Bioactivities of Fas ligand-expressing retroviral particles

Culture supernatants from retroviral packaging cells carrying the human Fas ligand (FasL) gene killed both human (Jurkat) and mouse (LB27.4) targets within 5 h of incubation. Cytotoxicity was found both in a fraction >/=500 kDa and a fraction between 50 and 500 kDa. Following ultracentrifugation, the activity in the >/=500-kDa fraction was concentrated in the pellet (FasL vector preparation (VP)), which was also infective when added to NIH-3T3 cells. Both Polybrene and poly-l -lysine significantly enhanced the cytotoxicity of FasL VP but not anti-Fas mAb, soluble FasL (sFasL), and cell-associated FasL. In the presence of Polybrene, FasL VP killed targets that are resistant to anti-Fas mAb and sFasL. The infectivity but not FasL cytotoxicity of FasL VP was sensitive to irradiation and heat shock. By contrast, cytotoxicity of FasL VP could be enhanced or inhibited depending on the doses of anti-FasL mAb. Interestingly, the infectivity of FasL VP was specifically enhanced by anti-FasL mAb, suggesting that a nonviral gene product could be used to regulate the behavior of the retroviral vector. Thus, in addition to expressing potent FasL cytotoxicity, the FasL VP exhibits unique properties heretofore not attributed to anti-Fas mAb, sFasL, and cell-associated FasL. Our study raises the possibility of using the retroviral gene-packaging technology to make powerful, versatile, and regulatable bioactive vesicles expressing a predetermined function of the protein encoded by the target gene.     

Involvement of TNF-related apoptosis-inducing ligand in human CD4+ T cell-mediated cytotoxicity

TNF-related apoptosis-inducing ligand (TRAIL) has been identified as a member of the TNF family that induces apoptosis in a variety of tumor cells, but its physiological functions are largely unknown. In the present study, we examined the expression and function of TRAIL in human CD4+ T cell clones by utilizing newly established anti-human TRAIL mAbs. Human CD4+ T cell clones, HK12 and 4HM1, exhibited perforin-independent and Fas ligand (FasL)-independent cytotoxicity against certain target cells, including T lymphoma (Jurkat) and keratinocyte (HaCaT) cell lines, which are susceptible to TRAIL-mediated cytotoxicity. In contrast to FasL, the expression of which was inducible upon anti-CD3 stimulation, TRAIL was constitutively expressed on HK12 and 4HM1 cells, and no further increase was observed after anti-CD3 stimulation. Spontaneous cytotoxic activities of resting HK12 and 4HM1 cells against Jurkat and HaCaT cells were blocked by anti-TRAIL mAb but not by anti-FasL mAb, and bystander cytotoxic activities of anti-CD3-stimulated HK12 and 4HM1 cells were abolished by the combination of anti-TRAIL and anti-FasL mAbs. These results indicate a differential regulation of TRAIL and FasL expression on human CD4+ T cell clones and that TRAIL constitutes an additional pathway of T cell-mediated cytotoxicity.     

Enhancement of human cord blood CD34+ cell-derived NK cell cytotoxicity by dendritic cells

NK cells and dendritic cells (DCs) are both important in the innate host defense. However, the role of DCs in NK cell-mediated cytotoxicity is unclear. In this study, we designed two culture systems in which human cord blood CD34(+) cells from the same donor were induced to generate NK cells and DCs, respectively. Coculture of the NK cells with DCs resulted in significant enhancement of NK cell cytotoxicity and IFN-gamma production. However, NK cell cytotoxicity and IFN-gamma production were not increased when NK cells and DCs were grown together separated by a transwell membrane. Functional studies demonstrated that 1) concanamycin A, a selective inhibitor of perforin/granzyme B-based cytolysis, blocked DC-stimulated NK cytotoxicity against K562 cells; and 2) neutralizing mAb against Fas ligand (FasL) significantly reduced DC-stimulated NK cytotoxicity against Fas-positive Jurkat cells. In addition, a marked increase of FasL mRNA and FasL protein expression was observed in DC-stimulated NK cells. The addition of neutralizing mAb against IL-18 and IL-12 significantly suppressed DC-stimulated NK cell cytotoxicity. Neutralizing IFN-gamma Ab almost completely inhibited NK cell cytotoxicity against Jurkat cells. These observations suggest that DCs enhance NK cell cytotoxicity by up-regulating both perforin/granzyme B- and FasL/Fas-based pathways. Direct interaction between DCs and NK cells is necessary for DC-mediated enhancement of NK cell cytotoxicity. Furthermore, DC-derived IL-18 and IL-12 were involved in the up-regulation of NK cell cytotoxicity, and endogenous IFN-gamma production plays an important role in Fas-mediated cytotoxicity.     

