Members of the tumor necrosis factor superfamily of receptors induce apoptosis by recruiting adaptor molecules through death domain interactions. The central adaptor molecule for these receptors is the death domain-containing protein Fas-associated death domain (FADD). FADD binds a death domain on a receptor or additional adaptor and recruits caspases to the activated receptor. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) signals apoptosis through two receptors, DR4 and DR5. Although there is much interest in TRAIL, the mechanism by which FADD is recruited to the TRAIL receptors is not clear. Using a reverse two-hybrid system we previously identified mutations in the death effector domain of FADD that prevented binding to Fas/CD95. Here we show that these mutations also prevent binding to DR5. FADD-deficient Jurkat cells stably expressing these FADD mutations did not transduce TRAIL or Fas/CD95 signaling. Second site compensating mutations that restore binding to and signaling through Fas/CD95 and DR5 were also in the death effector domain. We conclude that in contrast to current models where the death domain of FADD functions independently of the death effector domain, the death effector domain of FADD comes into direct contact with both TRAIL and Fas/CD95 receptors.

Although oxidative stress has been implicated in acute acetaminophen-induced liver failure and in chronic liver cirrhosis and hepatocellular carcinoma (HCC), no common underlying metabolic pathway has been identified. Recent case reports suggest a link between the pentose phosphate pathway (PPP) enzyme transaldolase (TAL; encoded by TALDO1) and liver failure in children. Here, we show that Taldo1-/- and Taldo1+/- mice spontaneously developed HCC, and Taldo1-/- mice had increased susceptibility to acetaminophen-induced liver failure. Oxidative stress in Taldo1-/- livers was characterized by the accumulation of sedoheptulose 7-phosphate, failure to recycle ribose 5-phosphate for the oxidative PPP, depleted NADPH and glutathione levels, and increased production of lipid hydroperoxides. Furthermore, we found evidence of hepatic mitochondrial dysfunction, as indicated by loss of transmembrane potential, diminished mitochondrial mass, and reduced ATP/ADP ratio. Reduced beta-catenin phosphorylation and enhanced c-Jun expression in Taldo1-/- livers reflected adaptation to oxidative stress. Taldo1-/- hepatocytes were resistant to CD95/Fas-mediated apoptosis in vitro and in vivo. Remarkably, lifelong administration of the potent antioxidant N-acetylcysteine (NAC) prevented acetaminophen-induced liver failure, restored Fas-dependent hepatocyte apoptosis, and blocked hepatocarcinogenesis in Taldo1-/- mice. These data reveal a protective role for the TAL-mediated branch of the PPP against hepatocarcinogenesis and identify NAC as a promising treatment for liver disease in TAL deficiency.

Dendritic cells (DCs) link innate and adaptive immunity, initiating and regulating effector cell responses. They ubiquitously express members of the LILR (ILT, LIR, CD85) family of molecules, some of which recognize self-HLA molecules, but little is known of their possible functions in DC biology. We demonstrate that the inhibitory receptor LILRB1 (ILT2, LIR1, CD85j) is selectively up-regulated during DC differentiation from monocyte precursors in culture. Continuous ligation of LILRB1 modulated cellular differentiation, conferred a unique phenotype upon the resultant cells, induced a profound resistance to CD95-mediated cell death, and inhibited secretion of cytokines IL-10, IL-12p70, and TGF-beta. These features remained stable even after exposure of the cells to bacterial LPS. Ligated DCs exhibited poor stimulatory activity for primary and memory T-cell proliferative responses, but this was substantially reversed by blockade of CD80 or its preferred ligand CTLA-4, or by depleting CD4(+) CD25(+) CD127(lo) regulatory T cells. Our findings suggest that ligation of LILRB1 on DCs by self-HLA molecules may play a key role in controlling the balance between the induction and suppression of adaptive immune responses.

OBJECTIVE

Pulmonary metastases continue to be a significant problem in osteosarcoma. Apoptosis dysfunction is known to influence tumor development. Fas (CD95, APO-1)/FasL is one of the most extensively studied apoptotic pathways. Because FasL is constitutively expressed in the lung, cells that express Fas should be eliminated by lung endothelium. Cells with low or no cell surface Fas expression may be able to evade this innate defense mechanism. The purpose of these studies was to evaluate Fas expression in osteosarcoma lung metastases and the effect of gemcitabine on Fas expression and tumor growth.

