Fas/Fas ligand expression and characteristics of primed CD45RO+ T cells in the inflamed mucosa of ulcerative colitis

BACKGROUND: Chronic immune activation in the colon is characteristic of ulcerative colitis (UC). Fas/Fas ligand (FasL) system is a mechanism responsible for activation-induced cell death (AICD), which maintains homeostasis within the immune system. Thus, Fas/FasL expression on activated colonic T cells of UC patients, as well as the susceptibility of such T cells to AICD was investigated in order to determine the role of activated colonic T cells in the long lasting inflammation in UC. METHODS: Fas, FasL, and CD45RO expression on peripheral blood and colonic T cells of UC patients were assayed by flow cytometry. Apoptosis of colonic T cells induced by anti Fas antibody was assessed using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) assay. RESULTS: The majority of colonic T cells expressed both CD45RO and Fas in the colonic mucosa, a situation that was quite different from that in the peripheral blood. The number of CD45RO+CD8+ and Fas+CD8+ T cells was significantly lower in UC patients than the controls, unlike the number of Fas+CD4+ T cells. In contrast, the number of both CD45RO+CD4+ and CD45RO+CD8+ T cells in UC mucosa expressing FasL was significantly higher than in the controls. While Fas mediated apoptosis of CD45RO+CD8+ T cells was higher in UC patients than the controls, the number of apoptotic CD45RO+CD4+ T cells from UC mucosa was not. CONCLUSIONS: In UC patients, CD45RO+CD4+ T cells are less sensitive to apoptotic signals mediated by Fas. These phenomena may contribute to the pathogenesis of UC.     

Activation-induced cell death of peripheral blood T lymphocytes in patients with primary Sjögren’s syndrome

Abstract

Activation-induced cell death (AICD) in T lymphocytes is important for the maintenance of peripheral tolerance. We studied AICD of peripheral blood T cells from patients with primary Sjögren’s syndrome (SS). AICD was induced in mitogen-activated T cells in vitro using mAb to CD3 or Fas (CD95). Cell death and proliferation, Fas and Fas ligand (FasL) expression, and soluble Fas and soluble FasL production were measured. Surface phenotypes and cytokine production of AICD-surviving cells and effects of cytokines on AICD were examined. Anti-CD3 mAb induced cell death is SS and normal T cells in the presence of exogenous interleukin (IL)-2. In the absence of IL-2 anti-CD3 mAb induced cell proliferation in SS and normal T cells. There was no significant difference in Fas/FasL expression and sFas/sFasL production between SS patients and normals. AICD-surviving cells consisted of more CD4⁺ T cells and less CD8⁺ T cells in SS compared to normals. AICD-surviving cells produced abundant interferon-γ and little IL-4. There was no significant difference in the effects of cytokines on AICD between SS patients and normals. These findings suggest that IL-2 is a critical factor for AICD. AICD works almost normally in SS T cells when sufficient IL-2 is present prior to T cell receptor re-stimulation.     

Activation-induced cell death of peripheral blood T lymphocytes in patients with primary Sjögren’s syndrome

Activation-induced cell death (AICD) in T lymphocytes is important for the maintenance of peripheral tolerance. We studied AICD of peripheral blood T cells from patients with primary Sjögren’s syndrome (SS). AICD was induced in mitogen-activated T cellsin vitro using mAb to CD3 or Fas (CD95). Cell death and proliferation, Fas and Fas ligand (FasL) expression, and soluble Fas and soluble FasL production were measured. Surface phenotypes and cytokine production of AICD-surviving cells and effects of cytokines on AICD were examined. Anti-CD3 mAb induced cell death is SS and normal T cells in the presence of exogenous interleukin (IL)-2. In the absence of IL-2 anti-CD3 mAb induced cell proliferation in SS and normal T cells. There was no significant difference in Fas/FasL expression and sFas/sFasL production between SS patients and normals. AICD-surviving cells consisted of more CD4⁺ T cells and less CD8⁺ T cells in SS compared to normals. AICD-surviving cells produced abundant interferon-γ and little IL-4. There was no significant difference in the effects of cytokines on AICD between SS patients and normals. These findings suggest that IL-2 is a critical factor for AICD. AICD works almost normally in SS T cells when sufficient IL-2 is present prior to T cell receptor re-stimulation.     

