High Fas ligand expression on lymphocytes in lesions of ulcerative colitis

Abstract

Background: The pathogenesis of ulcerative colitis is unclear, but cytotoxic T lymphocytes infiltrating the mucosa have been implicated in mucosal damage. The Fas ligand (FasL), expressed on cytotoxic T lymphocytes, induces apoptosis in cells expressing Fas.

Aim: To analyse FasL expression in affected colonic mucosa to ascertain Fas-FasL interaction in ulcerative colitis.

Methods: FasL mRNA was quantified in colonic mucosal specimens from healthy subjects and patients with ulcerative colitis or Crohn's disease, using the competitive reverse transcription polymerase chain reaction. FasL mRNA localisation was determined by in situ hybridisation. Expression of Fas in colonic mucosa was analysed immunohistochemically. Phenotypes of lamina propria lymphocytes that expressed FasL were analysed by flow cytometry.

Results: FasL mRNA was strongly expressed in active ulcerative colitis lesions, but not in those associated with active Crohn's disease or active proctitis-type ulcerative colitis. In situ hybridisation showed that FasL mRNA expression occurred in mononuclear cells infiltrating lesions. Fas was expressed in epithelial cells in ulcerative colitis and Crohn's disease, and in normal subjects. Cytometry showed that FasL was expressed in CD3 lymphocytes infiltrating the lamina propria in active lesions.

Conclusions: FasL is expressed in CD3 lymphocytes infiltrating into ulcerative colitis but not Crohn's disease lesions, suggesting that Fas-FasL induced apoptosis participates in the mucosal damage of ulcerative colitis.

Figure 1

Protocols for the quantification of Fas ligand (FasL) transcripts. The procedures involved reverse transcription (RT), polymerase chain reaction (PCR) amplification, and digestion by AlwNI for FasL PCR products.

Figure 2

Amount of Fas ligand (FasL) transcript in terms of ulcerative colitis activity. Levels of FasL transcript were determined in normal colonic mucosa, colonic mucosa with active and inactive ulcerative colitis and active Crohn's disease relative to CTLL-2 derived FasL transcript. The relative amount of FasL transcript in each colon sample was corrected on the basis of the amount of RNA in the sample correlated to the amount of 28S RNA, determined by quantitative PCR.

Figure 3

Relative amounts of Fas ligand (FasL) transcripts and areas involved. Relative FasL transcripts were analysed in rectal mucosal specimens of patients with active ulcerative colitis (n=15) and inactive ulcerative colitis (n=5), on the basis of ulcerative colitis involvement in the colon. Patients with active ulcerative colitis were divided into three subgroups: those with proctitis (n= 5), left sided colitis (n=5), and total colitis (n= 5).

Figure 4

A comparison of Fas ligand (FasL) transcripts between uninvolved mucosa and involved lesions in the same patients in six active ulcerative colitis lesions. Involved lesions showed a significant increase in FasL transcripts in all cases (p<0.05, v uninvolved).

Figure 5

Expression and localisation of Fas ligand (FasL) mRNA in colon specimens, as detected by in situ hybridisation (ISH). ISH was performed with an FasL antisense RNA probe in sections from an active ulcerative colitis lesion (A, B), normal colonic mucosa (C), and an active Crohn's disease lesion (D). Representative data are shown. The results were similar in four subjects in each group. Original magnification: A, C and D, × 143; B, × 715.

Figure 6

Immunohistochemical detection of Fas in colonic mucosa. Sections from active ulcerative colitis lesions (A, C) and normal colon (B, D) were stained with mouse monoclonal antibody against human Fas. Original magnification: × 129.

Figure 7

Flow cytometric analysis of Fas ligand (FasL) expression on CD3 lamina propria lymphocytes (LPLs). Expression of FasL on CD3 LPLs isolated from (A) involved colonic lesions of ulcerative colitis, (B) active lesions of Crohn's disease, and (C) control colon mucosa was examined by immunofluorescence and FACS analysis. LPLs were stained with anti-human FasL antibody (solid line) or an isotype-matched control antibody (dotted line). Representative data from five or six cases each are shown. (D) Percentage of FasL expressing cells in CD3 LPLs.