Dendritic cells undergo rapid apoptosis in vitro during antigen-specific interaction with CD4+ T cells

The terminal fate of dendritic cells (DC) remains relatively uncertain. In this study, we tested the hypothesis that DC undergo apoptosis after Ag-specific interaction with T cells. When splenic DC isolated from BALB/c mice were cocultured with HDK-1 T cells (a keyhole limpet hemocyanin (KLH)-specific CD4+ Th1 clone) in the presence of KLH, they showed conspicuous cell death as measured by propidium iodide (PI) uptake and chromatin condensation, whereas they remained relatively intact when incubated with either T cells or KLH alone. Likewise, the long term DC line XS52, which was established from BALB/c mouse epidermis, also died rapidly (within 2 h), and they exhibited characteristic DNA laddering when cocultured with HDK-1 T cells in the presence of KLH. RT-PCR and FACS analyses revealed the expression of CD95 (Fas) by XS52 DC and of CD95 ligand (CD95L) (Fas ligand) by activated HDK-1 T cells, suggesting a functional role for these molecules. In fact, anti-CD95L mAb inhibited partially (50%) T cell-mediated XS52 cell death, and coupling of surface CD95 with anti-CD95 mAb triggered significant XS52 cell death, but only in the presence of cycloheximide. Thus, ligation of CD95 (on DC) with CD95L (on T cells) is one, but not the only, mechanism by which T cells induce DC death. Finally, DC isolated from the CD95-deficient mice were found to be significantly more efficient than DC from control mice in their capacity to induce delayed type hypersensitivity responses in vivo. We propose that T cell-induced DC apoptosis serves as a unique down-regulatory mechanism that prevents the interminable activation of T cells by Ag-bearing DC.     

Differential susceptibility to CD95 (Apo-1/Fas) and MHC class II-induced apoptosis during murine dendritic cell development

Disappearance of antigen presenting cells (APC) from the lymph node occurs following antigen specific interactions with T cells. We have investigated the regulation of CD95 (Apo-1/Fas) induced apoptosis during murine dendritic cell (DC) development. Consistent with the moderate levels of CD95 surface expression and low, or absent, MHC class II expression, immature DC in bone marrow cultures were highly sensitive to CD95 induced apoptosis, but insensitive to class II mediated apoptosis. In contrast, mature splenic, epidermal and bone marrow derived DC were fully resistant to CD95 induced cell death, but sensitive to class II induced apoptosis. Although caspase 3 and 8 activation was detected in immature DC undergoing CD95L-induced apoptosis, the pan-caspase inhibitor zVAD-fmk did not inhibit the early events of CD95-induced mitochondrial depolarisation or phosphatidyl serine exposure and only partially inhibited the killing of immature DC. In contrast, zVAD-fmk was completely effective in preventing CD95L mediated death of murine thymocytes. Collectively, these data do not support a major role of CD95: CD95L ligation in apoptosis of mature DC, but rather emphasise the existence of distinct pathways for the elimination of DC at different stages of maturation.     

Muscle precursor cells isolated from aged rats exhibit an increased tumor necrosis factor-α response

Improving muscle precursor cell (MPC, muscle-specific stem cells) function during aging has been implicated as a key therapeutic target for improving age-related skeletal muscle loss. MPC dysfunction during aging can be attributed to both the aging MPC population and the changing environment in skeletal muscle. Previous reports have identified elevated levels of tumor necrosis factor-α (TNF-α) in aging, both circulating and locally in skeletal muscle. The purpose of the present study was to determine if age-related differences exist between TNF-α-induced nuclear factor-kappa B (NF-κB) activation and expression of apoptotic gene targets. MPCs isolated from 32-month-old animals exhibited an increased NF-κB activation in response to 1, 5, and 20 ng mL⁻¹ TNF-α, compared to MPCs isolated from 3-month-old animals. No age differences were observed in the rapid canonical signaling events leading to NF-κB activation or in the increase in mRNA levels for TNF receptor 1, TNF receptor 2, TNF receptor-associated factor 2 (TRAF2), or Fas (CD95) observed after 2 h of TNF-α stimulation. Interestingly, mRNA levels for TRAF2 and the cell death-inducing receptor, Fas (CD95), were persistently upregulated in response to 24 h TNF-α treatment in MPCs isolated from 32-month-old animals, compared to 3-month-old animals. Our data indicate that age-related differences may exist in the regulatory mechanisms responsible for NF-κB inactivation, which may have an effect on TNF-α-induced apoptotic signaling. These findings improve our understanding of the interaction between aged MPCs and the changing environment associated with age, which is critical for the development of potential clinical interventions aimed at improving MPC function with age.     