Trypanosoma cruzi infection selectively renders parasite-specific IgG+ B lymphocytes susceptible to Fas/Fas ligand-mediated fratricide

The control of B cell expansion has been thought to be solely regulated by T lymphocytes. We show in this study that Trypanosoma cruzi infection induces up-regulation of both Fas and Fas ligand (FasL) molecules on B cells and renders them susceptible to B cell-B cell killing (referred to as fratricide throughout this paper) mediated via Fas/FasL. Moreover, by in vivo administration of anti-FasL blocking mAb we demonstrate that Fas-mediated B cell apoptosis is an ongoing process during this parasitic infection. We also provide evidence that B cells that have switched to IgG isotype are the preferential targets of B cell fratricide. More strikingly, this death pathway selectively affects IgG(+) B cells reactive to parasite but not self Ags. Parasite-specific but not self-reactive B cells triggered during this response are rescued after either in vitro or in vivo FasL blockade. Fratricide among parasite-specific IgG(+) B lymphocytes could impair the immune control of T. cruzi and possibly other chronic protozoan parasites. Our results raise the possibility that the blockade of Fas/FasL interaction in the B cell compartment of T. cruzi-infected mice may provide a means for enhancing antiparasitic humoral immune response without affecting host tolerance.     

Augmentation by anti-T3 antibody of the lymphokine-activated killer cell-mediated cytotoxicity

This study showed that a mAb (145-2C11) against the T3 epsilon-chain of the TCR complex augmented the cytotoxic activity of the lymphokine-activated killer (LAK) effectors. The LAK cells were induced by culturing normal spleen cells with purified human rIL-2. Adding alpha T3 at the effector phase of the cytotoxic reactions augmented the LAK-mediated cytotoxicity. The alpha T3-augmented LAK killing was seen only with tumor targets, and there was no increase of killing against Con A-induced lymphoblasts. The augmentation effect was dose dependent on both the amounts of alpha T3 and the number of LAK cells added. A very low concentration of alpha T3 (1/10,000 dilution of culture supernatants) was sufficient to induce alpha T3-augmented LAK-mediated cytotoxicity. Human rIL-2 at 10 to 30 U/ml was sufficient to generate LAK cells for maximal alpha T3 augmentation, whereas 300 to 1000 U/ml of IL-2 were needed to generate maximal LAK activity when tested in the absence of alpha T3. LAK cells generated for longer periods of time showed a progressive increase of alpha T3-augmented cytotoxicity. For some targets, the alpha T3-augmented LAK killing was FcR dependent as evidenced by the ability of alpha FcR mAb 2.4G2 to inhibit, and for others it was not inhibited. The alpha T3-augmented killing did not correlate with the FcR expression on target cells as defined by 2.4G2. The LAK cells were both Lyt-2+ and Lyt-2-, but the LAK cells involved in alpha T3-augmented killing were exclusively Lyt-2+. Preincubation of LAK cells with alpha T3, but not preincubation of targets with alpha T3, resulted in augmented killing suggesting that the alpha T3 effect was unrelated to an antibody-dependent cell-mediated cytotoxicity. Our findings indicate that alpha T3 is a potent reagent to augment the cytotoxic reaction of LAK cells. These results suggested that a relationship might exist between the T3 complex and the cytotoxic activity of a subpopulation of Lyt-2+ LAK cells.     

Different signaling pathways induced by alpha-CD3 monoclonal antibody versus alloantigen on the basis of differential ornithine sensitivity.

