamaki, B. Diamond, and K. M. Das. 1985. A human colon specific antigen reactive with ulcerative colitis colon tissue-bound IgG; immunohistochemical localization by the use of a monoclonal antibody (abstract). Gastroenterology 90: 1679 5. Takahashi, F. and K. M. Das. 1985. Isolation and characterization of a colonic autoantigen specifically recognized by colon tissue-bound immunoglobulin G from idiopathic ulcerative colitis. 3. Clin. Invest. 76: 311-318 6. Lagercrantz, R., S. Hammarstrom, P. Perlmann, and B. B. Gustafsson. 1966. Immunological studies in ulcerative colitis. III. Incidence of antibodies to colon-antigen in ulcerative colitis and other gastrointestinal diseases. Clin. Exp. Zmmunol. 1: 263-276 7. Perlmann, P. and 0. Broberger. 1963. In vitro studies of ulcerative colitis. II. Cytotoxic action of white blood cells from patients on human fetal colon cells. 3. Exp. Med. 117: 717-733

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Autoantibodies to colonic cells and subcellular fractions in inflammatory bowel disease: do they exist?

Previous observations have purported to demonstrate circulating antibodies which bind to colon epithelial cells. However, the significance and reprodu...
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