Gene transfer of Fas ligand induces tumor regression in vivo

The Fas–Fas ligand (FasL) system plays an important role in the induction of lymphoid apoptosis and has been implicated in the suppression of immune responses. Herein, we report that gene transfer of FasL inhibits tumor cell growth in vivo. Although such inhibition is expected in Fas⁺ tumor cell lines, marked regression was unexpectedly observed after FasL gene transfer into the CT26 colon carcinoma that does not express Fas. Infection by an adenoviral vector encoding FasL rapidly eliminated tumor masses in the Fas⁺ Renca tumor by inducing cell death, whereas the elimination of Fas⁻ CT26 cells was mediated by inflammatory cells. Analysis of human malignancies revealed Fas, but not FasL, expression in a majority of tumors and susceptibility to FasL in most Fas⁺ cell lines. These findings suggest that gene transfer of FasL generates apoptotic responses and induces potent inflammatory reactions that can be used to induce the regression of malignancies.     

溃疡性结肠炎肠黏膜组织CD8、Fas/FasL和Bcl-2/Bax的表达及关系

目的:研究溃疡性结肠炎(UC)肠黏膜CD8 T细胞Fas／FasL、Bcl-2／Bax蛋白表达以及相互关系,探讨细胞凋亡机制在UC发病中的作用．方法:采用免疫组化SP法检测60例UC肠黏膜组织以及60例正常肠黏膜组织CD8,Fas／FasL, Bcl-2／Bax蛋白表达．结果:CD8阳性细胞在上皮间的浸润在UC 组为52％,正常组为78％,两组相比差异显著 (P<0．01);急性期较缓解期也显著减少(20％ vs 74％,P<0．01)．CD8在UC患者急性期黏膜固有层的表达为80％,高于缓解期的34％(P= 0．0006)．Fas在UC上皮中表达62％,正常组织 30％,两组相比差异显著(P<0．01);急性期高于缓解期(84％ vs 45％,P<0．01)．FasL在固有膜炎性细胞中的表达UC组为62％,正常组7％, 两组相比差异显著(P<0．00;急性期(88％)高于缓解期(43％),而且CD8与FasL在黏膜固有层炎性细胞的表达呈正相关(X2=7．3,P<0．01)． Bcl-2／Bax在UC肠黏膜上皮的表达率与正常组相近,差异无显著性．结论:UC肠黏膜组织Fas／FasL表达增强, Bcl-2／Bax表达无明显变化,CD8细胞与UC急性期Fas／FasL表达相关．     

Selective loss of peripheral blood CD45RO+ T lymphocytes correlates with increased levels of serum cytokines and endothelial cell-derived soluble cell adhesion molecules in patients with chronic idiopathic neutropenia of adults

 The present study was designed to investigate the hypothesis that selective loss of peripheral blood CD45RO⁺ T lymphocytes in patients with chronic idiopathic neutropenia of adults (CINA), previously reported from our laboratory, may be due to enhanced extravasation into the tissues. Serum levels of endothelial cell-derived soluble cell adhesion molecules (sELAM, sICAM and sVCAM), usually used as indicators of endothelial cell activation, were measured in 73 CINA patients and 32 healthy volunteers using a micro-ELISA method. We found that patients had markedly elevated concentrations of all three soluble cell adhesion molecules studied compared to the controls, and serum levels of sELAM, sICAM and, more importantly, sVCAM correlated inversely with the numbers of both CD4⁺/CD45RO⁺ and CD8⁺/CD45RO⁺ T cell subsets. Using a micro-ELISA method, we also measured serum levels of two endothelial cell activators, interleukin (IL)-1β and TNF-α, and found that CINA patients had significantly higher cytokine concentrations than control subjects. Serum levels of IL-1β and TNF-α correlated positively with the values of all three soluble cell adhesion molecules and inversely with the numbers of CD4⁺/CD45RO⁺ and CD8⁺/CD45RO⁺ T cell subsets. Moreover, we measured serum levels of the chemokine RANTES by a micro-ELISA technique and found that CINA patients also had elevated concentrations of the molecule compared to controls. Serum RANTES correlated positively with IL-1β, TNF-α, sICAM, sVCAM and sELAM and inversely with the numbers of both CD4⁺/CD45RO⁺ and CD8⁺/CD45RO⁺ T cell subsets. These findings strongly suggest that CINA patients have an activated endothelium to which CD45RA⁺ and CD45RO⁺ T cells tether and roll, but firm adhesion and transendothelial migration are restricted to CD45RO⁺ T cell subsets, as endothelial VCAM-1 interacts with the vascular leukocyte adhesion molecule-4 (VLA-4) constitutively expressed on CD45RO⁺ but not on CD45RA⁺ T cells. Subsequent subendothelial and tissue migration of CD45RO⁺ T cells may be facilitated by the chemokine RANTES, which acts mainly on CD45RO⁺ T cells. We concluded that selective loss of peripheral blood CD45RO⁺ T lymphocytes in CINA patients is probably due, at least in part, to enhanced extravasation of both CD4⁺/CD45RO⁺ and CD8⁺/CD45RO⁺ T cell subsets into the tissues. Received: February 18, 1998 / Accepted: June 10, 1998     

