Immunohistochemical analysis of the distribution of measles related antigen in the intestinal mucosa in inflammatory bowel disease

Abstract

Background: Measles virus is implicated in the aetiology of Crohn's disease. This measles hypothesis is mainly supported by immunohistochemical findings that the measles related antigen is present in the intestine of patients with Crohn's disease. Recently we isolated this antigen from the intestine of a patient with Crohn's disease using a molecular cloning technique and produced the monoclonal antibody against it (designated 4F12).

Aim: To discover whether the measles related antigen is uniquely present in Crohn's disease.

Subjects/methods: Colonic mucosa samples from 20 patients with Crohn's disease, 20 with ulcerative colitis, 11 with non-inflammatory bowel disease (IBD) colitis, and nine controls were immunohistochemically stained with the anti-measles monoclonal antibody 4F12. The numbers of positive cells, the ratio of positive cells to nucleated cells, and the staining intensity of the positive cells were compared. Furthermore, the distribution of the measles antigen in other human organs was examined.

Results: Both the number of positive cells and the ratio of positive cells to nucleated cells were significantly increased in Crohn's disease, ulcerative colitis, and non-IBD colitis compared with controls (p<0.05) but were similar among the three disease groups. The staining intensity of the positive cells was also similar among the three disease groups. Small numbers of positive cells were observed in the oesophagus, stomach, duodenum, jejunum, and lung.

Conclusions: The presence of the measles related antigen in the colonic mucosa was not unique to Crohn's disease. These results, together with the observation that such a measles related antigen was derived from host protein, do not support the hypothesis that measles virus causes Crohn's disease.

Figure 1

Staining intensity of positive cells: (A) strong; (B) moderate; (C) weak. No positive cells are observed in the negative control sample (D).

Figure 2

Multiple alignment of the nucleotide sequences between the positive clone and AA449055. Nucleotide sequences of non-homologous regions are shown in bold and underlined.

Figure 3

Deduced amino acids sequence of the positive clone. Each amino acid is shown using the single letter code.

Figure 4

SDS/PAGE of the positive clone expressed by Escherichia coli as a fusion protein with β-galactosidase, and western blot analysis of the fusion protein (the positive clone and β-galactosidase) and anti-measles monoclonal antibodies.

Figure 5

Immunofluorescence study of reaction of measles virus infected cells with 4F12. Syncytium in measles virus infected Vero cells is clearly stained with 4F12.

Figure 6

Double immunohistochemical study with MAS 182r and 4F12. Virtually all positive cells are doubly stained with MAS 182r and 4F12 (A). Doubly stained cells appear as a mixture of dark blue and brown. Cells stained singly with either 4-chloro-1-naphthol (dark blue) or diaminobenzidine (brown) are shown in (B).

Figure 7

SDS/PAGE of the positive clone expressed as a fusion protein with GST, and western blot analysis of the fusion protein and anti-measles monoclonal antibodies.

Figure 8

Numbers of positive cells in the colonic mucosa. The number of positive cells for each sample is shown as a closed circle. The mean and SE of the numbers of positive cells in each group is shown as an open circle and vertical bar. IBD, inflammatory bowel disease.

Figure 9

Ratio of positive cells to nucleated cells expressed as a percentage. The percentage for each sample is shown as a closed circle, and the mean and SE for each group is shown as an open circle and vertical bar.

Figure 10

Proportions of the staining intensities in each group. The proportion staining strongly is shown as black, that staining moderately as dark grey, and that staining weakly as grey.