Genomic amplification of a decoy receptor for Fas ligand in lung and colon cancer

Fas ligand (FasL) is produced by activated T cells and natural killer cells and it induces apoptosis (programmed cell death) in target cells through the death receptor Fas/Apol/CD95. One important role of FasL and Fas is to mediate immune-cytotoxic killing of cells that are potentially harmful to the organism, such as virus-infected or tumour cells. Here we report the discovery of a soluble decoy receptor, termed decoy receptor 3 (DcR3), that binds to FasL and inhibits FasL-induced apoptosis. The DcR3 gene was amplified in about half of 35 primary lung and colon tumours studied, and DcR3 messenger RNA was expressed in malignant tissue. Thus, certain tumours may escape FasL-dependent immune-cytotoxic attack by expressing a decoy receptor that blocks FasL.     

TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis in Fas ligand-resistant melanoma cells and mediates CD4 T cell killing of target cells

We have previously shown that melanoma cells were resistant to apoptosis induced by TNF family members Fas ligand (FasL), TNF-alpha, and CD40L. FasL also was not involved in CD4 T cell-mediated killing of melanoma cells. In the present study, we have tested melanoma cells for their susceptibility to apoptosis induced by human TNF-related apoptosis-inducing ligand (TRAIL) and the ability of a mAb against TRAIL to inhibit apoptosis and CD4 CTL-mediated killing of melanoma and Jurkat target cells. The results show that TRAIL-induced apoptosis in cells from 7 of 10 melanoma cell lines tested as well as in Jurkat T cells. Susceptibility to apoptosis was increased in some of the cell lines by treatment with cyclohexamide or actinomycin D. The melanoma cells were resistant to apoptosis induced by FasL, TNF-alpha, and CD40L. mAb M180 against TRAIL inhibited apoptosis induced by TRAIL. It was also found to inhibit CD4 CTL-mediated killing of Jurkat T cells as well as autologous and allogeneic melanoma cells. The degree of inhibition produced by the mAb varied between different clones of CTL and according to the susceptibility of the target cells to TRAIL-induced apoptosis. These results suggest that TRAIL is an important mediator of cell death induced by CTL and may have an important therapeutic role against human melanoma.     

Membrane Fas ligand kills human peripheral blood T lymphocytes, and soluble Fas ligand blocks the killing

It has been believed that the Fas expressed on human peripheral blood T cells (PBT) is nonfunctional, because these cells are insensitive to agonistic anti-Fas/Apo-1 mAbs that efficiently kill in vitro-activated T cells and many Fas-expressing cell lines. Here, we demonstrate that membrane-bound Fas ligand (FasL) kills both fresh and in vitro-activated PBT, indicating that the Fas expressed on fresh PBT is functional. In contrast, soluble FasL kills only the latter. Naive T cells in umbilical cord blood do not express Fas, but can be induced to express Fas by IFN-gamma or by a combination of IL-2 and anti-CD28 mAb, after which they acquire sensitivity to membrane but not to soluble FasL. Soluble FasL inhibited the killing of fresh PBT by membrane FasL. These results indicate that the shedding of FasL from the membrane is a mechanism for downregulating at least part of its killing activity.     

Fas ligand-mediated depletion of CD4 and CD8 lymphocytes by monomeric HIV-1-gp120

In order to determine in what condition and by what mechanism gp 120 can deplete not only CD4 but also CD8 T cells, an in vitro system was established in which peripheral blood lymphocytes from healthy donors were treated with recombinant gp 120. We found that gp 120 can deplete both CD4 and CD8 T cells when they have recently been activated and are exposed to IL-2-deficient conditions. Bioassay of the Fas ligand (FasL) demonstrated augmented expression and release of soluble FasL by CD4 T cells in the supernatant of this culture. The administration of anti-FasL mAb and anti-Fas mAb, both of which exhibit neutralizing activity, completely abolished the depletion of these two T cell populations in culture. Based on these findings, we concluded that FasL depletes Fas antigen expressing CD4 and CD8 T cells by programmed cell death.     