Mechanism of counterattack of colorectal cancer cell by Fas/Fas ligand system

AIM: To determine the role of Fas/Fas ligand (FasL) in the immune escape of colon cancer cells. METHODS: Immunohistochemistry was used to observe the expression of Fas and FasL in the tissues of colon cancer patients. In situ hybridization was used to detect the localization of FasL mRNA expression in cancer tissues. Terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) assay and CD45 staining were performed to detect the apoptosis of tumor-infiltrating lymphocytes (TILs). Co-culture assays of colon cancer cells (SW480) and Jurkat cells (Fas-sensitive cells) were performed to observe the counterattack of colon cancer cells to lymphocytes. RESULTS: Of 53 cases of colon carcinomas, 23 cases (43.4%) expressed Fas which was significantly lower as compared to the normal colonic mucosa (73.3%, P<0.01), and 45 cases (84.9%) of colon carcinomas expressed FasL, whereas only two cases (3.75%) in normal mucosa expressed FasL. FasL expression in the colon cancer cells was found to be associated with increased cell death of TILs. The apoptotic rate of TIL in the FasL-positive staining regions of tumor cells was significantly higher than that in the FasL-negative staining region (54.84±2.79% vs 25.73±1.98%, P<0.01). The co-culture of SW480 cells and Jurkat cells confirmed the function of FasL on the SW480 cells. The apoptotic rates of Jurkat cells were found to be related with the amount of SW480 cells. CONCLUSION: Colon cancer cells can escape the immune surveillance and killing via decreasing Fas expression, and can counterattack the immune system via increasing FasL expression. Fas/FasL can serve as potential targets for effective antitumor therapy.     

An Inverse Correlation of Human Peripheral Blood Regulatory T Cell Frequency with the Disease Activity of Ulcerative Colitis

Evidence suggests that CD4⁺CD25⁺ regulatory T cells play a crucial role in the suppression of intestinal inflammation. However, their role in the suppression of inflammatory bowel disease has not yet been addressed. We examined the proportion of regulatory T cells in inflammatory bowel disease. First, we isolated CD4⁺CD45RO⁺CD25⁺ T cells from the peripheral blood of healthy persons and showed that these cells suppressed T cell proliferation profoundly and expressed FoxP3 abundantly, revealing that they are regulatory cells. Then the proportion of CD45RO⁺CD25⁺ in peripheral blood CD4⁺ T cells was analyzed in patients and healthy controls by flow cytometry. CD4⁺CD45RO⁺CD25⁺ T cell frequency was significantly lower in active ulcerative colitis than in the control and inactive ulcerative colitis. CD4⁺CD45RO⁺CD25⁺ T cell frequency was inversely correlated with the clinical and endoscopic severity of ulcerative colitis. These results suggest that a deficiency of regulatory T cells is associated with the progression of ulcerative colitis. This work was supported in part by Health and Labour Science Research Grants from the Japanese Ministry of Health, Labour and Welfare and Research on Measures for Intractable Disease.     

Expression of Fas/Fas ligand (FasL) and its involvement in infiltrating lymphocytes in hepatocellular carcinoma (HCC)

