To study the putative agent(s) related to Crohn disease, we intraperitoneally in injected mesenteric lymph node homogenates from four patients with active Crohn disease into 10-week-old athymic (nu/nu) mice. Control mice (nu/nu) were injected with homogenates of mesenteric lymph nodes from two patients with ulcerative colitis and four patients undergoing elective cholecystectomy, and with a homogenate of a cervical lymph node containing sarcoid granuloma. Thirty-four mice received filtered or unfiltered homogenates from Crohn disease lymph nodes. Thirty-two mice received homogenates or filtrates from lymph nodes of control patients. Four mice from the group injected with Crohn disease homogenates from four different patients developed generalized lymphadenopathy due to lymphoma 10-28 weeks after th injection. Two additional mice developed lymphadenopathy due to plasma cell hyperplasia. None of the control mice developed lymphomas or lymphadenopathy. Two lymphomas were homogenized, filtered, and injected intraperitoneally into a second group of nu/nu mice, which also developed lymphoma within 8 weeks of injection. Two lymphomas were cultured in vitro and B cell sur?ACE MARKERS WERE IDENTIFIED. Indirect immunofluorescence studies in two lymphomas showed cytoplasmic staining of lymphoma cells with sera from 10 patients with active Crohn disease but not with sera from 13 control subjects, including 6 with ulcerative colitis and 7 with other gastrointestinal disorders. These results suggest that a transmissible factor present in Crohn disease lymph nodes produces lymphoma in nu/nu mice. Furthermore, sera of Crohn disease patients contain an antibody that recognizes an "antigen(s)" in the murine lymphoma.
We detected in human colon extracts a 40 kDa protein(s) that specifically reacts with tissue-bound IgG obtained from the colon of patients with ulcerative colitis or CCA-IgG. Using the hybridoma technology, we developed monoclonal antibodies to this 40 kDa protein. The specific immunoreactivity of one of the monoclonal antibodies (7E12H12, IgM isotype) against the 40 kDa protein was demonstrated both by ELISA and by immunotransblot. Competitive binding experiments showed that CCA-IgG inhibits the binding of 7E12H12 to the 40 kDa protein, suggesting the recognition of common epitope(s) on the 40 kDa protein by the monoclonal antibody and CCA-IgG. 7E12H12 was used to determine cellular localization of the 40 kDa protein. Biopsy tissue specimens from colon, esophagus, stomach, duodenum, jejunum, ileum, liver, pancreas, lungs, kidneys, salivary, and mammary glands were obtained. Tissue specimens were fixed in 4% paraformaldehyde or in 10% formalin. Sections were sequentially incubated with the hybridoma supernatant, biotinylated anti-mouse IgM, avidin-biotin-peroxidase complex, and 3,3'-diaminobenzidine. An unrelated hybridoma supernatant was used as control. The monoclonal antibody exclusively recognized colonic epithelial cells both in the crypt and on the luminal surface. Immunoreactivity was present on the plasma membrane chiefly along the basolateral areas of the cells. Plasma membrane localization of the 40 kDa protein was confirmed by immunoelectron microscopy. All colonic mucosal biopsy specimens from both adult and fetal colon reacted with the monoclonal antibody. None of the biopsy specimens from stomach, duodenum, jejunum, ileum, liver, pancreas, or non-gastrointestinal tissue reacted with the antibody, confirming the organ specificity of the 40 kDa protein. The interaction between this colonic epithelial membrane protein and the CCA-IgG may play an important role in the pathogenesis of ulcerative colitis.
SUMMARYWe studied the M. paratuberculosis-induced proliferation and suppressor cell generation by peripheral blood lymphocytes from patients with inflammatory bowel disease. Peripheral blood lymphocytes were separated from 33 patients with Crohn's disease, 18 with ulcerative colitis, nine with other intestinal diseases, and five with autoimmune disorders. Proliferation ofperipheral blood lymphocytes from normal individuals in response to 10 /^g/ml of M. paratuberculosis antigen was reduced by depletion of CD4+ T cells. The ability of M. paratuberculosis antigen to suppress eoncanavalin A-induced proliferation (expressed as a percentage suppression) was reduced by depletion of CD8+ T cells. This suppression was the same whether peripheral blood lymphocytes were from normal individuals, patients with intestinal diseases other than inflammatory bowel disease, or patients with autoimmune disorders (47+ 14%, 44 + 24%, and 30 + 26%, respectively). In contrast, the suppression induced by M. paratuberculosis for patients with Crohn's disease and ulcerative colitis (66 + 22% and 67 + 22%) was much greater than that for normal individuals (P < 0 001), In particular, lymphocytes from patients with active Crohn's disease demonstrated little proliferation in response to this antigen but marked suppressor activity (79±13%), How the immunomodulatory effects of this antigen relate to the pathogenesis of the inflammatory bowel diseases remains to be determined.
In patients with ulcerative colitis a colon tissue bound IgG and serum antibodies against an Mr 40000 colonic protein(s) has been identified.
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