Previously we reported that 10 mM ornithine (Orn) selectively inhibits the development of CD8+ CTL in MLC. Herein we show that induction by alpha-CD3 mAb of CD8+ killer cells which manifest antibody-redirected cytotoxicity (ARC) of FcR+ targets is not Orn sensitive. Orn resistance was independent of activation kinetics or alpha-CD3 mAb concentration. alpha-CD3 mAb added to the cytotoxicity assay did not reveal a cytolytic potential in CTL from an Orn-treated MLC when the target cells bore both the inducing alloantigen and FcR. Addition of alpha-CD3 mAb to MLC failed to overcome Orn inhibition of CTL and yet induced ARC activity in the same culture. Expression of mRNA for pore-forming proteins (PFP) and granzyme B was inhibited by Orn in CTL but not in ARC killer cells. Our results demonstrate differences in the T cell activation process stimulated by alloantigen or alpha-CD3 mAb.     

Comparative studies on FcR (FcRII, FcRIII, and FcR alpha) functions of murine B cells

Distribution of FcR II, FcRIII, and FcR alpha on murine splenic B cells was examined by using FITC-labeled heat-aggregated IgG of each subclass and IgA. Almost 60 to 80% of B cells expressed both FcRII and FcRIII. However, FcR alpha was expressed on only a small proportion (6%) of B cells that co-expressed FcRII. By inhibition assays with the use of cold IgG of each subclass and IgA in addition to anti-FcRII mAb (2.4G2), it was found that IgG1, IgG2a, and IgG2b utilized the same receptor (FcRII), whereas IgG3 and IgA bound only to their unique receptors, FcRIII and FcR alpha, respectively. Immune complexes IC prepared by IgG1, IgG2a, IgG2b, and IgA anti-TNP mAb with TNP-coupled SRBC inhibited the polyclonal Ig secretion and proliferative responses of B cells stimulated with either IL-4 or LPS. The inhibition of B cell activation was associated with the blockade of the membrane depolarization. Moreover, IC prepared by these antibodies caused production of suppressive B cell factor (SBF) as is the case with rabbit IgG antibody to SRBC, and SBF thus prepared regulated antibody responses in an isotype-nonspecific manner. In contrast, no inhibition for these responses or production of SBF was attained by the IC of IgG3 antibody. We concluded that FcRII and FcR alpha mediates a suppressive signal for B cells by acting on the initial step of activation, whereas FcRIII lacks this activity.     

Functional differences between the 40 kDa and 50 to 70 kDa IgG Fc receptors on human neutrophils revealed by elastase treatment and antireceptor antibodies

Most previous studies of IgG FcR on neutrophils (PMN) have focused on a single FcR of Mr = 50 to 70 kDa, which is recognized by mAb 3G8 and anti-Leu-11a. In the course of studying the effects of extracellular proteases on PMN receptor expression and function, we found that treatment with human leukocyte elastase reduced the expression of this FcR on the PMN surface by as much as 85% in flow cytometric studies, but did not inhibit ingestion of IgG-coated particles or O2- production induced by multivalent IgG complexes, and caused only a 35% decrease in IC binding to PMN. Since a second FcR with Mr = 40 kDa recognized by mAb IV-3, recently has been identified on PMN, we sought to determine if this FcR was resistant to elastase and thus accounted for the elastase stability of IgG-mediated PMN functions. Elastase treatment that reduced 3G8 binding by 85% caused no decrease in binding of mAb IV-3. For non-elastase-treated PMN, mAb IV-3 against the 40 kDa FcR caused as much as 79 +/- 7% inhibition of IgG-induced O2- production, whereas mAb 3G8 against the 50 to 70 kDa FcR caused only 32 +/- 5% inhibition. In contrast, for IC-binding, mAb IV-3 caused only 15 +/- 6% inhibition, whereas mAb 3G8 caused as much as 80 +/- 9% inhibition, a reversal of their relative effects on O2- production. In parallel studies with elastase-treated PMN, mAb IV-3 actually blocked more IC binding than did mAb 3G8, 55 +/- 4% vs 40 +/- 6%, respectively, presumably because most of the 50 to 70 kDa FcR molecules had been cleaved. The effect of the two mAb together in blocking IC binding was additive, whereas for blocking of O2- production, mAb 3G8 added little or nothing to the effect of mAb IV-3 alone. Direct 125I-labeled Ab binding studies with intact PMN revealed seven times as many 50 to 70 kDa as 40kDa FcR, 110,200 +/- 9600 and 15,100 +/- 700 sites/cell, respectively. Our findings suggest that the elastase-resistant 40 kDa FcR is primarily responsible for IgG-mediated activation of human PMN, whereas the elastase-sensitive 50 to 70 kDa FcR predominates in IC binding, by virtue of its numerical superiority, but does not directly activate the cell. The latter may serve to hold IgG-coated microorganisms or other multivalent IC in place at the PMN surface, enhancing contact with the 40 kDa FcR and thus facilitating cell activation in a cooperative manner.     