Human melanoma-reactive CD4+ and CD8+ CTL clones resist Fas ligand-induced apoptosis and use Fas/Fas ligand-independent mechanisms for tumor killing

Tumor cells have been shown recently to escape immune recognition by developing resistance to Fas-mediated apoptosis and acquiring expression of Fas ligand (FasL) molecule that they may use for eliminating activated Fas+ lymphocytes. In this study, we report that tumor-specific T lymphocytes isolated from tumor lesions by repeated in vitro TCR stimulation with relevant Ags (mostly represented by normal self proteins, such as MART-1/Melan A and gp100) can develop strategies for overcoming these escape mechanisms. Melanoma cells (and normal melanocytes) express heterogeneous levels of Fas molecule, but they result homogeneously resistant to Fas-induced apoptosis. However, CD4+ and CD8+ CTL clones kill melanoma cells through Fas/FasL-independent, granule-dependent lytic pathway. In these lymphocytes, Ag/MHC complex interaction with TCR does not lead to functional involvement of FasL, triggered, on the contrary, by T cell activation with nonspecific stimuli such as PMA/ionomycin. Additionally, melanoma cells express significant levels of FasL (detectable on the cell surface only after treatment with metalloprotease inhibitors), although to a lesser extent than professional immune cells such as Thl clones. Nevertheless, antimelanoma CTL clones resist apoptosis mediated by FasL either in soluble form or expressed by Thl lymphocytes or FasL+ melanoma cells. These results demonstrate that CD4+ and CD8+ antimelanoma T cell clones can be protected against Fas-dependent apoptosis, and thus be useful reagents of immunotherapeutic strategies aimed to potentiate tumor-specific T cell responses.     

Involvement of APO2 ligand/TRAIL in activation-induced death of Jurkat and human peripheral blood T cells

The interaction of Fas with Fas ligand (FasL) mediates activation-induced cell death (AICD) of T hybridomas and of mature T lymphocytes. The TNF/TNF receptor system also plays a significant role in AICD of mature T cells and in the maintenance of peripheral tolerance. We previously demonstrated that in human Jurkat leukemia cells, AICD is triggered mainly by the rapid release of preformed FasL upon TCR stimulation. In the present work, we show that the cytotoxic cytokine APO2 ligand (APO2L; also known as TRAIL) is constitutively expressed as an intracytoplasmic protein in Jurkat T cells and derived sublines. APO2L is also detected in fresh human peripheral blood mononuclear cells (PBMC) from a significant number of donors, and the amount of both FasL and APO2L substantially increases upon blast generation. A neutralizing anti-APO2L monoclonal antibody (mAb) partially suppresses the cytotoxicity induced by supernatants of phytohemagglutinin (PHA)-prestimulated Jurkat or human PBMC on non-activated Jurkat cells, indicating that APO2L is released by these cells and contributes to AICD. A combination of neutralizing anti-APO2L and anti-Fas mAb blocks around 60 % of the toxicity associated with supernatants from PHA-activated human PBMC. These results show that FasL and APO2L account for the majority of cytotoxic activity released during AICD, and suggest that additional uncharacterized factors may also contribute to this process.     

Human papillomavirus infection and esophageal cancer: a nationwide seroepidemiologic case-control study in Sweden