Mature but not immature Fas ligand (CD95L)-transduced human monocyte-derived dendritic cells are protected from Fas-mediated apoptosis and can be used as killer APC

Several in vitro and animal studies have been performed to modulate the interaction of APCs and T cells by Fas (CD95/Apo-1) signaling to delete activated T cells in an Ag-specific manner. However, due to the difficulties in vector generation and low transduction frequencies, similar studies with primary human APC are still lacking. To evaluate whether Fas ligand (FasL/CD95L) expressing killer APC could be generated from primary human APC, monocyte-derived dendritic cells (DC) were transduced using the inducible Cre/Loxp adenovirus vector system. Combined transduction of DC by AdLoxpFasL and AxCANCre, but not single transduction with these vectors, resulted in dose- and time-dependent expression of FasL in >70% of mature DC (mDC), whereas <20% of immature DC (iDC) expressed FasL. In addition, transduction by AdLoxpFasL and AxCANCre induced apoptosis in >80% of iDC, whereas FasL-expressing mDC were protected from FasL/Fas (CD95/Apo-1)-mediated apoptosis despite coexpression of Fas. FasL-expressing mDC eliminated Fas(+) Jurkat T cells as well as activated primary T cells by apoptosis, whereas nonactivated primary T cells were not deleted. Induction of apoptosis in Fas(+) target cells required expression of FasL in DC and cell-to-cell contact between effector and target cell, and was not dependent on soluble FasL. Induction of apoptosis in Fas(+) target cells required expression of FasL in DC, cell-to-cell contact between effector and target cell, and was not dependent on soluble FasL. The present results demonstrate that FasL-expressing killer APC can be generated from human monocyte-derived mDC using adenoviral gene transfer. Our results support the strategy to use killer APCs as immunomodulatory cells for the treatment of autoimmune disease and allograft rejection.     

Human anergic CD4+ T cells can act as suppressor cells by affecting autologous dendritic cell conditioning and survival.

T cell suppression exerted by regulatory T cells represents a well-established phenomenon, but the mechanisms involved are still a matter of debate. Recent data suggest that anergic T cells can suppress responder T cell activation by inhibiting Ag presentation by dendritic cells (DC). In this study, we focused our attention on the mechanisms that regulate the susceptibility of DC to suppressive signals and analyzed the fate of DC and responder T cells. To address this issue, we have cocultured human alloreactive or Ag-specific CD4+ T cell clones, rendered anergic by incubation with immobilized anti-CD3 Ab, with autologous DC and responder T cells. We show that anergic T cells affect either Ag-presenting functions or survival of DC, depending whether immature or mature DC are used as APC. Indeed, MHC and costimulatory molecule expression on immature DC activated by responder T cells is inhibited, while apoptotic programs are induced in mature DC and in turn in responder T cells. Ligation of CD95 by CD95L expressed on anergic T cells in the absence of CD40-CD40L (CD154) interaction are critical parameters in eliciting apoptosis in both DC and responder T cells. In conclusion, these findings indicate that the defective activation of CD40 on DC by CD95L+ CD154-defective anergic T cells could be the primary event in determining T cell suppression and support the role of CD40 signaling in regulating both conditioning and survival of DC.     

Dendritic cells exposed to MVA-based HIV-1 vaccine induce highly functional HIV-1-specific CD8(+) T cell responses in HIV-1-infected individuals

Currently, MVA virus vectors carrying HIV-1 genes are being developed as HIV-1/AIDS prophylactic/therapeutic vaccines. Nevertheless, little is known about the impact of these vectors on human dendritic cells (DC) and their capacity to present HIV-1 antigens to human HIV-specific T cells. This study aimed to characterize the interaction of MVA and MVA expressing the HIV-1 genes Env-Gag-Pol-Nef of clade B (referred to as MVA-B) in human monocyte-derived dendritic cells (MDDC) and the subsequent processes of HIV-1 antigen presentation and activation of memory HIV-1-specific T lymphocytes. For these purposes, we performed ex vivo assays with MDDC and autologous lymphocytes from asymptomatic HIV-infected patients. Infection of MDDC with MVA-B or MVA, at the optimal dose of 0.3 PFU/MDDC, induced by itself a moderate degree of maturation of MDDC, involving secretion of cytokines and chemokines (IL1-ra, IL-7, TNF-α, IL-6, IL-12, IL-15, IL-8, MCP-1, MIP-1α, MIP-1β, RANTES, IP-10, MIG, and IFN-α). MDDC infected with MVA or MVA-B and following a period of 48 h or 72 h of maturation were able to migrate toward CCL19 or CCL21 chemokine gradients. MVA-B infection induced apoptosis of the infected cells and the resulting apoptotic bodies were engulfed by the uninfected MDDC, which cross-presented HIV-1 antigens to autologous CD8(+) T lymphocytes. MVA-B-infected MDDC co-cultured with autologous T lymphocytes induced a highly functional HIV-specific CD8(+) T cell response including proliferation, secretion of IFN-γ, IL-2, TNF-α, MIP-1β, MIP-1α, RANTES and IL-6, and strong cytotoxic activity against autologous HIV-1-infected CD4(+) T lymphocytes. These results evidence the adjuvant role of the vector itself (MVA) and support the clinical development of prophylactic and therapeutic anti-HIV vaccines based on MVA-B.     