Interaction of Fas ligand and Fas expressed on osteoclast precursors increases osteoclastogenesis

We incidentally found that osteoclast precursors and mature osteoclasts express Fas ligand (FasL) as well as Fas, which was confirmed by flow cytometry, immunofluorescent staining, and RT-PCR. The aim of this study was to determine the role of FasL in differentiation and cell death of osteoclasts. To study the role of FasL in osteoclastogenesis, neutralizing anti-FasL mAb or rFasL was added during receptor activator of NF-kappaB ligand (RANKL)-induced osteoclastogenesis using bone marrow-derived macrophages. Neutralization of endogenous FasL by anti-FasL mAb decreased osteoclastogenesis, whereas rFasL enhanced osteoclast differentiation in a dose-dependent manner. In addition, rFasL up-regulated the secretion of osteoclastogenic cytokines, such as IL-1beta and TNF-alpha, and the activation of NF-kappaB. Functional blocking of IL-1beta and TNF-alpha using IL-1 receptor antagonist and soluble TNFR confirmed that those cytokines mediated the effect of FasL on osteoclastogenesis. The osteoclast precursors were relatively resistant to rFasL-induced apoptosis especially before RANKL treatment, resulting in minimal cell loss by rFasL treatment during osteoclastogenesis. Although rFasL increased the cell death of mature osteoclasts, growth factor withdrawal induced much more cell death. However, anti-FasL mAb did not affect the survival of mature osteoclasts, suggesting that the endogenous FasL does not have a role in the apoptosis of osteoclasts. Finally, in contrast to the effect on apoptosis, rFasL-assisted osteoclastogenesis was not mediated by caspases. In conclusion, FasL has a novel function in bone homeostasis by enhancing the differentiation of osteoclasts, which was not considered previously.     

Mechanisms of nonspecific cytotoxic cell regulation of apoptosis: cytokine-like activity of Fas ligand

The role of FasL/FasR pathways of immunoregulation of programmed cell death in teleost cytotoxic innate immunity has not been previously examined. In the present study, constitutive cytosolic soluble FasL (sFasL) was detected in anterior kidney (AK), peripheral blood (PBL) and liver NCC obtained from tilapia. Ligation of NCC by tumour cells caused the release of sFasL that was associated with lysis of HL-60 targets in 14 h killing assays. Evidence that sFasL mediated this activity was that anti-(human) FasL inhibited tilapia and catfish (cf.) NCC lysis of FasR+ HL-60 tumour cells. Inhibition was concentration dependent. Lysis of IM-9 targets (12% positive for FasR) by (cf.) anterior kidney and PBL NCC was only partially inhibited by anti-FasL mab. Activated NCC from both species were negative for the expression of membrane FasL and FasR. These data confirmed that NCC lyse sensitive tumour cells by multiple effector pathways. Pretreatment of (FasR+) HL-60 cells with anti-FasR mab completely inhibited cf. cytotoxicity at low (100:1) E:T ratios. Anti-FasR mab did not inhibit the lysis of IM-9 targets by cf. NCC. This study demonstrated that for catfish and tilapia, initial target cell conjugate formation was required; however, the terminal killing mechanism depended on at least two different pathways of cytotoxicity. One pathway depended on the release of preformed soluble FasL by activated NCC in the presence of FasR positive target cells. A second pathway has yet to be determined.     

