(1) The presence and functional status of surface expression of Fas receptor (FasR) and its ligand (FasL) on tumor and tumor-infiltrating lymphocytes (TIL) in gastric carcinoma (n=36) from the primary locus, metastatic gastric carcinoma (n=30) from malignant ascites, benign gastric mucosa (n=30) for the control were investigated. The quantitative analysis was based on percentage of positive cells by a flow cytometry. The results showed that membrane-bound FasL molecule was constitutively expressed on primary and metastatic gastric carcinomas as well as normal gastric epithelium in nearly all the patients. Especially, metastatic carcinoma showed to aberrantly express FasL molecule. Whereas, FasR expression ranged from minimal or absence in primary and metastatic gastric carcinomas, suggesting that the carcinoma might be rendered less sensitive toward FasR-induced killing. Apoptotic tumor cells detected by TUNEL were hardly identified in primary and metastatic carcinomas. In the analysis
of TIL, the expression of FasR and FasL, and apoptotic TIL could not usually be observed in primary gastric carcinoma. In metastatic carcinoma, however, there was significant overexpression of FasR and FasL on immune TIL associated with a higher frequency of apoptotic cell death detected by TUNEL. The results suggest that metastatic carcinoma expressing FasL, but not FasL^+ primary carcinoma might evade the immune attack by apoptotic depletion of activated TIL through the FasR/FasL systems. These provide the direct and quantitative evidences to support the FasR counterattack and/or paracrine fratricide as a mechanism of tumor-immune escape in vivo in human cancer.
(2) We investigated the presence and functional status of TRAIL and its receptors, DR4, DR5 and DcR2 on tumor as well as tumor-infiltrating lymphocytes (TIL) in primary (n=37), and metastatic gastric carcinoma from malignant ascites (n=37) by a flow cytometry. In addition, phenotypic proportions of major T-cell subsets or B-cells in TIL were also determined. Membrane-bound TRAIL/its receptors are constitutively expressed at high levels in primary and metastatic carcinomas in nearly all the patients. Apoptotic tumor cells detected by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick and labeling (TUNEL) were barely identified in primary and metastatic carcinomas. TIL in primary carcinoma showed very low level of expression of TRAIL/its receptors and TUNEL-positive cells. In metastatic carcinoma, however, there was significant overexpression of TRAIL/its receptors in TIL associated with a higher frequency of apoptotic cell death detected by TUNEL. The TIL within metastatic carcinoma, but not within primary carcinoma revealed the increased proportions of CD3^+ T cells bearing CD8^+CD11b^-, CD8^+CD11b^+, and CD4^+CD62L^-, CD4^+CD62L^+ surface phenotype in patients. These results suggest that TRAIL^+ and DcR2^+ metastatic carcinoma from malignant ascites could not only have resistance to DR4/DR5-induced apoptosis, but also might take the TRAIL-mediated counterattack against activated CD3^+ T cells. These functions of the cancer cells would neutralize host immune responses at effector phase, and accelerate further invasion and/or metastasis of carcinoma through the escape from immune attack.
(3) The expression of Fas receptor (FasR) and FasL of the gastric epithelium and infiltrating mucosal lymphocytes (IML) was investigated in cell suspensions of gastric biopsies from patients with Hp-positive (n=42) and with Hp-negative (n=18) gastritis by FACS using mAbs. Furthermore, CD-antigens of IML were analyzed by flow cytometry. To quantify apoptosis of the epithelium and IML, TUNEL method was done by FACS. The membrane-bound FasL was constitutively expressed on freshly isolated gastric epithelium and IML. Overexpression of FasR/ FasL was seen in Hp-infected mucosal epithelium and IML. The percentages of CD3^+CD19^-, CD8^+CD11b^- and CD4^+CD62L^- T cells, and CD3^-CD19^+ B cells in IML from Hp-positive patients were significantly higher than those from Hp-negative patients (p<0.01). There was significantly higher frequency of apoptotic cell death detected by TUNEL in both epithelium and IML in Hp-infected mucosa (p<0.05). Hp-infection results in enhanced immune state within gastric mucosa, followed by induction of activated CD3^+T and CD19^+B cells triggered by Hp-antigens. FasR/FasL are involved in an apoptosis of epithelium in Hp-associated gastritis to some extent. However, rather more specified findings are increased apoptotic depletion of IML associated with FasL overexpression by Hp-positive gastric epithelium. The results provide the quantitative evidence of FasR counterattack against IML in vivo as a mechanism of immune privilege of glandular stomach infected with Hp. The substantial mucosal injury of Hp-associated gastritis is mainly occurred in IML rather than in gastric epithelium. These may help to explain the so-called African enigma. In addition, the situations would also allow persistent long-term Hp-infection without elimination of Hp from the host.
(4) TNF-related apoptosis-inducing ligand (TRAIL) and its receptors have recently been known to be responsible for apoptotic signaling molecules. However, little is known about the TRAIL-mediated apoptosis in human glandular stomach. Biopsies from 66 patients (H. pylori-negative, 28 ; H. pylori-positive, 38) were investigated for phenotypic distribution of the TRAIL/its receptors, DR4/DR5 and DcR2 on mucosal epithelium, and infiltrating mucosal lymphocytes using flow cytometry. Apoptosis of the cells was examined by TUNEL. Also, phenotypic distribution of CD antigens on infiltrating major T- and B-lymphocytes were determined. Membrane-bound TRAIL/its receptors are constitutively expressed in all patients with high levels in gastric mucosal epithelium. In particular, these protein molecules are overexpressed in mucosal lymphocytes coupled with increased proportions of CD19^+ B cells, and CD3^+ T cells bearing CD8^+CD11b^- and CD4^+CD62L^- surface phenotype in H. pylori-positive gastric mucosa. Frequency of apoptotic epithelium and infiltrating lymphocytes in H. pylori-associated gastritis is significantly greater than those of H. pylori-negative normal mucosa (p<0.01). The findings from this study suggest that apoptosis of gastric epithelial cells and infiltrating mucosal lymphocytes is possibly induced by activation of the TRAIL/its receptors system in H. pylori-associated gastritis. Less