Ellen C Ebert1, Xin Geng, Manisha Bajpai, Zui Pan, Eric Tatar, Kiron M Das. 1. Department of Medicine and Physiology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Crohn's and Colitis Center of NJ, New Brunswick, New Jersey 08903, USA. ebertec@umdnj.edu
Abstract
OBJECTIVES: Tropomyosins (TMs) are cytoskeletal microfilament proteins present in all eukaryotic cells. Human TM isoform 5 (hTM5) is the predominant isoform in colonic epithelial cells. Antibodies against hTM5 are found both in the sera and in the mucosa of patients with ulcerative colitis (UC) but not Crohn's disease (CD). We investigated whether anti-hTM5 autoantibodies are pathogenic. METHODS: Normal-appearing colonic mucosal biopsy specimens were incubated with autologous serum. After 45 min, deposition of the complement component C3b was identified by indirect immunofluorescence assay (IFA). Additional specimens were incubated with autologous serum fixed in formalin, and their architecture was examined by hematoxylin and eosin (H&E) staining. RESULTS: For 79% of UC patients, autologous serum caused C3b staining along the colonic epithelium. Recombinant hTM5 or anti-hTM5 monoclonal antibody blocked serum-induced C3b deposition. Immunoglobulin G (IgG) antibody and affinity-purified anti-hTM5 IgG antibody from UC sera with complement caused C3b deposition, indicating specificity of hTM5 as an autoantigen. When analyzed by H&E staining, sera obtained from 71% of UC patients caused a significant loss of epithelium. This process was inhibited by Fc fragments, indicating that it is complement mediated. With medium, normal, or CD serum, there was no C3b deposition or morphological changes of the colonic epithelium, indicating disease specificity. The ileal mucosa was not affected by UC sera, suggesting specificity for the colon. In UC mucosa, expression of hTM5 increased. CONCLUSIONS: hTM5 acts as an autoantigen in UC. hTM5-specific IgG autoantibody in sera from UC patients induces C3b deposition and destruction of colonic epithelial cells, suggesting a direct pathogenic effect. If used as a diagnostic test to distinguish UC from CD, IFA would have 79% sensitivity and 100% specificity. Development of blocking antibodies may lead to novel therapies.
OBJECTIVES: Tropomyosins (TMs) are cytoskeletal microfilament proteins present in all eukaryotic cells. Human TM isoform 5 (hTM5) is the predominant isoform in colonic epithelial cells. Antibodies against hTM5 are found both in the sera and in the mucosa of patients with ulcerative colitis (UC) but not Crohn's disease (CD). We investigated whether anti-hTM5 autoantibodies are pathogenic. METHODS: Normal-appearing colonic mucosal biopsy specimens were incubated with autologous serum. After 45 min, deposition of the complement component C3b was identified by indirect immunofluorescence assay (IFA). Additional specimens were incubated with autologous serum fixed in formalin, and their architecture was examined by hematoxylin and eosin (H&E) staining. RESULTS: For 79% of UC patients, autologous serum caused C3b staining along the colonic epithelium. Recombinant hTM5 or anti-hTM5 monoclonal antibody blocked serum-induced C3b deposition. Immunoglobulin G (IgG) antibody and affinity-purified anti-hTM5 IgG antibody from UC sera with complement caused C3b deposition, indicating specificity of hTM5 as an autoantigen. When analyzed by H&E staining, sera obtained from 71% of UC patients caused a significant loss of epithelium. This process was inhibited by Fc fragments, indicating that it is complement mediated. With medium, normal, or CD serum, there was no C3b deposition or morphological changes of the colonic epithelium, indicating disease specificity. The ileal mucosa was not affected by UC sera, suggesting specificity for the colon. In UC mucosa, expression of hTM5 increased. CONCLUSIONS:hTM5 acts as an autoantigen in UC. hTM5-specific IgG autoantibody in sera from UC patients induces C3b deposition and destruction of colonic epithelial cells, suggesting a direct pathogenic effect. If used as a diagnostic test to distinguish UC from CD, IFA would have 79% sensitivity and 100% specificity. Development of blocking antibodies may lead to novel therapies.
Authors: S Hosomi; N Oshitani; N Kamata; M Sogawa; H Okazaki; T Tanigawa; H Yamagami; K Watanabe; K Tominaga; T Watanabe; Y Fujiwara; K Maeda; K Hirakawa; T Arakawa Journal: Clin Exp Immunol Date: 2010-11-19 Impact factor: 4.330