Alveolar and peritoneal macrophages bear three distinct classes of Fc receptors for IgG

The FcR for IgG on the plasma membrane of cells of the mononuclear phagocyte system mediate a number of different biologic responses such as phagocytosis, pinocytosis, superoxide generation, and antibody-dependent cytotoxicity. In the interest of understanding the pathophysiology of these processes we have begun to characterize the FcR for IgG on two readily available sources of macrophages--the lung and the peritoneum--using antireceptor mAb. We find that all three of the distinct classes of FcR for IgG which have been described in man are present on both pulmonary and peritoneal macrophages. Most monocytes, we suggest, bear low numbers of Fc gamma RIII whereas a small subpopulation of monocytes expresses substantial numbers of Fc gamma RIII. Furthermore, we find that two different forms of Fc gamma RIII differ in their capacity to bind anti-Fc gamma RIII mab 3G8 in the presence of human IgG. Human IgG does not block the binding of mAb 3G8 to neutrophils, but it does block 3G8 binding to macrophages and large granular lymphocytes; this finding correlates with the expression of the two Fc gamma RIII genes, I and II, in man. Studies aimed at illuminating the molecular mechanisms of Fc gamma R-mediated processes in macrophages will require consideration of the receptors of all three classes.     

Transmembrane signaling during natural killer cell-mediated cytotoxicity. Regulation by protein kinase C activation

NK cells can mediate either FcR-dependent cytotoxicity against antibody-coated target cells or direct cytotoxicity against a variety of tumor cells. We used homogeneous, cloned populations of CD16+/CD3- human NK cells to characterize and compare the transmembrane signaling mechanisms used during these alternative forms of cytotoxicity. Cross-linkage of NK cell FcR with anti-FcR (anti-CD16) mAb or direct binding to NK-sensitive tumor targets resulted in a rapid release of inositol phosphates and increases in [Ca2+]i. The receptor-dependent [Ca2+]i increase (as monitored in indo-1 loaded NK cells by flow cytometry) consisted of an initial release of calcium from intracellular stores, followed by a sustained influx of calcium across the plasma membrane. To assess the potential regulatory feedback role of protein kinase C (PKC) activation in these proximal signaling events, NK cells were pretreated with either PKC-activating phorbol esters, nonactivating phorbol ester homologs, or synthetic diacylglycerols. Brief pretreatment with activating phorbol esters rapidly inhibited, in a concentration-dependent manner, both phosphoinositide hydrolysis and increases in [Ca2+]i induced by FcR ligation, whereas pretreatment with an inactive phorbol ester had no effect. This acute inhibitory effect was not explained by FcR down-regulation, which occurred with more prolonged exposure to phorbol esters. In contrast, the phosphoinositide turnover and [Ca2+]i increase in NK cells stimulated with NK-sensitive tumor targets were not affected by prior exposure to PKC-activating phorbol esters. This differential regulatory effect of phorbol ester on proximal signaling was paralleled by a corresponding effect on cytotoxicity, i.e., phorbol ester-induced activation of PKC inhibited FcR-dependent cytotoxicity, but did not alter direct cytotoxicity against NK-sensitive tumor cells. These results indicate that PKC activation can differentially regulate alternative forms of NK cell-mediated cytotoxicity by rapidly and specifically desensitizing the FcR.     

Signal transduction during human natural killer cell activation: inositol phosphate generation and regulation by cyclic AMP

NK cells mediate both direct cytotoxicity against a variety of tumor cells and indirect (FcR-dependent) cytotoxicity against antibody-coated targets. When cloned human NK cells (CD16+/CD3-) were exposed to NK-sensitive targets for 30 min, the level of inositol phosphates rose two to five times above background. The rise in inositol phosphates induced by NK-sensitive targets was paralleled by an increase in intracellular free calcium concentration ([Ca2+]i). A panel of tumor cells that were resistant to NK cell lysis did not stimulate significant levels of inositol phosphate production and did not induce an elevation of intracellular free calcium. Ligation of the FcR (CD16) with the mAb 3G8 also triggered phosphoinositide turnover. Kinetic experiments demonstrated that stimulation by either susceptible target cells or by FcR ligation led to rapid (less than 1 min) generation of the Ca2+-mobilizing second messenger, inositol trisphosphate, with slower accumulation of inositol bisphosphate and inositol monophosphate. Previous studies have demonstrated that activation of the cAMP-dependent second messenger pathway strongly inhibits NK cell-mediated cytotoxic functions. Treatment of NK effector cells with forskolin to elevate intracellular cAMP levels resulted in a concentration-dependent inhibition of phosphoinositide hydrolysis induced by both NK-sensitive targets and 3G8-mediated FcR ligation. These results suggest that phosphoinositide turnover represents a critical early event in the human NK cell cytolytic process. Moreover, the potent inhibitory effect of cAMP on NK cell cytotoxicity may be explained by the uncoupling of NK receptors from phospholipase C-mediated phosphoinositide hydrolysis.     