RESULTS

Using the K7M2 murine osteosarcoma model, Fas expression was quantified using immunohistochemistry. High levels of Fas were present in primary tumors, but no Fas expression was present in actively growing lung metastases. Blocking the Fas pathway using Fas-associated death domain dominant-negative delayed tumor cell clearance from the lung and increased metastatic potential. Treatment of mice with aerosol gemcitabine resulted in increased Fas expression and subsequent tumor regression.

CONCLUSIONS

We conclude that corruption of the Fas pathway is critical to the ability of osteosarcoma cells to grow in the lung. Agents such as gemcitabine that up-regulate cell surface Fas expression may therefore be effective in treating osteosarcoma lung metastases. These data also suggest that an additional mechanism by which gemcitabine induces regression of osteosarcoma lung metastases is mediated by enhancing the sensitivity of the tumor cells to the constitutive FasL in the lung.

Plasma membrane lipid microdomains have been considered as a sort of 'closed chamber', where several subcellular activities, including CD95/Fas-mediated proapoptotic signaling, take place. In this work we detected GD3 and GM3 gangliosides in isolated mitochondria from lymphoblastoid CEM cells. Moreover, we demonstrated the presence of microdomains in mitochondria by immunogold transmission electron microscopy. We also showed that GD3, the voltage-dependent anion channel-1 (VDAC-1) and the fission protein hFis1 are structural components of a multimolecular signaling complex, in which Bcl-2 family proteins (t-Bid and Bax) are recruited. The disruption of lipid microdomains in isolated mitochondria by methyl-beta-cyclodextrin prevented mitochondria depolarization induced by GD3 or t-Bid. Thus, mitochondrion appears as a subcompartmentalized organelle, in which microdomains may act as controllers of their apoptogenic programs, including fission-associated morphogenetic changes, megapore formation and function. These results disclose a new scenario in which mitochondria-associated lipid microdomains can act as regulators and catalysts of cell fate.

BACKGROUND

Recent evidence suggests that co-clustering of Fas/CD95 death receptor and lipid rafts plays a major role in death receptor-mediated apoptosis.

RESULTS

By a combination of genetic, biochemical, and ultrastructural approaches, we provide here compelling evidence for the involvement of lipid raft aggregates containing recruited Fas/CD95 death receptor, Fas-associated death domain-containing protein (FADD), and procaspase-8 in the induction of apoptosis in human T-cell leukemia Jurkat cells by the antitumor drug edelfosine, the prototype compound of a promising family of synthetic antitumor lipids named as synthetic alkyl-lysophospholipid analogues. Co-immunoprecipitation assays revealed that edelfosine induced the generation of the so-called death-inducing signaling complex (DISC), made up of Fas/CD95, FADD, and procaspase-8, in lipid rafts. Electron microscopy analyses allowed to visualize the formation of raft clusters and their co-localization with DISC components Fas/CD95, FADD, and procaspase-8 following edelfosine treatment of Jurkat cells. Silencing of Fas/CD95 by RNA interference, transfection with a FADD dominant-negative mutant that blocks Fas/CD95 signaling, and specific inhibition of caspase-8 prevented the apoptotic response triggered by edelfosine, hence demonstrating the functional role of DISC in drug-induced apoptosis. By using radioactive labeled edelfosine and a fluorescent analogue, we found that edelfosine accumulated in lipid rafts, forming edelfosine-rich membrane raft clusters in Jurkat leukemic T-cells. Disruption of these membrane raft domains abrogated drug uptake and drug-induced DISC assembly and apoptosis. Thus, edelfosine uptake into lipid rafts was critical for the onset of both co-aggregation of DISC in membrane rafts and subsequent apoptotic cell death.

CONCLUSIONS

This work shows the involvement of DISC clusters in lipid raft aggregates as a supramolecular and physical entity responsible for the induction of apoptosis in leukemic cells by the antitumor drug edelfosine. Our data set a novel framework and paradigm in leukemia therapy, as well as in death receptor-mediated apoptosis.

OBJECTIVE

To examine the role of apoptosis in the pathogenesis of Sjögren syndrome (SS), a chronic autoimmune disease characterized by the infiltration of mononuclear cells in the salivary and lacrimal glands, leading to the destruction of the parenchymal tissue.

METHODS

A detailed search via MEDLINE (PubMed) and Biosis, covering the period from January 1994 to July 2002, was accomplished, combining the key terms SS and apoptosis. A qualitative review of the articles was undertaken and the obtained information was summarized.