Background. This study was conducted to examine the expression of Fas/Fas ligand (FasL), to elucidate its relationship with tumor-infiltrating lymphocytes (TILs), and to detect possible gene mutation of Fas/FasL in patients with hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC). Methods. Indirect immunohistochemical staining was performed on formalin-fixed, paraffin-embedded sections of liver biopsy and surgery specimens from five normal livers, and from the livers of 30 patients with HCC. Fas/FasL mRNA-expressing cells and apoptotic cells were detected by in situ hybridization and DNA nick end labeling (TUNEL), respectively. We also performed polymerase chain reaction (PCR)-amplifying and direct sequencing for the Fas/FasL gene. Results. Fas/FasL and its mRNA were localized on the membrane or in the cytoplasm in some HCC cells, as well as hepatocytes. Their expression was enhanced in areas with infiltrating inflammatory cells in the noncancerous regions of liver tissue and on the margins of the cancerous tissue. The positivity rate for TUNEL was elevated along these margins. The labeling index of Fas/FasL was lower in the cancerous liver tissue than in the surrounding noncancerous region (P < 0.01), and tended to decrease in proportion to the malignancy of tumor cells; Fas/FasL expression was not found on poorly differentiated type cancer cells. Fas(−)/FasL(+), FasL-mRNA(+) HCC cells were seen in one specimen of moderately differentiated type. Some CD8+T lymphocytes were TUNEL-positive around the cancerous region. In this study, cancerous and noncancerous tissues in HCC revealed no genetic mutations in any exons of Fas/FasL. Conclusions. These findings suggest that Fas/FasL expression was decreased in proportion to the malignancy of tumor cells, and that infiltrating CD8+T lymphocytes play a role in apoptosis in HCC. The apoptosis in HCC could be regulated by the suppression of Fas/FasL expression, or, sometimes, by the enhancement of FasL expression. Received: October 5, 2000 / Accepted: February 23, 2001     

Associations of circulating CXCR3–PD-1+CD4+T cells with disease activity of systemic lupus erythematosus

Abstract

Objectives: Which helper CD4⁺ T cell subset contributes to autoantibodies generation and severity of end-organ involvement in lupus patients remains to be explored. Our research aims to investigate the roles of circulating Tfh (cTfh) cell subsets and corresponding CXCR5^– Th cells in lupus patients and their correlation with SLE disease activity index 2000 (SLEDAI).

Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from blood of systemic lupus erythematosus (SLE) patients as well as healthy donors. The proportion of Th cell subsets classified from cell surface markers (CD45RO, CXCR5, CXCR3, CCR6, PD-1, ICOS, and CCR7) is detected by flow cytometry.

Results: We found no difference in the frequency of CD45RO⁺CXCR5⁺CD4⁺ T cells between SLE patients and health controls. As previously reported, SLE patients showed an increase in the percentage of CXCR5⁺PD-1⁺, CXCR5⁺ICOS⁺PD-1⁺ and CXCR5⁺CCR7^loPD-1^hi cTfh subset, however, none of these populations had correlation with SLEDAI. Therefore, we further investigated the CXCR5^– subsets, and surprisingly we found that the frequency of CXCR3^–PD-1⁺ subset was correlated with SLEDAI, ds-DNA IgG, anti-nucleosome antibody, C3, and C4 independent of CXCR5. Consistently, CXCR3^–PD-1⁺CD45RO⁺CD4⁺T cells expressed factors associated with B-cell-help for the autoantibody production.

Conclusion: CXCR3^–PD-1⁺CD4⁺T cells are a sensitive indicator to assess SLE disease activity and might contribute B cell help and the generation of autoantibodies in patients.     

Immunohistochemical characterization of lymphocytes in microscopic colitis

Background and AimsMicroscopic colitis (MC), encompassing the subgroups collagenous colitis (CC) and lymphocytic colitis (LC), is characterized by macroscopically normal or near-normal colonic mucosa, and an increased number of intraepithelial lymphocytes (IELs) and mononuclear cell infiltration in the underlying lamina propria (LP), in addition to an increased collagen layer in CC. This study aimed to characterize the inflammatory cells involved in mucosal inflammation, using immunohistochemistry.MethodsParaffin-embedded biopsies from 23 untreated patients with MC (CC = 13, LC = 10) and 17 controls were stained with antibodies against CD3, CD4, CD8, CD20, CD30, Foxp3, CD45RO and Ki67. Computerized image analysis was used to calculate areas of stained lymphocytes in the surface and crypt epithelia as well as in the LP.ResultsIn CC and LC, an increase of predominantly CD8⁺ lymphocytes was seen in both the epithelium and the lamina propria, whereas a decreased amount of CD4⁺ lymphocytes was found in the lamina propria. CD45RO⁺ and Foxp3⁺ cells were more abundant in all areas in both patient groups compared to controls, as were CD20⁺ areas, although more scarce. Ki67⁺ areas were only more abundant in the epithelium, whereas CD30⁺ areas were more abundant in the lamina propria of both patient groups compared to controls.ConclusionsThis study confirms an increased amount of CD8⁺ lymphocytes in the epithelium. Lymphocytic proliferation and activation markers were more abundant, whereas a decreased amount of CD4⁺ lymphocytes was seen in the LP. Further studies are needed to reveal the underlying mechanism(s).     