BACKGROUND: Despite being immunogenic, gastric cancers overcome antitumour immune responses by mechanisms that have yet to be fully elucidated. Fas ligand (FasL) is a molecule that induces Fas receptor mediated apoptosis of activated immunocytes, thereby mediating normal immune downregulatory roles including immune response termination, tolerance acquisition, and immune privilege. Colon cancer cell lines have previously been shown to express FasL and kill lymphoid cells by Fas mediated apoptosis in vitro. Many diverse tumours have since been found to express FasL suggesting that a "Fas counterattack" against antitumour immune effector cells may contribute to tumour immune escape. AIM: To ascertain if human gastric tumours express FasL in vivo, as a potential mediator of immune escape in stomach cancer. SPECIMENS: Thirty paraffin wax embedded human gastric adenocarcinomas. METHODS: FasL protein was detected in gastric tumours using immunohistochemistry; FasL mRNA was detected in the tumours using in situ hybridisation. Cell death was detected in situ in tumour infiltrating lymphocytes using terminal deoxynucleotidyl transferase mediated dUTP nick end labelling (TUNEL). RESULTS: Prevalent expression of FasL was detected in all 30 resected gastric adenocarcinomas examined. In the tumours, FasL protein and mRNA were co-localised to neoplastic gastric epithelial cells, confirming expression by the tumour cells. FasL expression was independent of tumour stage, suggesting that it may be expressed throughout gastric cancer progression. TUNEL staining disclosed a high level of cell death among lymphocytes infiltrating FasL positive areas of tumour. CONCLUSIONS: Human gastric adenocarcinomas express the immune downregulatory molecule, FasL. The results suggest that FasL is a prevalent mediator of immune privilege in stomach cancer.     

Involvement of the Fas (CD95) system in peripheral cell death and lymphoid organ development

Fas-mediated apoptosis is a form of cell death that operates through a Fas-Fas ligand (FasL) interaction. In this study we investigated the role of the Fas system during development of normal and Fas-mutated lymphocytes. Irradiated RAG2-/-recipients were reconstituted with bone marrow cells from B6 and lpr mice (Fas defective) or from B6 and gld mice (FasL defective), and analyzed for long-term development. The results showed a primary role of the Fas system in peripheral cell death and thymic colonization. In the periphery, the interaction in vivo between Fas+ and Fas-T cell populations indicated that cellular homeostasis was defective. Indeed, we observed a FasL-mediated cytotoxic effect on normal-derived T cells, explaining the dominance of lpr T cells in the mixed chimeras. The Fas mutation affected neither cell activation nor cell proliferation, as the effector (Fas-) and target (Fas+) cells behaved similarly with regard to activation marker expression and cell cycle status. However, Fas-T cells failed to seed the periphery and the thymus in the long term. We suggest that this could be due to the fact that FasL is involved in the structural organization of the lymphoid compartment.     

Is epsilon-aminocaproic acid as effective as aprotinin in reducing bleeding with cardiac surgery?: a meta-analysis

Infection of BALB/c mice with Trypanosoma cruzi resulted in up-regulated expression of Fas and Fas ligand (FasL) mRNA by splenic CD4+ T cells, activation-induced CD4+ T cell death (AICD), and in Fas: FasL-mediated cytotoxicity. When CD4+ T cells from infected mice were co-cultured with T. cruzi-infected macrophages, onset of AICD exacerbated parasite replication. CD4+ T cells from T. cruzi-infected FasL-deficient BALB gld/gld mice had no detectable AICD in vitro and their activation with anti-TCR did not exacerbate T. cruzi replication in macrophages. However, infection of BALB gld/gld mice with T. cruzi resulted in higher and more prolonged parasitemia, compared to wild-type mice. Secretion of Th2 cytokines IL-10 and IL-4 by CD4+ T cells from infected gld mice was markedly increased, compared to controls. In addition, in vivo injection of anti-IL-4 mAb, but not of an isotype control mAb, reduced parasitemia in both gld and wild-type mice. These results indicate that, besides controlling CD4+ T cell AICD and parasite replication in vitro, an intact Fas: FasL pathway also controls the host cytokine response to T. cruzi infection in vivo, being required to prevent an exacerbated Th2-type immune response to the parasite.     