RESULTS

Apoptosis of the acinar and ductal epithelial cells of the salivary and lacrimal glands has been proposed as a possible mechanism responsible for the impairment of secretory function. Apoptotic cell death may be induced by either cytotoxic T cells through the release of proteases, such as perforin and granzyme B, or the interaction of Fas ligand (FasL/CD95L), expressed by T lymphocytes, with Fas (Apo-1/CD95) on epithelial cells. The increased rate of apoptosis of epithelial cells in SS may result from either the imbalance between the down-regulated apoptosis-inhibitor Bcl-2 and the up-regulated apoptosis-inducer Bax, or the autocrine and/or paracrine Fas/FasL interaction. Lymphocytes infiltrating the salivary glands are blocked in their ability to commit to apoptosis, despite the expression of the apoptosis-inducer Fas. The expression of Bcl-2 in these cells may explain their resistance to apoptosis, resulting in a prolonged production of proinflammatory cytokines and autoantibodies, as well as in their longer survival that may result in the late development of lymphoma in some SS patients. Studies of the SS-like sialoadenitis of nonobese diabetic (NOD) mice with severe combined immunodeficiency (NOD.scid) suggest that the primary defect responsible for the initiation of SS resides in the epithelial cells that undergo apoptosis mediated by the autocrine Fas/FasL interaction. This first not-immune-mediated phase of target cell destruction is followed by a lymphocyte-dependent autoimmune aggression, which leads to extensive tissue damage and subsequent loss of secretory function. Apoptosis of the salivary epithelial cells has been shown in other animal models of SS and in cell lines in vitro. Apoptosis may also play a role in the pathogenesis of some extraglandular manifestations of SS, such as interstitial nephritis and peripheral CD4(+) lymphocytopenia.

CONCLUSIONS

The possible role of apoptosis in SS is suggested from the literature review. A better understanding of the mechanisms responsible for the epithelial cell apoptosis may allow the discovery of new therapeutic strategies. By inhibiting programmed cell death, the glandular damage and the subsequent impairment of the secretory function should be reduced.

A recent report described the expression of Fas ligand (FasL) by melanoma cells as an important mechanism involved in the immune evasion by tumors [M. Hahne et al., Science (Washington DC), 274: 1363-1366, 1996]. To investigate the expression of FasL by melanomas, we screened a panel of early-passage cell lines by functional assay and reverse transcriptase-PCR. Using conditions designed to replicate those in the original report, we did not find functional FasL on any of the 19 human melanoma lines established at the National Cancer Institute. Furthermore, we additionally evaluated our melanoma lines using reverse transcriptase-PCR and found that 0 of the 26 human melanoma cell lines expressed FasL mRNA. FasL mRNA was abundantly expressed by anti-melanoma T-cell lines after activation. These data do not support a role for FasL expression in the escape of melanoma cells from immune destruction.

We recently reported that nuclear factor-kappa B (NF-κB) promotes DNA damage-triggered apoptosis in glioblastoma, the most common brain tumor. In the present study, we investigated the role of NF-κB in death receptor-mediated apoptosis. Here, we identify a novel pro-apopotic function of NF-κB in TRAIL- and CD95-induced apoptosis. Inhibition of NF-κB by overexpression of the dominant-negative IκBα-superrepressor (IκBα-SR) significantly decreases tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)- or CD95-induced apoptosis. Vice versa, activation of NF-κB via overexpression of constitutively active IκB kinase complex (IKK)β (IKK-EE) significantly increases TRAIL-mediated apoptosis. Intriguingly, NF-κB inhibition reduces the recruitment of Fas-associated death domain and caspase-8 and formation of the death-inducing signaling complex (DISC) upon stimulation of TRAIL receptors or CD95. This results in reduced TRAIL-mediated activation of caspases, loss of mitochondrial potential and cytochrome c release in IκBα-SR-expressing cells. In comparison, NF-κB inhibition strongly enhances TNF-α-mediated apoptosis. Comparative studies revealed that TNF-α rapidly stimulates transcriptional activation and upregulation of anti-apoptotic proteins, whereas TRAIL causes apoptosis before transcriptional activation. Thus, this study demonstrates for the first time that NF-κB exerts a pro-apoptotic role in TRAIL- and CD95-induced apoptosis in glioblastoma cells by facilitating DISC formation.

