Role of Perforin and Granzyme B of Cytotoxic T Lymphocyte in the Onset of Peptic Ulcer Formation

Role of Perforin and Granzyme B of Cytotoxic T Lymphocyte in the Onset of Peptic Ulcer Formation

Tadashi Ohara

, Tetsuo Morishita

, Hidekazu Suzuki

, Tatsuhiro Masaoka

, and Hiromasa Ishii

Key words.

Cytotoxic T lymphocyte, Perforin, Granzyme B, H. pylori, Apop- tosis, Peptic ulcer

Introduction

Attention has been focused on apoptosis in terms of its involvement in the pathogenesis of various diseases including hepatitis, graft-versus-host disease (GVHD), and multiple sclerosis [1–3]. Some pathways are known to be impli- cated in cytotoxic T lymphocyte (CTL)-mediated apoptotic induction: per- forin/granzyme pathway (based on granules stored in cytoplasmic azurophile granules of CTLs), Fas/Fas ligand (FasL), tumor necrosis factor (TNF)/TNF receptor (TNFR), or TNF-related apoptosis-inducing ligand (TRAIL) path- ways based on type II transmembrane proteins belonging to the TNF family expressed on the cell surface. Among them, the perforin/granzyme and Fas/FasL pathways are considered the two major routes of apoptosis. The aim of this study is to investigate whether apoptosis associated with peptic ulcer formation may occur via either or both of these two pathways.

Methodology

Patients

Thirty-two patients with active peptic ulcer at the A

–A

stage with and without Helicobacter pylori infection were enrolled this study (gastric ulcer:

21 ; duodenal ulcer: 11). Helicobacter pylori infection was diagnosed by enzyme-linked immunosorbent assay and the urea breath test (UBT) method.

99

1Department of Internal Medicine, Tokyo Dental College, Chiba, Japan

2Department of Internal Medicine, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan

Experimental protocol was as follows: (1) detection of apoptotic cells by in situ fluorescence (TUNEL method), (2) examination of the induction of per- forin, Fas, and Fas ligand (FasL) by reverse transcription–polymerase chain reaction, (3) examination of the induction of caspase 3 by immunoblotting, and (4) examination of the detection of perforin, granzyme B, and caspase 3 by immunohistochemistry (light and electron microscopy staining).

Results

Expression of apoptotic cells was observed from the lamina propria to the epithelium in all samples taken from the marginal zone of peptic ulcers with or without H. pylori infection by the in situ fluorescence TUNEL method (Fig.

1 ). Reverse transcription–polymerase chain reaction findings showed the expression of perforin and Fas in all samples with and without H. pylori infec-

Fig. 1. Expression of apoptotic cells in the marginal zone of gastric ulcer by in situ fluorescence tunel method

tion; however, FasL expression was never seen constantly (Fig. 2).

Immunoblotting analysis showed constantly expression of caspase 3 in all samples regardless of H. pylori infection (Fig. 3). Immunohistochemical analysis revealed that perforin and granzyme B localized in the lesion from the CTL cytoplasm, between the proper mucous membrane and epithelium, to the target cell (Fig. 4). Moreover, caspase 3 was seen in granzyme B- expressing CTL and its surrounding interstitium (Fig. 5). Immunoelec- tron microscopy provided images of perforin released from cytoplasmic azurophile granules of CTL towards the target cells (Fig. 6a), and images of the target cells showed an early apoptotic appearance with a tight spherical body change and oscula in the cell membrane (Fig. 6b). There were also images of granzyme B that invaded the target cells through oscula to induce apoptosis of blood cells in cytoplasm, and in turn other cells, causing exfoli- ation (Fig. 7a,b).

Discussion

There are many reports on the relation between peptic ulcers and apoptosis, but reports on the pathway of apoptosis involved in peptic ulcers are limited [4]. Investigation of the effector of mucosal injury has never yet been reported except for our study [5]. By all means, the present clinical investigation alone

( I )

( II )

265 bp (Fas) 231 bp (FasL)

399 bp (perforin)

Fig. 2. Expression of Fas, FasL, and perforin in gastric and duodenal mucosa infected with or without Helicobacter pylori (HP) reverse transcription–polymerase chain rearta by RT-PCR

86

41.5

33.4

19.5

Fig. 3. Examination of caspase 3 by immunoblotting in gastric and duodenal mucosa with or without H. pylori infection. MW, molecular weight

Fig. 4. Expression of perforin granzyme B by immunohistochemistry in the marginal zone of gastric ulcer

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cannot determine whether the observed phenomena may mean onset of mucosal damage or the course of regeneration. However, interestingly enough, it was suggested that CTL might be an effector (trigger) for mucosal damage irrespective of H. pylori infection. If it could be demonstrated that a certain substance might induce CTL with a common epitope in both groups irrespective of H. pylori infection, it would signify the possibility that there

granzyme B caspase 3

Fig. 5. Expression of granzyme B caspase 3 by immunohistochemistry in the marginal zone of gastric ulcer

Fig. 6a,b. Electron microscopic findings of perforin, which attacks the target cell by immunohistochemistry

a b

may be an unknown antigen responsible for peptic ulcers. If such a substance could be determined, it may be a significant discovery in terms of investiga- tion of peptic ulcers, and further development can be expected. Whether we studied CTL derived from CD4 or CD8 using a flow cytometry in mononu- clear cells obtained from mucosal biopsy specimens endoscopically, we could analyze it only for very few mononuclear cells. We have a schedule of exami- nation concerning this unsolved problem using other methods. Thus we are planning further investigation of these various problems and questions using an experimental animal model and other clinical examinations based on these results.

References

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2. Hattori K, Hirano T, Miyajima H, et al (1999) Differential effects of anti-Fas ligand and anti-tumor necrosis factor alpha antibodies on acute graft-versus-host disease patholo- gies. Blood 93:2738–2747

Fig 7a,b. Electron microscopic findings of granzyme B, which attacks the target cell by immunohistochemistry. ER, rough endoplasmic reticular

a b

3. Sabelko Downers KA, Cross AH, Russell JH (1999) Dual role for Fas ligand in the initi- ation of and recovery from experimental allergic encephalomyelitis. J Exp Med 189:

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