Novel antiallergic agents. Part I: Synthesis and pharmacology of pyrimidine amide derivatives

Cytolytic T cells use two mechanisms to kill virally infected cells, tumor cells, or other potentially autoreactive T cells in short-term in vitro assays. The perforin/granule exocytosis mechanism uses preformed cytolytic granules that are delivered to the target cell to induce apoptosis and eventual lysis. FasL/Fas (CD95 ligand/CD95)-mediated cytolysis requires de novo protein synthesis of FasL by the CTL and the presence of the death receptor Fas on the target cell to induce apoptosis. Using a CD8(+) CTL clone that kills via both the perforin/granule exocytosis and FasL/Fas mechanisms, and a clone that kills via the FasL/Fas mechanism only, we have examined the requirement of intra- and extracellular Ca2+ in TCR-triggered cytolytic effector function. These two clones, a panel of Ca2+ antagonists, and agonists were used to determine that a large biphasic increase in intracellular calcium concentration, characterized by release of Ca2+ from intracellular stores followed by a sustained influx of extracellular Ca2+, is required for perforin/granule exocytosis. Only the sustained influx of extracellular Ca2+ is required for FasL induction and killing. Thapsigargin, at low concentrations, induces this small but sustained increase in [Ca2+]i and selectively induces FasL/Fas-mediated cytolysis but not granule exocytosis. These results further define the role of Ca2+ in perforin and FasL/Fas killing and demonstrate that differential Ca2+ signaling can modulate T cell effector functions.     

Fas-Fas ligand-based interactions between tumor cells and tumor-specific cytotoxic T lymphocytes: a lethal two-way street

In the current study, we investigated the repercussions of the interaction between tumor cells (LSA) and the tumor-specific cytotoxic T lymphocyte (CTL) (PE-9) when both expressed Fas and Fas ligand (FasL). The CTL clone, PE-9, expressed high levels of Fas and FasL upon activation through the T-cell receptor (TCR). Furthermore, the activated PE-9 cells used both perforin- and FasL-based pathways to kill Fas-positive (Fas+) LSA tumor cells. Interestingly, LSA tumor cells also constitutively expressed FasL but not perforin, and killed Fas+ PE-9 CTLs and Fas+ but not Fas-negative (Fas-) activated T cells and thymocytes, as detected using the JAM test. PE-9 CTLs, cultured for 24 hours in the presence of cell lysates of FasL-bearing LSA cells but not FasL-deficient P815 cells, exhibited significant apoptosis as detected using the TUNEL method. Moreover, another FasL+ T-cell lymphoma line, EL-4, induced apoptosis in Fas+ but not in Fas- T cells in a similar fashion. The current study demonstrates for the first time that not only can the tumor-specific CTL mediate Fas-based killing of tumor cells, but FasL+ tumor cells can kill the Fas+ tumor-specific CTL. Thus, the survival of the tumor or the host may depend on which cell can accomplish this task more efficiently. The current study also suggests that FasL-based killing of CTLs by specific tumor cells may constitute a major limiting factor in successful immunotherapy.     

Death of solid tumor cells induced by Fas ligand expressing primary myoblasts

Anticancer therapy for solid tumors suffers from inadequate methods for the localized administration of cytotoxic agents. Fas ligand (FasL) has been reported to be cytotoxic to a variety of cells, including certain tumor cell lines. We therefore postulated that myoblasts could serve as non-transformed gene therapy vehicles for the continuous localized delivery of cytotoxic anticancer agents such as FasL. However, contrary to previous reports, fluorescence activated cell sorting (FACS) analyses revealed that both primary mouse and human myoblasts express Fas, the receptor for FasL. To avoid self-destruction and test the cytotoxic potential of myoblasts, the cells were isolated from mice deficient in Fas (lpr/lpr), the mouse counterpart of human autoimmune lymphoproliferative syndrome (ALPS). These primary mouse myoblasts were transduced with a retroviral vector encoding mouse FasL and expression of a biologically active and soluble form of the molecule was confirmed by the apoptotic demise of cocultured Fas-expressing Jurkat cells, the standard in the field. To test whether the lpr myoblasts expressing FasL could be used in anticancer therapy, human rhabdomyosarcoma derived cell lines were assayed for Fas and then tested in the apoptosis coculture assay. The majority of Fas-expressing muscle tumor cells were rapidly killed. Moreover, FasL expressing myoblasts were remarkably potent; indeed well characterized cytotoxic antibodies to Fas were only 20% as efficient at killing rhabdomyosarcoma cells as FasL expressing myoblasts. These findings together with previous findings suggest that primary myoblasts, defective in Fas but genetically engineered to express FasL, could function as potent anticancer agents for use in the localized destruction of solid tumors in vivo by three synergistic mechanisms: (1) directly via Fas/FasL mediated apoptosis, (2) indirectly via neutrophil infiltration and immunodestruction, and (3) as allogeneic inducers of a bystander effect via B and T cells.     