Apoptosis via the CD95/FasL (CD95L) pathway plays an important role in allograft rejection. Heme oxygenase 1 (HO-1), a stress-responsive cytoprotective molecule, may be essential in preventing graft rejection. We used Ad-HO-1 gene transfer to analyze HO-1-mediated effects in a rat allogeneic orthotopic liver transplantation (OLT) model. The cytotoxicity to Fas-bearing YAC-1 target cells and frequency of terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling-positive (TUNEL(+)) cells in vitro were diminished in Ad-CD95L + Ad-HO-1-transfected cells, as compared with Ad-CD95L + Ad-beta-gal controls (p < 0.001). AdHO-1 gene transfer prevented <10-day rejection of dark agouti (DA) livers in Lewis (LEW) rats (survival >32 days), and diminished apoptosis. Unlike Ad-beta-gal OLTs, which showed signs of severe acute rejection, OLTs in the Ad-HO-1 group exhibited mild to moderate rejection and improved function. These beneficial effects were abrogated after adjunctive treatment with tin protoporphyrin (SnPP), an HO-1 antagonist. Intragraft expression of HO-1 and antiapoptotic gene products (Bcl-xl/Bag-1) was enhanced in Ad-HO-1-transduced OLTs, in association with selectively depressed expression of helper T cell type 1 cytokines (interleukin 2 and interferon gamma), as compared with Ad-beta-gal controls. To deliver CO, one of the downstream HO-1 mediators, allogeneic OLT recipients were exposed to methylene chloride. Such treatment prolonged survival to >47 days, diminished apoptosis, and preserved hepatic architecture/function. Thus, Ad-HO-1 gene transfer prevents CD95/FasL-mediated apoptosis, and significantly prolongs allogeneic OLT survival via a downstream HO-1-CO signaling pathway.

The inhibitor-of-apoptosis (IAP) proteins are a novel family of antiapoptotic proteins that are thought to inhibit cell death via direct inhibition of caspases. Here, we report that human malignant glioma cell lines express XIAP, HIAP-1 and HIAP-2 mRNA and proteins. NAIP was not expressed. IAP proteins were not cleaved during CD95 ligand (CD95L)-induced apoptosis, and loss of IAP protein expression was not responsible for the potentiation of CD95L-induced apoptosis when protein synthesis was inhibited. LN-18 cells are highly sensitive to CD95-mediated apoptosis, whereas LN-229 cells require co-exposure to CD95L and a protein synthesis inhibitor, CHX, to acquire sensitivity to apoptosis. Adenoviral XIAP gene transfer blocked caspase 8 and 3 processing in both cell lines in the absence of CHX. Apoptosis was blocked in the absence and in the presence of CHX. However, XIAP failed to block caspase 8 processing in LN-229 cells in the presence of CHX. There was considerable overlap of the effects of XIAP on caspase processing with those of BCL-2 and the viral caspase inhibitor crm-A. These data define complex regulatory mechanisms for CD95-mediated apoptosis in glioma cells and indicate that there may be a distinct pathway of death receptor-mediated apoptosis that is readily activated when protein synthesis is inhibited. The constitutive expression of natural caspase inhibitors may play a role in the resistance of these cells to apoptotic stimuli that directly target caspases, including radiochemotherapy and immune-mediated tumor cell lysis.

ABSTRACT. End-stage renal failure (ESRF) and chronic hemodialysis (HD) induce a state of immunodeficiency that involves T cell-mediated responses. A decreased T cell number combined with a reduced T cell lifespan and an increased T cell activation might play a role in the immune impairment associated with ESRF and chronic HD. Increased T cell activation associated with immunodeficiency suggests that activated T cells may be driven to apoptosis. To test this hypothesis, CD3+ T cell activation (CD69) and apoptosis (annexin V, CD95 (Fas), and DNA fragmentation) were analyzed in a case control study after blood draw sampling (ex vivo), in culture conditions, and after phytohemagglutinin or anti-CD3 stimulation. Ex vivo evaluation of T cells showed an increased number of activated CD69+ T cells in chronic HD patients (142 +/- 5 cells/mm3) compared with patients with ESRF (115 +/- 2 cells/mm3, P = 0.04) and controls (74 +/- 2 cells/mm3, P = 0.0006). These data were confirmed in culture conditions and after stimulation. Similarly, annexin V and CD95 (Fas)-positive T cells were more numerous in both patient groups than in controls, irrespective of the experimental conditions (P < or = 0.005 for both markers), and their percentage was always significantly higher in chronic HD patients than in patients with ESRF. The amount of DNA fragmentation was also significantly higher in the cultured resting T cells of chronic HD patients (37 +/- 3%) than in those of patients with ESRF (25 +/- 3%) and controls (20 +/- 2%) (P = 0.01). Percentage of cultured resting T cells expressing both CD69 and annexin V markers was higher in chronic HD patients (17 +/- 4%) than in patients with ESRF (10 +/- 4%) and controls (6 +/- 2%), (P = 0.005). After stimulation (phytohemagglutinin or anti-CD3), CD69+ T cell apoptosis increased by 2.4-fold in chronic HD patients compared with 1.8-fold in patients with ESRF and only 1.2-fold in controls (P = 0.001). T cells from chronic HD patients and patients with ESRF thus showed an aberrant state of early activation that contrasted with an increased proportion of annexin V and CD95 (Fas)-positive T cells engaged in apoptosis, as confirmed by DNA fragmentation. Increased susceptibility to early activated T cell apoptosis is not only associated with uremia, but is also enhanced by HD procedure. This may account for the T lymphopenia, progressive immunodeficiency, and increased infection risk seen in these patients.