Cutting edge: RANTES regulates Fas ligand expression and killing by HIV-specific CD8 cytotoxic T cells.

Based on the previous observation that RANTES mediates the cytotoxic activity of human HIV-specific CD8+ T cells via the chemokine receptor CCR3, we studied the effect of this chemokine on different effector CD8+ cytolytic cells requiring Fas/Fas ligand (FasL) or perforin-dependent pathway. In CTLs derived from PBMCs of HIV-infected patients, both the spontaneous and the RANTES-induced cytotoxicity were inhibited by anti-FasL neutralizing Abs. In contrast, allogeneic CTLs or NK cells killing through perforin were not affected by RANTES and anti-FasL Ab. Accordingly, RANTES enhanced the expression of FasL in a concentration- and time-dependent manner in HIV-specific CTLs, whereas anti-RANTES Ab decreased markedly FasL expression. Finally, cell surface expression of FasL protein in HIV-specific CTLs was also up-regulated by eotaxin, a selective ligand for CCR3. Our observations show that the action of RANTES via CCR3 is necessary to regulate FasL expression on HIV-specific CD8+ T cells that kill through the Fas/FasL pathway.     

Expression and function of TNF-related apoptosis-inducing ligand on murine activated NK cells.

TNF-related apoptosis-inducing ligand (TRAIL), a new member of TNF family, induces apoptotic cell death of various tumor cells. We recently showed that TRAIL mediates perforin- and Fas ligand (FasL)-independent cytotoxic activity of human CD4+ T cell clones. In the present study, we investigated the expression and function of TRAIL on murine lymphocytes by using newly generated anti-murine TRAIL mAbs. Although freshly isolated T, B, or NK cells did not express a detectable level of TRAIL on their surface, a remarkable level of TRAIL expression was induced preferentially on CD3- NK1.1+ NK cells after stimulation with IL-2 or IL-15. In contrast, TRAIL expression was not induced by IL-18, whereas it efficiently potentiated lymphokine-activated killer activity of NK cells. In addition to perforin inactivation and neutralization of FasL by anti-FasL mAb, neutralization of TRAIL by anti-TRAIL mAb was needed for the complete inhibition of IL-2- or IL-15-activated NK cell cytotoxicity against mouse fibrosarcoma L929 target cells, which were susceptible to both FasL and TRAIL. These results indicated preferential expression of TRAIL on IL-2- or IL-15-activated NK cells and its potential involvement in lymphokine-activated killer activity.     

Hypericin photo-induced apoptosis involves the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and activation of caspase-8

Hypericin (HYP) is a photosensitizing pigment from Hypericum perforatum that displays cytotoxic effects in neoplastic cell lines. Therefore, HYP is presently under consideration as a new anticancer drug in photodynamic therapy. Here, we investigated the mechanism of action of HYP photo-induced apoptosis of Jurkat cells compared to the cytostatic drug paclitaxel (PXL). Both photoactivated HYP and PXL similarly increased the activity of caspase-8 and caspase-3, and drug-induced apoptosis of Jurkat cells was completely blocked by inhibitors of caspase-8 (Z-IETD-FMK) and caspase-3 (Z-DEVD-FMK). The involvement of death receptors was analyzed using neutralizing monoclonal antibodies against Fas (SM1/23), FasL (NOK-2) and TNF-R1 (MAB225), and a polyclonal rabbit anti-human TNF-related apoptosis-inducing ligand (TRAIL) antiserum. TRAIL antibody blocked TRAIL-induced and HYP photo-induced, but not PXL-induced apoptosis of Jurkat cells. In contrast, PXL-induced, but not HYP-induced apoptosis was blocked by the SM1/23 and NOK-2 antibodies. Anti-TNF-R1 antibody had no effect. These findings suggest that HYP photo-induced apoptosis of Jurkat cells is mediated in part by the TRAIL/TRAIL-receptor system and subsequent activation of upstream caspases.     