Expression of Apo-1/Fas (CD95), Bcl-2, Bax and Bcl-x in myeloma cell lines: relationship between responsiveness to anti-Fas mab and p53 functional status

The down-regulation of apoptosis may be an essential mechanism for tumour cell expansion in slowly proliferating tumours such as multiple myeloma. We studied eight myeloma cell lines for the presence of Bcl-2, which inhibits apoptosis, of Bax, which counteracts Bcl-2, of Bcl-x(L) and Bcl-x(S), which act in an anti- and pro-apoptotic fashion, respectively, and of Apo-1/Fas, which induces programmed cell death, when activated by the Apo-1/Fas ligand or the relevant monoclonal antibody (mab). All cell lines constitutively expressed homogenous amounts of Bcl-2, but displayed different amounts of Bax and Bcl-x proteins. The Apo-1/Fas antigen could be detected in seven out of eight myeloma lines, but expression levels varied considerably. The relative expression levels of Apo-1/Fas correlated with that of Bax, but not with that of Bcl-2 or Bcl-x subtypes. Furthermore, the effectiveness of the Apo-1/Fas mab was associated with the relative expression levels of the Apo-1/Fas and with that of the Bax antigen, but not with that of the Bcl-2 and Bcl-x antigens. We further showed that wild-type p53 function is not required for Apo-1/Fas-induced apoptosis, nor is it necessary for the expression of Bax or Apo-1/Fas antigens in myeloma. In conclusion, our results suggest a p53-independent co-regulation of Apo-1/Fas and Bax, as well as a role for Bax in Apo-1/Fas-induced apoptosis in myeloma.     

Sensitization of human bladder cancer cells to Fas-mediated cytotoxicity by cis-diamminedichloroplatinum (II)

PURPOSE: The resistance of bladder cancer to anticancer chemotherapeutic drugs is a major problem. Several immunotherapeutic approaches have been developed to treat drug-resistant tumor cells. The Fas antigen (Fas)-Fas ligand pathway is involved in cytotoxic T lymphocyte and natural killer cell-mediated cytotoxicity. Like the Fas ligand, anti-Fas monoclonal antibody (mAb) induces apoptosis in tumor cells expressing Fas. Several anticancer drugs also mediate apoptosis and may share with Fas common intracellular pathways leading to cell killing. We reasoned that treatment of drug-resistant cancer cells with a combination of anti-Fas mAb and drugs might overcome their resistance. This study has investigated whether anticancer drugs synergize with anti-Fas mAb in cytotoxicity against bladder cancer cells. MATERIALS AND METHODS: Cytotoxicity was determined by a 1-day microculture tetrazolium dye assay. Synergy was assessed by isobolographic analysis. RESULTS: Treatment of the T24 human bladder cancer cell line with anti-Fas mAb in combination with 5-fluorouracil, mitomycin C or methotrexate did not overcome resistance to these agents. However, treatment of T24 tumor cells with a combination of anti-Fas mAb and cisdiamminedichloroplatinum (II) (CDDP) resulted in a synergistic cytotoxic effect. In addition, the CDDP-resistant T24 line (T24/CDDP) was sensitive to treatment with a combination of anti-Fas mAb and CDDP. Synergy by combination of anti-Fas mAb and CDDP was also achieved in three other bladder cancer lines and four freshly derived human bladder cancer cells. The combination of anti-Fas mAb and carboplatin also resulted in a synergistic cytotoxic effect on T24 cells; however, the combination of anti-Fas mAb and trans-diamminedichloroplatinum (II) resulted in an additive cytotoxic effect. Treatment with CDDP enhanced the expression of Fas on T24 cells. The synergy achieved in cytotoxicity with anti-Fas mAb and CDDP was also achieved in apoptosis. Incubation of T24 cells with anti-Fas mAb increased the intracellular accumulation of CDDP. Treatment of freshly isolated bladder cancer cells with CDDP enhanced their susceptibility to lysis by autologous lymphocytes. CONCLUSIONS: This study demonstrates that combination treatment of bladder cancer cells with anti-Fas mAb and CDDP overcomes their resistance. Synergy was achieved with established CDDP-resistant bladder cancer cells and freshly isolated bladder cancer cells. In addition, the sensitization required low concentrations of CDDP, thus supporting the potential in vivo application of combination of CDDP and immunotherapy in the treatment of CDDP- and/or immunotherapy-resistant bladder cancer.     