Expression of CD4+ forkhead box P3 (FOXP3)+ regulatory T cells in inflammatory bowel disease

OBJECTIVE: Forkhead box P3 (FOXP3) plays an important role in the development and function of CD4⁺ regulatory T (Treg) cells. In this study the percentage of CD4⁺ FOXP3⁺ Treg cells in peripheral blood mononuclear cells (PBMC) and the frequency of Treg cells in the colonic mucosa of patients with inflammatory bowel disease (IBD) were investigated. METHODS: The percentage of CD4⁺FOXP3⁺ Treg cells in PBMC was analyzed by flow cytometry. Immunohistochemistry was used to examine the FOXP3⁺ cells in the inflamed mucosa. Real‐time polymerase chain reaction and Western blot were used to detect the expressions of FOXP3 mRNA and protein in PBMC and mucosal biopsy specimens of IBD patients, respectively. RESULTS: Together with the decrease of percentage of Treg cells in PBMC, we found that the frequency of Treg cells increased significantly in inflamed mucosa of active or inactive Crohn's disease (CD) and ulcerative colitis (UC). The expressions of FOXP3 mRNA and protein increased in inflamed mucosa when compared with those in healthy controls, especially the FOXP3 mRNA in patients with active CD or UC. Interestingly, the expression of FOXP3 protein in active UC was higher than that in active CD. CONCLUSIONS: There was a decrease of CD4⁺FOXP3⁺ Treg cells in peripheral blood and an accumulation of Treg cells in inflamed mucosa. These data suggested that the suppressive function of Treg cells may be partially inhibited and this could be an important factor in the recurrence of disease, especially in UC.     

Systemic and local evidence of increased Fas-mediated apoptosis in ulcerative colitis

BACKGROUND AND AIMS: Recent studies suggest that Fas-mediated apoptosis is involved in the pathogenesis of inflammatory bowel disease (IBD). This study was conducted to clarify whether soluble forms of Fas (sFas) and Fas ligand (sFasL) are concerned with inflammation in IBD. METHODS AND PATIENTS: Concentration of serum sFas and sFasL was measured by enzyme-linked immunosorbent assay in 10 patients with ulcerative colitis (UC), 10 with Crohn's disease (CD) in both active and remission stages, and 20 controls. Expression of Fas and sFas in colonic mucosa was examined by western blot. Distribution of Fas and FasL in colonic mucosa was examined by immunohistochemistry in 20 UC, 20 CD, and 10 non-IBD colitis patients and in 10 controls. Apoptotic cells were examined by TUNEL. RESULTS: Concentration of systemic sFas was significantly lower in active UC than controls. The number of FasL-containing cells was significantly higher in active UC than in remission UC, non-IBD colitis, and controls. Apoptotic cells were increased in active UC. CONCLUSIONS: Our results demonstrate that systemic and local Fas-mediated apoptosis is promoted in UC, which might be involved in the pathogenesis in UC.     

CD8+CD122+CD49dlow regulatory T cells maintain T-cell homeostasis by killing activated T cells via Fas/FasL-mediated cytotoxicity