Fas is a cell-surface protein which belongs to the tumor-necrosis-factor-receptor family. Signals through Fas are able to induce apoptosis in sensitive cells, and thus modalities for regulating the level of Fas expression on tumor cells are needed. We have studied cellular responses to gamma irradiation. The level of p53 tumor-suppressor protein was found to be elevated 3 hr after irradiation of p53wild-type MCF-7 breast-carcinoma cells. Interestingly, accumulation of p53 was followed by up-regulation of surface Fas levels between 4 and 8 hr after irradiation. The level of Fas up-regulation was dependent on dose and, whereas elevation in the level of p53 was transient, enhancement of Fas expression was stable. Fas up-regulation occurred coincidentally with induction of G1 cell-cycle arrest, a post-irradiation phenomenon known to be dependent on wild-type-p53 activity. We studied 9 other tumor lines, 2 with wild-type p53, 5 with mutant p53, and 2 expressing no p53. All lines expressing wild-type p53 were found to arrest in G1 and to up-regulate Fas after irradiation. In contrast, all 7 p53null and p53mutant lines failed not only to arrest their cell cycles in G1 phase, but also to up-regulate Fas levels in response to treatment. These findings demonstrate a direct correlation between wild-type-p53 activity and Fas up-regulation after treatment with ionizing radiation, strongly suggesting that post-irradiation Fas up-regulation is dependent on wild-type-p53 activity. Since low doses of radiation were sufficient to modulate Fas expression, up-regulation of the Fas death receptor may have clinical implications following radiotherapy.

Epidemiological studies have established that heavy alcohol consumption in persons with chronic hepatitis C virus (HCV) infection is associated with advanced liver disease, including cirrhosis. The aims of this study were to evaluate the relationship between alcohol consumption and hepatocyte apoptosis in HCV-infected patients and to determine the role of Fas in HCV-mediated apoptosis. Liver tissue from 44 HCV-infected patients with variable alcohol consumption, and 10 normal control subjects who did not consume alcohol was examined for hepatocyte apoptosis, proliferation and Fas expression. Alcohol consumption was assessed using the 'Lifetime Drinking History' alcohol questionnaire. HCV RNA, alanine aminotransferase (ALT) and ferritin were also assessed in addition to demographic data. Hepatocyte apoptosis was significantly greater in HCV-infected patients compared to controls. Expression of Fas (CD95) was found in HCV patients but not in controls. The degree of Fas expression correlated with hepatocyte apoptosis as detected by terminal UTP nick end labelling (TUNEL). Active ethanol consumption led to a significant increase in hepatocyte apoptosis. Fas expression correlated with fibrosis in HCV-infected patients who were not actively drinking ethanol. In summary, HCV leads to increased apoptotic cell death in hepatocytes. Programmed cell death can be further up-regulated by active ethanol consumption. The correlation between Fas expression and TUNEL supports the hypothesis that the Fas-Fas ligand interaction is the major mechanism for HCV-induced hepatocyte apoptosis.

