Purification of Fibroblast-Derived Celiac Disease Autoantigen Molecules1
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003 1-3998/93/3404-0420$03.00/0 PEDIATRIC RESEARCH Vol. 34. No. 4. 1993 Copyright 0 1993 International Pediatric Research Foundation. Inc. Prinrcd in L'. S...I. Purification of Fibroblast-Derived Celiac Disease Autoantigen Molecules1 AULlS MARTTINEN AND MARKKU MAKI Cni~~cr.sirj*(?/'Tumprrc~. Dc>purrmcJnr c?fC'linicul hfc~dicinc. Tc~iskonric, 35. SF-33101 Tunlperc,. finlund ABSTRACT. We have recently purified autoantigen poly- bodies known as ARA and EMA (3). The occurrence of these peptides reacting with celiac disease patient sera IgA from autoantibodies is highly sensitive and specific for gluten-sensitive the extracellular noncollagenous matrix compartment of enteropathy in CD and dermatitis herpetiformis (4-7). Positivity fetal lung tissue. These molecules trigger the production for these antibodies is also genetically determined (8). of different tissue antibodies, the so-called antireticulin and CD and dermatitis herpetiformis patient sera IgA detect mol- antiendomysium antibodies in celiac disease. In the present ecules not only in rodent but also in primate tissues, including report, we show that fibroblasts synthesize and secrete human jejunum, in a pattern very closely resembling the con- celiac disease autoantigen molecules. The secretion prod- nective tissue fibrillar staining pattern produced by silver-im- uct, reactive with IgA from celiac disease patients, is a pregnation staining (9. 10). Karpati et al. (10) showed that in large-molecular-weight protein aggregate. When the pro- these molecules the reticulin antigen was not detected directly tein complex was treated with 4 M guanidinium hydro- on collagen fibers but was associated with amorphous material chloride and 0.1% SDS, 11 monocomponent polypeptides surrounding these fibers (6). could be detected by PAGE. Of these, four single polypep- In close contact with the intestinal epithelial cells. separated tides with molecular weights of 17 000-39 500 and isoelec- from them by the basement membrane. lies a continuous sheath tric points of 5.0-7.0 were observed to react with IgA of fibroblasts that forms part of the connective tissue infrastruc- separated from sera of children with celiac disease. The ture of the lamina propria (1 1-13). Fibroblasts are also known polypeptide molecules produced by fibroblasts in vitro to synthesize extracellular matrix proteins, collagen and procol- bound to antireticulin and antiendomysium antibodies but lagen (14, 15). and noncollagenous proteins, many of which have not to antigliadin antibodies. The present observations autocrine or paracrine functions (16-19). Berman et a/. (20) show that tissue antibodies found to be specifically asso- showed that fibroblasts produced extracellular fibers that stained ciated with celiac disease are generated against a synthesis positively with silver-impregnation reticulin stain. Fibroblasts product of fibroblasts, a cell-type known to synthesize a have also been shown to express reticulin molecules (21). number of biologically active polypeptides. The fibroblast- In the present study, we hypothesized that CDAP are synthe- derived extracellular matrix proteins and the formed auto- sized by fibroblasts, and our results show that fibroblasts do antibodies may be important in the pathogenesis of gluten- synthesize noncollagenous proteins that, in genetically deter- sensitive enteropathy. (Pediatr Res 34:420-423, 1993) mined individuals, become autoantigens when gluten ingestion commences. We further show the CDAP molecules to be secreted Abbreviations by fibroblasts as part of a large protein complex. ARA, antireticulin antibody MATERIALS AND METHODS AGA, antigliadin antibody CD, celiac disease Fltioroimm~inostainingofctrlt~rrc~d/ibroblas~la!~c~r. Fibroblasts CDAP, celiac disease autoantigen protein were removed from fetal lung tissue (3) and stored in liquid EMA, antiendomysium antibody nitrogen. Approximately 0.5 x 10' cells were cultured in 4-mL NCRC, noncollagenous reticulin component culture vessels in 1.5 mL of cell culture medium containing 10% of FCS. The culture was incubated at 37°C in atmosphere con- taining 5% COz for 2 d. The medium was then discarded, and the cells were acetone-fixed and fluoroimmunostained using purified CD patient sera IgA as previously described (21). For The etiology and pathogenesis of CD, or gluten-sensitive en- control purposes, fluoroimmunostaining was also performed teropathy, is not well understood, despite a large body of infor- using IgA purified from sera from healthy subjects. mation accumulated in recent decades (I, 2). However, we have Evaluation of fibroblast synthesis product accttmulation into recently proposed a new way of thinking and hypothesized that culture medium. Approximately 5 x lo6 fetal lung fibroblasts the gluten-sensitive enteropathy might be attributable to an were divided into five culture vessels of 50 mL and cultured for autoimmune mechanism in which antibodies are generated 4 d as described above with tritiated amino acid mixture (Amer- aeainst the ~atient'sown tissue material. We have identified sham, Buckinghamshire, England) with a total radioactivity of 2 e;tracellular'matrix noncollagenous protein molecules that spe- pCi. Samples of 0.5 mL were removed from the media once a cifically react with CD patient sera IgA (3). In CD, these identified day to evaluate the appearance of tritiated protein material in human protein molecules trigger the production of tissue anti- the culture medium. The samples were gel filtrated in 9 x 1. I- Received October 26. 