The Fas/FasL (CD95/CD178) system is required for immune regulation; however, it is unclear in which cells, when, and where Fas/FasL molecules act in the immune system. We found that CD8⁺CD122⁺ cells, which are mostly composed of memory T cells in comparison with naïve cells in the CD8⁺CD122⁻ population, were previously shown to include cells with regulatory activity and could be separated into CD49d^low cells and CD49d^high cells. We established in vitro and in vivo experimental systems to evaluate the regulatory activity of CD122⁺ cells. Regulatory activity was observed in CD8⁺CD122⁺CD49d^low but not in CD8⁺CD122⁺CD49d^high cells, indicating that the regulatory cells in the CD8⁺CD122⁺ population could be narrowed down to CD49d^low cells. CD8⁺CD122⁻ cells taken from lymphoproliferation (lpr) mice were resistant to regulation by normal CD122⁺ Tregs. CD122⁺ Tregs taken from generalized lymphoproliferative disease (gld) mice did not regulate wild-type CD8⁺CD122⁻ cells, indicating that the regulation by CD122⁺ Tregs is Fas/FasL-dependent. CD122⁺ Tregs taken from IL-10–deficient mice could regulate CD8⁺CD122⁻ cells as equally as wild-type CD122⁺ Tregs both in vitro and in vivo. MHC class I-missing T cells were not regulated by CD122⁺ Tregs in vitro. CD122⁺ Tregs also regulated CD4⁺ cells in a Fas/FasL-dependent manner in vitro. These results suggest an essential role of Fas/FasL as a terminal effector of the CD122⁺ Tregs that kill activated T cells to maintain immune homeostasis.Fas (CD95) and its ligand FasL (CD178) compose a unique system that is strongly related to programmed cell death (1). FasL has been considered one of the effector molecules involved in the killing of target cells by cytotoxic T lymphocytes (CTLs) (2). When Fas binds to FasL, it induces downstream signal transduction pathways that subsequently activate cell death induction pathways (3, 4). Thus, the Fas/FasL system appears to act as an effector for CTL-mediated killing of virus-infected or cancer cells, similar to the perforin–granzyme system (5, 6). However, because Fas-mutant (lpr) and FasL-deficient (gld) mice show lymphoproliferative changes, it has been suggested that the Fas/FasL system is important for suppression/regulation of activated effector T cells (7, 8).Molecular mechanisms after Fas activation have been thoroughly investigated, and the signal transduction pathway has been largely elucidated (9, 10). However, research on the cellular events that use the Fas/FasL system has progressed comparatively slowly. There are only a few relevant reports in this respect, mostly of human CD4⁺Foxp3⁺ Tregs that use the Fas/FasL system for their regulatory activity. [In this article, we use the term “Treg(s)” for any kind of T cells that show immune regulatory activity.] To complicate matters further, some contradictory reports claim that such Fas/FasL-engaging Tregs do not exist (8, 11, 12). No reliable reports suggesting that CD8⁺ T cells use the Fas/FasL system for their regulatory action are available. Therefore, it is not clear precisely which subset of T cells express FasL or where Fas/FasL-dependent CD8⁺ Tregs, if such cells exist, are located and at which point they function. Thus, the ultimate pathophysiological role of the Fas/FasL system is still unknown.Regulation of the immune reaction is of pivotal importance for maintaining health in multicellular organisms. In highly developed animals, Tregs, a subset of T lymphocytes especially known as CD4⁺CD25⁺Foxp3⁺ cells, are the main regulators of the immune response (13–16). However, it is not quite clear whether CD8⁺ regulatory T cells exist; there are only few and contradictory reports on their existence, in contrast to the reports on CD4⁺ Tregs. A population of predominantly CD8⁺ suppressor T cells has been described and debated in the 1980s (17, 18). In the 2000s, we found and reported that the cells of central memory phenotype (CD8⁺CD122⁺) also possess the regulatory function, and some other reports regarding CD8⁺ Tregs with some contradictory findings have been published. To date, more than 10 CD8⁺ Treg populations with different markers have been reported (19).One of the best characterized CD8⁺ Treg populations is the CD8⁺CD122⁺ Treg (122⁺ Treg) population. To confirm the existence of CD8⁺ Tregs, markers that may be possible to distinguish Tregs from other T cells were examined. We previously found that CD49d can separate CD8⁺CD122⁺ cells into at least two subsets (CD49d^low and CD49d^high) (20). To judge which cells have a stronger regulatory activity, we prepared two experimental systems: an in vitro system, based on cell culture, and an in vivo system, based on adoptive transfer of T cells.Interestingly, CD8⁺ Tregs express CD122 (IL-2/IL-15 receptor β chain) in contrast to CD4⁺ Tregs, which express CD25 (IL-2 receptor α chain), indicating the fundamental importance of IL-2 at the development/maturation of both CD4⁺ and CD8⁺ Tregs. Suzuki and coworkers generated CD122-deficient mice using gene targeting (21) and used them to identify 122⁺ Tregs (22). In this study, which is a follow-up study to our previous report, we performed in vitro and in vivo experiments to gain further understanding of these cells. We found that CD49d might be a good marker for classifying CD8⁺ T cells into naïve, resting T cells (CD62L⁺CD122⁻), effector memory T cells (CD62L⁻), and T cells of central memory phenotype (CD122⁺CD49d^low), by using multicolor staining of CD62L, CD122, and CD49d. To our knowledge, this is the first report on the role of the Fas/FasL system in the action of CD8⁺CD122⁺ Tregs (122⁺ Tregs) (22–25). Additionally, we show that CD8⁺ Tregs are included in the CD49d^low population, which corresponds to the central memory T-cell population, and that their mechanism of suppression depends on the cytotoxicity mediated by the Fas and FasL interaction.     