Chronic hepatitis C virus infection causes B cell lymphoproliferative disorders that include type II mixed cryoglobulinemia and lymphoma. This virus drives the monoclonal expansion and, occasionally, the malignant transformation of B cells producing a polyreactive natural Ab commonly encoded by the V(H)1-69 variable gene. Owing to their property of producing natural Ab, these cells are reminiscent of murine B-1 and marginal zone B cells. We used anti-Id Abs to track the stages of differentiation and clonal expansion of V(H)1-69(+) cells in patients with type II mixed cryoglobulinemia. By immunophenotyping and cell size analysis, we could define three discrete stages of differentiation of V(H)1-69(+) B cells: naive (small, IgM(high)IgD(high)CD38(+)CD27(-)CD21(high)CD95(-)CD5(-)), "early memory" (medium-sized, IgM(high)IgD(low)CD38(-)CD27(+)CD21(low)CD95(+)CD5(+)), and "late memory" (large-sized, IgM(low)IgD(low-neg)CD38(-)CD27(low)CD21(low-neg)CD5(-)CD95(-)). The B cells expanded in cryoglobulinemia patients have a "memory" phenotype; this fact, together with the evidence for intraclonal variation, suggests that antigenic stimulation by hepatitis C virus causes the unconstrained expansion of activated V(H)1-69(+) B cells. In some cases, these cells replace the entire pool of circulating B cells, although the absolute B cell number remains within normal limits. Absolute monoclonal V(H)1-69(+) B lymphocytosis was seen in three patients with cryoglobulinemia and splenic lymphoma; in two of these patients, expanded cells carried trisomy 3q. The data presented here indicate that the hepatitis C virus-driven clonal expansion of memory B cells producing a V(H)1-69(+) natural Ab escapes control mechanisms and subverts B cell homeostasis. Genetic alterations may provide a further growth advantage leading to an overt lymphoproliferative disorder.

OBJECTIVE

The chemokine receptor CXCR3, involved in chemotaxis, is expressed on normal and malignant B cells and plasma cells. Recent data suggest that CXCR3-binding chemokines may also regulate proliferation and survival in endothelial cells through the interaction with two distinct isoforms of CXCR3 (CXCR3-A and CXCR3-B).

METHODS

We evaluated the potential expression of CXCR3 isoforms in myeloma cells, also investigating whether CXCR3 expression is affected by cell cycle and apoptosis. Furthermore, we assessed the effect of CXCR3 activation on myeloma cell proliferation and survival.

RESULTS

We found that CXCR3 is widely expressed on human myeloma cell lines and freshly purified myeloma cells. The presence of both CXCR3 isoforms, CXCR3-A and CXCR3-B, was observed in myeloma cells with different ratios of expression. Interestingly, we found that CXCR3 expression in myeloma cell was cell cycle dependent and that myeloma growth factors inhibited CXCR3 expression in myeloma cells. On the other hand, we found that FAS (CD95)-mediated apoptosis up-regulated CXCR3 expression. A similar behavior was observed for the CXCR3-binding chemokines. Finally we found that the activation of CXCR3 on myeloma cells by CXCL10/IP-10 partially blunted FAS-mediated apoptosis in myeloma cells that express CXCR3-A and that high concentrations of CXCL10/IP-10 inhibit myeloma cell proliferation. INTERPRETATION AND CONCLUSIONS Our data indicate that myeloma cells express the CXCR3 system with patterns correlated to cell cycle and apoptosis and that CXCR3 activation may affect myeloma cell survival and proliferation.

To investigate HIV-1-related B cell disorders, the quantity of CD27 positive (CD27+) B cells and their CD38, CD95, and bcl-2 intensities were examined by flow cytometry analysis in 16 drug-naive patients, 27 highly active antiretroviral therapy (HAART)-treated patients, and 20 uninfected controls. CD27+ B cells have been recognized as memory B cells. The mean percentage of CD27+ B cells was significantly lower in drug-naive patients (11.9%) and in HAART-treated patients (16.1%) than in controls (31.4%) (p < 0.01). The intensities of CD38 and CD95 on CD27+ B cells were higher in drug-naive patients than in controls (p < 0.01 in CD95). The intensity of CD95 on CD27+ B cells in HAART-treated patients was lower than that of drug-naive patients, but significantly higher than that of controls (p < 0.01). The intensity of bcl-2 on CD27+ B cells was equivalent among the three groups. These findings suggest that disturbance of peripheral B cell composition, exemplified by CD27+ B cell reduction, exists in both drug-naive and HAART-treated HIV-1-infected patients. In addition, the augmented apoptotic state of CD27+ B cells found in HAART-treated patients with undetectable viral loads, indicated by CD95 elevation, suggests that some HIV-1-related B cell disorders last for years after effective antiviral therapy.

OBJECTIVE

Hepatic stellate cells play an important role in liver fibrogenesis, and hepatic stellate cell death may be involved in the termination of this response.