Cutting edge: CD4+ T cells kill CD8+ T cells via Fas/Fas ligand-mediated apoptosis

To explore the possibility that CD4+ T cells, described to mediate the elimination of themselves or B lymphocytes, could also mediate the elimination of CD8+ T cells, we analyzed apoptotic phenomena in cocultures of CD4+ and CD8+ autologous T cell lines. The data show that CD8+ T cells were lysed by activated CD4+ helper T cells by a Fas/FasL-mediated mechanism. CD4+ T cells were not lysed by activated CD8+ T cells, although Fas and FasL were equally expressed and anti-Fas Abs induced apoptosis in both CD4+ and CD8+ T cell populations. The results allowed us to speculate that CD4+ T cells not only help CD8+ T lymphocytes to mature into effector killer cells and to sustain this function but can also limit their growth.     

Protein kinase C regulates Fas (CD95/APO-1) expression

Fas (CD95/APO-1) is a transmembrane protein of the TNF/neuron growth factor receptor family. Ligation of Fas by specific Abs or Fas ligand (FasL/CD95 ligand) induces rapid apoptotic cell death in a variety of cell types. Despite progress in understanding the death signals transduced from Fas, very little is known with regard to the mechanisms by which Fas expression is regulated. Using our previously established murine T cell hybridoma model A1.1, we show that specific protein kinase C (PKC) inhibitors could block activation-induced Fas expression and apoptosis. The activation of PKC with PMA or 1-oleoyl-2-acetyl-sn-glycerol could mimic the TCR signal by inducing the expression of Fas but not FasL. PKC-dependent Fas expression was also observed in several murine and human tumor cell lines. Since the inhibition of Ca2+ redistribution by an inhibitor of intracellular Ca2+ mobilization, 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride, inhibited TCR-induced FasL but not Fas, the expression of Fas appears to be independent of Ca2+ mobilization. Significantly, expression of the newly identified Fas-regulatory gene, TDAG51, was found to be dependent upon the activity of PKC. PKC activation only induced Fas expression in cells expressing wild-type TDAG51. Thus, Fas expression is likely mediated by PKC through TDAG51.     

Differential sensitivity of CD4+ and CD8+ T lymphocytes to the killing efficacy of Fas (Apo-1/CD95) ligand+ tumor cells in B chronic lymphocytic leukemia