Impact of intrarectal chromofungin treatment on dendritic cells-related markers in different immune compartments in colonic inflammatory conditions

BACKGROUNDChromofungin (CHR: chromogranin-A 47-66) is a chromogranin-A derived peptide with anti-inflammatory and anti-microbial properties. Ulcerative colitis (UC) is characterized by a colonic decrease of CHR and a dysregulation of dendritic CD11c⁺ cells.AIMTo investigate the association between CHR treatment and dendritic cells (DCs)-related markers in different immune compartments in colitis.METHODSA model of acute UC-like colitis using dextran sulphate sodium (DSS) was used in addition to biopsies collected from UC patients.RESULTSIntrarectal CHR treatment reduced the severity of DSS-induced colitis and was associated with a significant decrease in the expression of CD11c, CD40, CD80, CD86 and interleukin (IL)-12p40 in the inflamed colonic mucosa and CD11c, CD80, CD86 IL-6 and IL-12p40 within the mesenteric lymph nodes and the spleen. Furthermore, CHR treatment decreased CD80 and CD86 expression markers of splenic CD11c⁺ cells and decreased NF-κB expression in the colon and of splenic CD11c⁺ cells. In vitro, CHR decreased CD40, CD80, CD86 IL-6 and IL-12p40 expression in naïve bone marrow-derived CD11c⁺ DCs stimulated with lipopolysaccharide. Pharmacological studies demonstrated an impact of CHR on the NF-κB pathway. In patients with active UC, CHR level was reduced and showed a negative linear relationship with CD11c and CD86.CONCLUSIONCHR has protective properties against intestinal inflammation via the regulation of DC-related markers and CD11c⁺ cells. CHR could be a potential therapy of UC.     

Activation-induced CD4+ T cell death in HIV-positive individuals correlates with Fas susceptibility, CD4+ T cell count, and HIV plasma viral copy number

The relevance of activation-induced cell death (AICD) of CD4+ T cells to AIDS pathogenesis is unknown. The present study investigates the relationship of AICD to a defined molecular mechanism regulating peripheral T cell homeostasis, Fas-mediated apoptosis, and clinical correlates of the pathogenesis of AIDS. Increased pokeweed mitogen (PWM)-induced AICD (22.8 versus 4.4%, p = 0.006) and Fas-mediated apoptosis (27.7 versus 12.0%, p = 0.002) of CD4+ T cells were observed in HIV+ versus HIV- individuals. Similarly, increased PWM-mediated AICD (16.2 versus 2.2%, p < 0.001) and Fas-mediated apoptosis (25.8 versus 7.6%, p = 0.005) were noted in CD8+ T cells from HIV+ versus HIV- individuals. PWM-induced AICD of CD4+ T cells was blocked (83% median specific inhibition) by Fas-blocking antibodies, whereas PWM-induced AICD of CD8+ T cells was Fas independent. Comparison between PWM- and anti-CD3-mediated AICD of CD4+ T cells indicated that PWM- and not CD3-induced AICD is Fas dependent. HIV+ individuals with an HIV RNA copy number of <30,000 copies/ml had lower PWM-induced AICD of CD4+ T cells than did those with an HIV RNA copy number of >30,000 copies/ml (5.7 versus 22.1%, p = 0.034), and PWM-induced AICD inversely correlated with CD4+ T cell count (R = -0.567, p = 0.043). Initiation of HAART decreased PWM-induced CD4+ T cell AICD from 24.4 to 9.4% posttreatment (p = 0.035). These results demonstrate that PWM-induced AICD of CD4+ T cells from HIV+ individuals is mediated by Fas/FasL, parallels the in vivo susceptibility of the CD4+ T cell to Fas-mediated apoptosis, and correlates with clinical markers of AIDS pathogenesis and response to HAART.     

Apoptosis in the intestinal mucosa of patients with inflammatory bowel disease: evidence of altered expression of FasL and perforin cytotoxic pathways