METHODS

Molecular mechanisms of hepatic stellate cell killing were studied in hepatic stellate cell/Kupffer cell cocultures.

RESULTS

Lipopolysaccharide stimulation of hepatic stellate cell/Kupffer cell cocultures, but not of hepatic stellate cell monocultures, induced profound alterations of hepatic stellate cell morphology and hepatic stellate cell death. Kupffer cell-induced hepatic stellate cell killing required hepatic stellate cell/Kupffer cell contacts and was prevented by dexamethasone, prostaglandin E(2), tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor 2 antagonists, and down-regulation of receptor-interacting protein, but not by antioxidants, tumor necrosis factor receptor, or CD95 antagonists. Hepatic stellate cell death was characterized by activation of caspases 3, 8, and 9, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling negativity, lack of gross calcium overload, and TRAIL trafficking to the plasma membrane. Inhibition of caspase 9, but not of caspases 3, 8, or 10, prevented hepatic stellate cell death. Lipopolysaccharide induced a dexamethasone- and prostaglandin E(2)-sensitive expression of TRAIL in Kupffer cells. TRAIL receptors 1 and 2, FLIP (caspase 8-inhibitory protein), and receptor-interacting protein were up-regulated during hepatic stellate cell transformation; however, TRAIL addition did not induce hepatic stellate cell death. Hepatic stellate cell susceptibility toward Kupffer cell-induced death paralleled receptor-interacting protein and TRAIL-receptor expression levels.

CONCLUSIONS

Activated Kupffer cell can effectively kill hepatic stellate cell by a caspase 9- and receptor-interacting protein-dependent mechanism, possibly involving TRAIL. The data may suggest a novel form of hepatic stellate cell death.

Soluble HLA class I molecules (sHLAs) have been identified in the serum of patients with inflammatory diseases, allografts and autoimmune diseases and in serum of healthy individuals. The biological significance of these molecules, particularly after allogeneic organ transplantation, has been enigmatic. Here we show that primary alloreactive CD8+ T cells interact with sHLA and undergo apoptosis in the absence of a second signal. Ligation of CD28 rescued T cells from death, implying that sHLAs induce apoptosis through selective stimulation of the T-cell receptor. CD95-L was upregulated after cytotoxic T lymphocytes were incubated with sHLAs, and cell death was blocked by a neutralizing anti-CD95-L antibody, suggesting that sHLAs induce endogenous mutual killing of activated T cells. These results provide a molecular basis for the capacity of sHLAs to downregulate T-cell responses, which may be especially relevant to organ transplantation.

Mouse 3T3 fibroblasts derived from fetuses lacking c-Jun were used to define an essential role of c-Jun, a main component of the transcription factor AP-1, in the cellular response to the alkylating agent methyl methanesulfonate (MMS). MMS represents the most potent and selective activator of the stress-induced kinases JNK/SAPK and p38, resulting in very efficient induction of c-Jun hyperphosphorylation and c-jun transcription. This agent induced apoptosis with high efficiency in wild-type cells but not in c-jun(-/-) cells. Resistance to apoptosis was accompanied by impaired expression of CD95 ligand (CD95-L), a well-known inducer of apoptosis. The addition of recombinant CD95-L restored apoptosis sensitivity in c-jun(-/-) fibroblasts. MMS-induced apoptosis in wild-type fibroblasts or human lymphocytes was strongly reduced by neutralizing CD95-L antibodies or transdominant negative FADD, confirming the importance of CD95 signalling in MMS-induced apoptosis. The loss-of-function approach in fibroblasts allowed the identification and dissection of c-Jun-dependent and -independent processes upstream or downstream of CD95 activation. We have found that c-Jun can act as a proapoptotic regulator in cells exposed to DNA damage via induction of CD95-L. Once activated, CD95-induced death signalling is not affected by the loss of c-Jun, demonstrating that only the initiation and not the execution of stress-induced apoptosis depends on c-Jun.