B-chronic lymphocytic leukemia (B-CLL) is characterized by cellular and humoral immune defects resulting in increased rates of infection and disturbed immune surveillance against cancer cells as well as by the expansion of slowly proliferating tumor cells. We found increased Fas receptor (FasR) expression in peripheral blood CD4+ and CD8+ cells of B-CLL patients compared with the equivalent cells of healthy donors. Although increased Fas receptor expression was significant in both T-lymphocytic subsets, only CD4+ cells from B-CLL patients underwent apoptosis after treatment with the agonistic Fas antibody CH11. In CD4+ cells of B-CLL patients, the Fas-sensitivity also correlated with a CD4+/CD8+ ratio below the lower threshold of healthy individuals (<1.0). By contrast, FasR expression in the CD19(+) fraction of B-CLL patients was downregulated compared with normal controls, and this was associated with an insensitivity to CH11-induced apoptosis. The B-CLL cell line EHEB as well as CD19(+) cells from B-CLL patients constitutively expressed Fas ligand (FasL). The FasL was functionally active, as the B-CLL cell line as well as T-cell-depleted CD19+ B-CLL fractions were able to kill target T-acute lymphatic leukemia (T-ALL) cells in vitro. This effect was inhibited by the antagonistic FasR-antibody ZB4, the neutralizing anti-FasL monoclonal antibody (MoAb) NOK-2 or by transfection of the caspase inhibitor crmA. These data point to the fact that expression of FasL on CD19(+) B-CLL cells, together with enhanced susceptibility of CD4+ T cells toward FasL-bearing effector cells, are causally linked to the relative reduction of CD4+ cells occurring during B-CLL progression. These findings could explain the inversion of the ratio of CD4+/CD8+ cell numbers, which may be causally linked to the immune deficiency observed in these patients and to the expansion of the neoplastic clone in B-CLL.     

Vascular dementia: dead or alive?

Allergic respiratory inflammation in target organs does not occur in any atopic (genetically susceptible) subject, since other not fully characterized factors can influence the subsequent development of overt clinical disease. Here are presented some recent developments in experimental animal and human research that can offer a new "non-classical" interpretation about the way by which allergens are recognized and allergic inflammation persists. These aspects of the immunopathogenesis of allergic diseases can now be viewed as organ-specific pathways, acting independently from other peripheral lymphoid organs. This is a consequence of new knowledge about the function of, and molecular interactions by, intraepithelial gammadelta T cells and CD1+ dendritic cells. The allergic subject, unlike the normal one, is equipped at the mucosal surface by particular T cell and antigen-presenting cell (APC) subsets that enable them to recognize undenatured proteic and non-proteic (glycolipidic) external structures of aerodispersed particles, presented in the context of CD1 molecules. Once initiated, the mucosal allergic reaction cannot be turned off in atopic individuals because CD4+ allergen-specific T cells lack surface Fas receptor. This defect, that impairs the so-called activation-induced programmed cell death (determined by Fas/FasL interaction), is caused by the local Th2-type cytokine milieu.     

Fas ligand is constitutively secreted by prostate cancer cells in vitro

LNCaP, DU145, and PC3 prostate carcinoma cells secrete the 27-kDa soluble Fas ligand (sFasL) into their local environment. sFasL arises from the 40-kDa membrane-bound form (mFasL), which can be found on the cell surface in the LNCaP line, as demonstrated by monoclonal antibody staining. mFasL was also found in extracts of all three cell lines, as demonstrated by Western blotting. FasL mRNA was detected not only in the cell lines, but in the normal prostate as well. sFasL protein could also be detected immunohistochemically in prostate secretions and in human semen. Cleavage of mFasL to sFasL could be inhibited by several matrix metalloprotease inhibitors without a change in the cellular levels of FasL. Prostate-derived sFasL is biologically active, as demonstrated by its induction of apoptosis in Fas-positive Ramos cells, which was detected by terminal deoxynucleotidyl transferase-mediated nick end labeling assay. Mitoxantrone induces cellular apoptosis in all three prostate cancer cell lines. Mitoxantrone treatment and doxorubicin treatment also cause up-regulation of Fas, the cell surface receptor for FasL, in LNCaP cells, but not in DU145 or PC3 cells. Furthermore, the up-regulation of Fas expression by mitoxantrone at a high concentration was potentiated by hydrocortisone. When FasL interacts with its Fas, the Fas-bearing cell undergoes apoptosis. When LNCaP cells were treated with mitoxantrone and incubated with an anti-FasL monoclonal antibody, apoptosis was partially blocked. This not only further suggests that the sFasL is biologically active, but that the up-regulation of Fas in the presence of sFasL accounts, in part, for the cytotoxicity of mitoxantrone.