Background and aims Abnormal apoptosis may result in the persistence of activated intestinal T-cells in inflammatory bowel disease (IBD). We investigated apoptosis in distinct mucosal compartments, and the expression of Fas/Fas ligand and perforin in the inflamed and non-inflamed intestinal mucosa of patients with IBD.Methods Colon specimens from 15 patients with ulcerative colitis (UC) and inflamed and non-inflamed mucosa from 15 patients with Crohns disease (CD) were analysed for densities and distribution of apoptotic cells determined by the terminal deoxynucleotidyltransferase-mediated dUDP-biotin nick-end labelling (TUNEL) method. Fas, FasL, and perforin-expressing cells were assessed by immunoperoxidase, and with anti-CD3, anti-CD20 and anti-CD68, by double immunofluorescence with confocal microscopy. Quantitative analysis was performed using a computer-assisted image analyser.Results Colonic lamina propria (LP) and epithelium from patients with UC showed higher rates of apoptosis than controls, but no difference was shown regarding patients with CD. In LP, co-expression of Fas was reduced with T-cells in inflamed CD mucosa, and with macrophages in all patients with IBD. No difference was found in the expression of Fas on B-cells. Rates of FasL-expressing cells in LP were higher in IBD than in controls, with no correlation with the rates of apoptosis. Rates of perforin-expressing cells in LP were greater in UC than in controls, and correlated to the rates of apoptosis. No difference was shown regarding the inflamed and non-inflamed CD mucosa. Rates of FasL and perforin-expressing intra-epithelial lymphocytes showed no difference among groups.Conclusions Increased expression of FasL in IBD colonic LP not parallelled by Fas on T-cells and macrophages may indicate a reduced susceptibility to the Fas/FasL-mediated apoptosis of lymphoid cells. Expression of perforin is correlated to the tissue damage, and may represent the enhancement of a distinct cytotoxic pathway in UC.Heitor S.P. Souza and Claudio J.A. Tortori contributed equally to this paper     

Fas/Fas配体在大肠癌发生和免疫逃逸及反击中的作用

目的　探讨Fas、Fas配体 (FasL)在大肠癌发生和免疫逃逸及反击中的作用。方法　应用免疫组织化学染色的方法 ,对 5 2例大肠癌、2 7例大肠腺瘤、2 8例溃疡性结肠炎、30例正常大肠组织及 2 3例大肠癌淋巴结转移癌组织进行Fas和FasL的检测。对 5 2例大肠癌用TUNEL法 ,检测不同FasL表达区肿瘤细胞的凋亡情况。结果　 (1)Fas在不同大肠组织中的表达规律是 :大肠癌中表达最低 ,其次是大肠腺瘤、溃疡性结肠炎 ,表达水平最高的是正常大肠组织。FasL在不同大肠组织中的表达规律与Fas正好相反 ,大肠癌组织中表达最高 ,其次是大肠腺瘤、溃疡性结肠炎 ,正常大肠组织中几乎不表达FasL。 (2 )Fas、FasL的表达与大肠癌的组织学类型无关 (P >0 0 5 ) ,与Dukes分期、分化程度、有无淋巴结转移有关 (P <0 0 5 )。 (3)在同一大肠癌组织切片中 ,FasL的表达不均匀 ;FasL表达阳性区(n =2 5 )肿瘤细胞的凋亡率 (81 2 % )比FasL阴性区 (n =2 5 ) (47 4 % )高 (P <0 0 1)。 (4)在 2 3例大肠癌淋巴结转移癌组织中 ,发现 2 3例转移癌FasL均强阳性表达 ,阳性细胞率均大于 75 %。结论　Fas、FasL在大肠癌发生和免疫逃逸及反击中起重要的作用     

Serum soluble Fas ligand levels and peripheral blood lymphocyte subsets in patients with drug-induced maculopapular rashes,dress, and viral exanthemas

BackgroundFatty acid synthetase (Fas)/Fas ligand (FasL)-dependent apoptotic pathways have been reported as being involved in the pathogenesis of drug-induced maculopapular rashes.ObjectiveWe investigated serum soluble FasL (sFasL) levels and peripheral blood lymphocyte subtypes to discriminate maculopapular drug eruptions (MPDE) from viral exanthema (VE).Patients/methodsChildren with confirmed MPDE (group I), VE (group II), and drug rashes with eosinophilia and systemic symptoms (DRESS) or drug-induced hypersensitivity syndrome (DIHS) (group III) were included. Serum sFasL levels and peripheral blood lymphocyte subtypes were analyzed in groups I–III on admission, and repeated twice (only once for group IV – controls).ResultsThere were no significant serum soluble FasL level differences among the groups for all the samples. In the initial samples, CD19⁺ cell numbers in group II were significantly higher than in group IV, and the CD4⁺/CD8⁺ ratio was higher than groups I and IV. In the second samples, CD4⁺ and CD19⁺ cell numbers were significantly higher in group II than group I. In the final samples, CD4⁺ cell numbers in group II were significantly higher than group I and group III. CD19⁺ cells numbers in group III were significantly lower than the other groups for all samples.ConclusionSerum sFasL levels were not found to be useful in discriminating viral exanthemas from other drug rashes. The significant differences between MPDE, VE, and DRESS were high CD4⁺ and CD19⁺ cell-count numbers in VE but low B-cell numbers in DRESS. This might be important for discriminating VE from DRESS, and the low B-cell count in early symptoms might be a useful predictor of DRESS development.