Statement of the Problem: In the intestine, dysregulated neutrophil (PMN) transmigration is a central component of inflammatory bowel disease ((IBD), consisting primarily of Crohn’s Disease and Ulcerative Colitis). The pathology of Ulcerative Colitis reveals an irregular, damaged epithelium populated by massive numbers of PMN frequently located at the apical (lumenal) intestinal surface. These migrating PMN appear to be attached to the epithelial surface and fail to release into the intestinal lumen. Consequently such PMN accumulate at the lumenal surface where they form crypt abscesses. It is not known why PMN fail to release into the intestinal lumen or how this leads to the formation of crypt abscesses. Interestingly, a majority of IBD patients possess antibodies reactive with their colonic epithelium. Thus, Fc-receptor mediated interactions likely contribute to the mucosal pathophysiology of IBD. Using an in vitro model of PMN transepithelial migration that includes cultured human intestinal epithelial cells and purified human PMN, we produced a monoclonal antibody (g82) that inhibits PMN transepithelial migration at level of the apical epithelial surface. We have determined that this antibody prevents release of PMN from the apical surface; an effect that is both Fc-receptor specific and antigen-dependent. The antigen is present on normal human colon and is markedly up regulated in IBD where crypt abscesses develop. National statistics indicate that there is a spike in the development of the IBD during the 5th and 6th decades and that an increasing number of African Americans are being diagnosed with the disease. Thus, the goal of this proposal is to determine if African American and Caucasian patients diagnosed with IBD produce antibodies that function in a similar manner as mAb g82. HYPOTHESIS 1 Apical epithelial antibodies are present in IBD patients and such antibodies contribute to dysregulated PMN transmigration Specific Aim 1: To evaluate the serum of IBD patients for apical epithelial antibodies and determine if such antibodies react with the antigen defined by mAb g82 Anti-colonic epithelial antibodies are a common finding in patients with IBD. 1) A panel of serum samples will be collected and analyzed from both African American and Caucasian patients (in the 5th and 6th decades) diagnosed with IBD. 2) Serum samples will be examined for specific binding to T84 cells and binding to frozen sections of human colon using ELISA and/or immunofluorescence. 3) Samples with staining profiles similar to that observed with antibody g82 will be tested for reactivity with epithelial cell immunoprecipitates of mAb g82. 4) Positive binding samples will be tested for inhibition of PMN transepithelial migration and effects on PMN cellular adhesion. HYPOTHESIS 2 Following transmigration, antigen-antibody adhesive interactions that occur at the lumenal epithelial surface result in crypt-like abscess formation and such adhesive interactions are modulated by inflammatory cytokines Specific Aim 2: To evaluate the effect that inflammatory cytokines have on the expression of the antigen and functional effects on mAb g82 Preliminary results indicate a considerable increase in mAb g82 immunofluorescence labeling of T84 cells after stimulation with interferon-g and in the colonic epithelium of IBD patients. 1) The effect of cytokine exposure to mAb g82 expression in T84 cell monolayers will be evaluated. 2) Functional effects that up regulation of the antigen defined by mAb g82 has on PMN transepithelial migration and cellular adhesion will then be evaluated once cytokine treatment conditions have been optimized.