Interleukin-21 enhances NK cell activation in response to antibody-coated targets

NK cells express an activating FcR (FcgammaRIIIa) that mediates Ab-dependent cellular cytotoxicity and the production of immune modulatory cytokines in response to Ab-coated targets. IL-21 has antitumor activity in murine models that depends in part on its ability to promote NK cell cytotoxicity and IFN-gamma secretion. We hypothesized that the NK cell response to FcR stimulation would be enhanced by the administration of IL-21. Human NK cells cultured with IL-21 and immobilized IgG or human breast cancer cells coated with a therapeutic mAb (trastuzumab) secreted large amounts of IFN-gamma. Increased secretion of TNF-alpha and the chemokines IL-8, MIP-1alpha, and RANTES was also observed under these conditions. NK cell IFN-gamma production was dependent on distinct signals mediated by the IL-21R and the FcR and was abrogated in STAT1-deficient NK cells. Supernatants derived from NK cells that had been stimulated with IL-21 and mAb-coated breast cancer cells were able to drive the migration of naive and activated T cells in an in vitro chemotaxis assay. IL-21 also enhanced NK cell lytic activity against Ab-coated tumor cells. Coadministration of IL-21 and Ab-coated tumor cells to immunocompetent mice led to synergistic production of IFN-gamma by NK cells. Furthermore, the administration of IL-21 augmented the effects of an anti-HER2/neu mAb in a murine tumor model, an effect that required IFN-gamma. These findings demonstrate that IL-21 significantly enhances the NK cell response to Ab-coated targets and suggest that IL-21 would be an effective adjuvant to administer in combination with therapeutic mAbs.     

Apoptosis-inducing membrane vesicles. A novel agent with unique properties

The CD95 ligand (FasL) transmembrane protein is found on activated T cells and cells outside the immune system. A well-known turnover process of membrane FasL is mediated by matrix metalloproteinase, which generates soluble FasL (sFasL). Here, we demonstrate that membrane FasL turnover occurs effectively through the release of membrane vesicles. Quantitative analysis indicates that this process is as effective as sFasL release for FasL-3T3 cells but somewhat less effective for FasL-expressing T cells. The apoptosis-inducing membrane vesicles display unique properties not found in FasL-expressing cells and sFasL. Unlike sFasL, vesicle-associated FasL remained bioactive, killing the same panel of targets that are susceptible to FasL-expressing cells. In contrast to FasL-expressing T cells, FasL-mediated killing by vesicles do not involve LFA-1/ICAM interaction and do not depend on de novo protein synthesis. These observations indicate that the release of FasL-bearing vesicles contributes to the turnover of cell-associated FasL, but the impact of the bioactive FasL-expressing vesicles on the function of cell-associated FasL is different from that of sFasL.     

CD95 ligand (CD95L) immunohistochemistry: a critical study on 12 antibodies

In recent years, some studies on the expression of CD95(Fas/APO-1) ligand (CD95L) in tissues or cells raised concerns about the specificity of the antibodies used. We therefore tested 12 CD95L antibodies for their reliability in immunocyto/histochemistry by (i) staining CD95L-transfected and control CV-1/EBNA cells and (ii) comparing staining patterns in immunohistochemically labeled tissue sections with the localization of CD95L+ cells in in situ hybridization. While G247-4, NOK-1, NOK-2, 4H9, and MIKE-1 stained CD95L-transfected cells and did not significantly bind to controls, G247-4 was the only antibody giving satisfying signals in tissue sections perfectly matching the distribution of CD95L+ cells by in situ hybridization. MAb 33, C-20, and N-20 comparably stained both transfected and control cells and showed considerable background or falsely positive staining in sections. MIKE-2, 8B8, A11, and 4A5 did not or only very faintly bind to either cells and, thus, were not tested on sections. We conclude that G247-4 is the only tested antibody that is recommendable for immunohistochemistry.     

Activated human T cells release bioactive Fas ligand and APO2 ligand in microvesicles.

Activation-induced cell death is a process by which overactivated T cells are eliminated, thus preventing potential autoimmune attacks. Two known mediators of activation-induced cell death are Fas(CD95) ligand (FasL) and APO2 ligand (APO2L)/TNF-related apoptosis-inducing ligand (TRAIL). We show here that upon mitogenic stimulation, bioactive FasL and APO2L are released from the T cell leukemia Jurkat and from normal human T cell blasts as intact, nonproteolyzed proteins associated with a particulate, ultracentrifugable fraction. We have characterized this fraction as microvesicles of 100-200 nm in diameter. These microvesicles are released from Jurkat and T cell blasts shortly (相似文献