1992: accepted April 29. 1993. cm Sephadex G-25 columns (Pharmacia, Uppsala, Sweden) to Correspondence and reprint requests: Aulis Marttinen, University of Tampere. remove tritiated amino acids from protein material of the culture Department of Clinical Medicine, PO Box 607. SF-33101 Tampere. Finland. medium. 'H radioactivity of the protein material was counted. The Celiac Disease Study Project is supported by the Medical Research Council. Academy of Finland (research contract 1061289) and by the Sigrid Juselius Sc>parationr!f:fihrohlast-synlhesizedprotein molecc.ltlc~s reacting Foundation. with CD patient sera IgA. After 4 d, the fibroblast culture media ' This paper is dedicated to Professor S. Auricchio on his 60th birthday. were collected and concentrated by dialyzing against 60 mM FIBROBLAST-DERIVED CD AUTOANTIGENS 42 1 potassium phosphate buffer. pH 6.9, containing 40% (wt/vol) Fixing and staining were performed according to the manufac- sucrose. Sucrose and the remaining free titrated amino acids were turer's instructions. removed from the dialysate by gel filtration with a 9 x I. I-cm Sephadex G-25 gel filtration column. The concentrated fibroblast culture medium was then gel filtrated using HPLC equipment RESULTS fitted with a 600 x 2 1.5-mm Spherogel TSK HPLC gel filtration Fluoroimmunostaining ofcu1tured.fihrohlast la.ver. Using ace- column (Beckman, Fullerton, CA), and 60 mM potassium phos- tone-fixed fibroblast layer and highly purified CD patient sera phate buffer, pH 6.9. as eluent. Fractions of 3.5 mL were col- IgA in the test, almost 100% of the cell monolayer stained lected and 0.5 mL from each fraction was used to count 'H positively (Fig. 1). Control sera IgA and the sera absorbed with radioactivity. Thereafter, 0.5 mL of each fraction was diluted to Jacalin gave negative staining. yield a protein concentration of 20 g/mL, using the method of Synthesis and secretion of CD alitoantigcw molec~~lesh19,fihro- Lowry for quantitative protein determination (22). For detection hla.sts. Continuous accumulation of'H-labeled fibroblast-synthe- of reactivity with CD patient sera IgA of the 'H-protein material sized proteins into the medium was observed during culture. The in the fractions, a Nunc Maxisorb ELISA plate (Nunc, Roskilde, radioactivity of the protein material was 180 dpm/mL after 4 d Denmark) was coated with 100 FL of each diluted fraction/well of culturing. The gel filtration curve in Figure 2 shows that about overnight at 4'C and blocked by I % gelatin solution for 1 h at 30% of the secreted 'H-labeled protein was found to be in 37°C. After washing. the plate was incubated with IgA (5 g/L) complex form with a molecular weight higher than 1 million. purified from sera from a child with CD with extremely high This complex, collected with fractions 3. 4, and 5, was highly EMA titer, using the Jacalin (Pierce, Rockford, IL) method (23). reactive with CD patient sera IgA. Other secreted proteins did Alkaline phosphatase-conjugated, a-chain-specific anti-human not react significantly with IgA (fractions 1, 2. and 6-23). IgA (Orion Diagnostiga, Helsinki, Finland) was used as the In drastic conditions, a major part of the tightly bound mon- second antibody, and 100 pg of phosphatase substrate/well were ocomponent polypeptides could be dissociated from the protein used for color development. OD was measured after 45 min complex (Fig. 3). After gel filtration of the dissociated material. incubation at 37°C. The rest of the fractions that gave an OD most of the radioactivity (indicating presence of fibroblast syn- higher than 0.2 were then pooled and concentrated by dialyzing thesis products) was detected in the fractions of the lower- against 40% sucrose solution for additional analysis. For control molecular-weight region (fractions 15- 19). The material in these measurement, ELISA analysis was performed similarly using a fractions was also reactive with patient sera IgA analyzed by plate coated with 100 pL/of corresponding diluted fraction of ELISA. The mixture of dissociated material contained at least medium per well without fibroblast culturing, treated and gel- l l different polypeptides with isoelectric points from 4.5 to 7.8 filtrated equally. The value of the OD of the control plate was and molecular weights from 16.5 to 39.5 (determined using subtracted from the corresponding analysis value. HPLC gel filtration) (Figs. 3 and 4). When that mixture was Pzrrification of f'monocomponent IRA reactive po1jpeptide.s. The applied to the affinity chromatography column with CD patient pooled fractions, containing 'H-labeled CD patient sera IgA reactive fibroblast-synthesized material, were concentrated by dialyzing against 40% sucrose solution.