Methods: Specific Aim 1: To examine the serum of African American and Caucasian IBD patients for anti-apical epithelial antibodies and determine if such antibodies react with the antigen defined by mAb g82. Rational: Investigations into the functional effect of anti-epithelial antibodies from IBD patients may contribute to the understanding of the pathogenesis of IBD and may provide a basis for therapeutic interventions. Research design Specific Aim 1: This aim will involve collecting serum samples from African American and Caucasian patients with active Ulcerative Colitis and Crohn’s disease. The serum samples will be properly classified. For example, serum samples from patients with active ulcerative colitis (without medication-treatment); serum samples from patients with active ulcerative colitis (with medication-treatment) including inflammatory diseases that do not involved the GI tract. Secondly, I will culture monolayers and 96 well plates (monolayers and tissue sections) of T84 intestinal epithelial cells and analyze them for reactivity to the collected serum samples using an Elisa assay and/or immunofluorescence labeling. Elisa—96 well plates with epithelial cells will be washed with Hanks (HBSS) balanced salt solution, which contains (in g/L): 0.185 CaCl2, 0.098 MgSO4, 0.4 KCL, 0.06 KH2PO4, 8.0 NaCl, 0.048 Na2PO4, 1 glucose, and 10 mM HEPES pH 7.4. A modified HBSS (-) is prepared just as above, but without CaCl2 and MgSO4. Plates are then blocked with 0.5% bovine serum albumin (BSA) in HBSS for 1 hr at room temp and washed with HBSS before the serum samples are added. A 10% solution of serum (primary antibody) in HBSS will be added to the plates and allowed to incubate for 2 h at 4°C. After 2 h, plates will be washed with HBSS before the secondary antibody is added. The secondary antibody (peroxidase conjugated goat anti-mouse Fc-specific) at a concentration of 1:5000 in 10% blocking buffer is allowed to incubate for 1 hr at 4°C. Plates will then be washed and developed using 2,2’-Azino-bis (3 ethylbenz-thiaoline-6-sulfonic acid) (ABTS). Immunofluorescence—96 well plates (monolayers/tissue sections on glass plates) will be fixed in cold ethanol for 30 min at 4°C. Plates will then be washed with HBSS and blocked with BSA. This will be followed by primary antibody for 2 h (10 mg/ml in 5% NGS) (4). Labeling with serum samples will also be performed on 4 mm, frozen tissue sections of human colonic mucosa, both normal and active IBD, which are obtained from fresh surgical resection specimens. After washing, 96 well plates will be incubated with FITC-conjugated secondary antibody for 1 hour at 20°C. To visualize nuclei, both monolayers and tissue sections will be labeled with propidium iodide (1:1000 dilution, 20 min, 20°C). Transmigration and cellular adhesion assays will be performed according to previous published methods. For serum samples, 5-10% sera in HBSS will be added to apical compartments (reservoirs) for both transmigration and cellular adhesion assays. Potential Pitfalls/Alternative Approaches The preliminary data indicate that both Crohn’s Disease and Ulcerative Colitis patients possess the antigen. Therefore, I do not anticipate a negative result in the expression of the antigen defined by mAb g82. However, if the serum samples do not identify auto antibodies, alternatively, IgG from the sera of Ulcerative Colitis patients could be directly isolated in a salt precipitation. The isolated IgG could then be used to make other antibodies and tested against serum samples from IBD patients. Specific Aim 2: To characterize the effect that inflammatory cytokines have on the expression and function of the antigen defined by mAb g82 and cytokine effects on the function of the antibody. Rational: These studies will enhance our understanding of the role of cytokine exposure in the expression and function of antigens present on the lumenal epithelial surface and how this contributes to the development of crypt abscesses. Research design for Specific Aim 2: T84 intestinal epithelial cells will be separately treated with inflammatory cytokines to determine if such antibodies have an effect on expression of the antigen and mAb g82. As indicated in Reaves et al. (2001), when confluent monolayers of T84 cells are treated with 28ng/ml of IFN-g for 48 hrs, the antigen defined by mAb g82 was substantially up regulated, as determined by immunofluorescence. Furthermore, the antigen was also substantially up regulated in patients diagnosed with active Ulcerative Colitis. I will begin by treating T84 and/or HT-29 cell monolayers with IFN-g to re-examine the expression profile of the antigen defined by mAb g82. Such monolayers will be treated with IFN-g for 48 hours (12). In parallel, I will also treat monolayers of T84 and/or HT-29 cells with tumor necrosis factor-a and interleukin-8. For these cytokines, monolayers will be treated with concentrations ranging from 5-30 ng of each cytokine. Monolayers will be examined at a minimum of 6-48 h to determined the proper conditions under which treatment will not have a detrimental effect on the epithelial cells. Once proper conditions have been determined, immunofluorescence assays will be used to expose differences in expression of the antigen defined by mAb g82. Based on the immunofluorescence experiments, transmigration and cellular adhesion experiments will be performed to determine functional effects on the role of antigen and mAb g82.