Hepatitis C virus (HCV) infection is one of the major causes of chronic liver disease worldwide. The critical role of innate as well as adaptive immunity has been reported in HCV persistence and liver injury. In the early phase of acute infection, HCV continues to replicate in the liver, suggesting the HCV capability of inhibiting innate immunity. The sustained, vigorous and multiepitope-specific CD4+ and CD8+ T cell responses are essential for spontaneous HCV clearance. HCV-specific CD8+ T cells are primary elements for HCV clearance by inducing hepatocyte apoptosis, in which Fas/CD95 is fundamentally involved. However, once HCV persistency develops, HCV utilizes multifaceted arms to subvert various immune effectors. During IFNalpha-based therapy, the enhancement of HCV-specific CD4+ T cell response followed by HCV eradication has been reported, however, it remains obscure whether the therapeutic HCV clearance is able to restore the durable immune competency to HCV. Further investigation is still warranted to establish the means to direct HCV-specific immune responses in the desired way.

In murine models, T-cell costimulation blockade of the CD28:B7 and CD154:CD40 pathways synergistically promotes immune tolerance after transplantation. While CD28 blockade has been successfully translated to the clinic, translation of blockade of the CD154:CD40 pathway has been less successful, in large part due to thromboembolic complications associated with anti-CD154 antibodies. Translation of CD40 blockade has also been slow, in part due to the fact that synergy between CD40 blockade and CD28 blockade had not yet been demonstrated in either primate models or humans. Here we show that a novel, nondepleting CD40 monoclonal antibody, 3A8, can combine with combined CTLA4Ig and sirolimus in a well-established primate bone marrow chimerism-induction model. Prolonged engraftment required the presence of all three agents during maintenance therapy, and resulted in graft acceptance for the duration of immunosuppressive treatment, with rejection resulting upon immunosuppression withdrawal. Flow cytometric analysis revealed that upregulation of CD95 expression on both CD4+ and CD8+ T cells correlated with rejection, suggesting that CD95 may be a robust biomarker of graft loss. These results are the first to demonstrate prolonged chimerism in primates treated with CD28/mTOR blockade and nondepletional CD40 blockade, and support further investigation of combined costimulation blockade targeting the CD28 and CD40 pathways.

Apoptotic changes of the nucleus induced by Fas (Apo1/CD95) stimulation are completely blocked by reducing intracellular ATP level. In this study, we examined the ATP-dependent step(s) of Fas-mediated apoptotic signal transduction using two cell lines. In SKW6.4 (type I) cells characterized by rapid formation of the death-inducing signaling complex on Fas treatment, the activation of caspases 8, 9, and 3, cleavage of DFF45 (ICAD), and release of cytochrome c from the mitochondria to the cytoplasm were not affected by reduction of intracellular ATP, although chromatin condensation and nuclear fragmentation were inhibited. On the other hand, in the Fas-mediated apoptosis of Jurkat (type II) cells, which is characterized by involvement of mitochondria and, thus, shares signal transduction mechanisms with apoptosis induced by other stimuli such as genotoxins, activation of the three caspases, cleavage of DFF45 (ICAD), and nuclear changes were blocked by reduction of intracellular ATP, whereas release of cytochrome c was not affected. These results suggested that the ATP-dependent step(s) of Fas-mediated apoptotic signal transduction in type I cells are only located downstream of caspase 3 activation, whereas the activation of caspase 9 by released cytochrome c is the most upstream ATP-dependent step in type II cells. These observations also confirm the existence of two pathways for Fas-mediated apoptotic signal transduction and suggest that the Apaf-1 (Ced-4 homologue) system for caspase 9 activation operates in an ATP-dependent manner in vivo.

Affinity maturation of the B cell response to antigen (Ag) takes place in the germinal centers (GCs) of secondary follicles. Two sequential molecular mechanisms underpin this process. First, the B cell repertoire is diversified through hypermutation of the immunoglobulin (Ig) variable region genes. Second, mutant B cell clones with improved affinity for Ag are positively selected by Ag and CD40 ligand (L). This selection step is contingent upon "priming" of GC B cells for apoptosis. The molecular means by which B cell apoptosis is initiated and controlled in the GC remains unclear. Here, we show that GC B cell apoptosis is preceded by the rapid activation of caspase-8 at the level of CD95 death-inducing signaling complex (DISC). We found that GC B cells ex vivo display a preformed inactive DISC containing Fas-associated death domain-containing protein (FADD), procaspase-8, and the long isoform of cellular FADD-like IL-1beta-converting enzyme-inhibitory protein (c-FLIP(L)) but not the CD95L. In culture, c-FLIP(L) is rapidly lost from the CD95 DISC unless GC B cells are exposed to the survival signal provided by CD40L. Our results suggest that (a) the death receptor signaling pathway is involved in the affinity maturation of antibodies, and (b) c-FLIP(L) plays an active role in positive selection of B cells in the GC.