CD44v7 ligation downregulates the inflammatory immune response in Crohn's disease patients by apoptosis induction in mononuclear cells from the lamina propria

Deletion of exon CD44v7 abrogates experimental colitis by apoptosis induction in intestinal mononuclear cells. Here we show that CD44v7 expression was upregulated upon CD40 ligation in human mononuclear cells, and examined whether ligation of CD44v7 also affects activation and apoptosis in lamina propria mononuclear cells (LPMC) from Crohn's disease (CD) patients. Thirty five patients with chronic inflammatory bowel disease (IBD), fourteen controls and four patients with diverticulitis were evaluated. CD44v7 was upregulated predominantly in the inflamed mucosa of CD patients. Furthermore, incubation with an anti-CD44v7 antibody induced apoptosis in LPMC isolated from inflamed mucosa of CD patients, but not from non-inflamed mucosa, from patients with ulcerative colitis (UC) or from normal controls. CD40 ligation and simultaneous incubation with anti-CD44v7 significantly downregulated CD80 in dendritic cells, thus inhibiting a critical second signal for naive T-cell activation. The apoptotic signal was mediated via the intrinsic mitochondrial pathway with decreased Bcl-2 and increased 7A6 (a mitochondrial membrane protein) expression. It was Fas independent and required caspases-3 and -9 activation. The process is highly specific for macrophage activation via CD40. These findings point to a novel mechanism of apoptosis induction in CD patients mediated by CD44v7 ligation.     

Apoptosis by CD95 (Fas)-dependent and -independent mechanisms in Peyer's patch lymphocytes in murine retrovirus-induced immunodeficiency syndrome.

CD95 (Fas)/CD95 ligand (CD95 L)-mediated apoptosis is thought to be involved in the delayed progression of murine AIDS (MAIDS) induced by LP-BM5 murine leukemia virus (MuLV). We show evidence of apoptosis in lymphocytes of Peyer's patches (PP) at the early stage of MAIDS. Both T and B cells in PP expressed CD95 at the early stage of MAIDS and decreased in number thereafter. The decrease in T cells was not evident in CD95-mutated lpr mice with MAIDS, suggesting that CD95/CD95 L interaction is involved in the apoptosis of T cells in PP during the course of MAIDS. On the other hand, the number of B cells was also decreased in PP of lpr mice with MAIDS. The proliferative ability of B cells in PP of MAIDS mice in response to immunoglobulin M cross-linking or lipopolysaccharide was severely impaired, while the B cells normally proliferated in response to anti-CD40 monoclonal antibody. These findings imply that aberrantly activated B cells in PP undergo apoptosis independently of the CD95/CD95 L system during the course of infection with MAIDS virus.     

Immature CD4- CD103+ rat dendritic cells induce rapid caspase-independent apoptosis-like cell death in various tumor and nontumor cells and phagocytose their victims

We previously reported the characterization of a MHC class II(low) CD4- CD103+ (CD4-) subset of dendritic cells (DC) in rat spleen that exhibit a Ca2+-, Fas ligand-, TRAIL- and TNF-alpha-independent cytotoxic activity against specific targets in vitro. In this study, we demonstrate that this DC subset was also found in lymph nodes. Freshly extracted and, therefore, immature CD4- DC exhibited a potent cytotoxic activity against a large panel of tumor cell lines as well as primary endothelial cells. The cytotoxic activity of immature CD4- DC required cell-to-cell contact and de novo protein expression. CD4- DC-mediated cell death resembled apoptosis, as evidenced by outer membrane phosphatidylserine exposure and nuclear fragmentation in target cells, but was caspase as well as Fas-associated death domain and receptor-interacting protein independent. Bcl-2 overexpression in target cells did not protect them against DC-mediated cell death. Immature CD4- DC phagocytosed efficiently apoptotic cells in vitro and, therefore, rapidly and specifically engulfed their victims following death induction. Maturation induced a dramatic down-regulation of the killing and phagocytic activities of CD4- DC. In contrast, CD4+ DC were both unable to kill target cells and to phagocytose apoptotic cells in vitro. Taken together, these data indicate that rat immature CD4- CD103+ DC mediate an unusual cytotoxic activity and can use this function to efficiently acquire Ag from live cells.     

Targeted NF-κB inhibition of asthmatic serum-mediated human monocyte-derived dendritic cell differentiation in a transendothelial trafficking model

Transendothelial trafficking model mimics in vivo differentiation of monocytes into dendritic cells (DC). The serum from patients with systemic lupus erythematosus promotes the differentiation of monocytes into mature DC. We have shown that selective inhibition of NF-κB by adenoviral gene transfer of a novel mutated IκBα (AdIκBαM) in DC contributes to T cell tolerance. Here we demonstrated for the first time that asthmatic serum facilitated human monocyte-derived DC (MDDC) maturation associated with increased NF-κB activation in this model. Furthermore, selective blockade of NF-κB by AdIκBαM in MDDC led to increased apoptosis, and decreased levels of CD80, CD83, CD86, and IL-12 p70 but not IL-10 in asthmatic serum-stimulated MDDC, accompanied by reduced proliferation of T cells. These results suggest that AdIκBαM-transferred MDDC are at a more immature stage which is beneficial to augment the immune tolerance in asthma.     

Catalytically active <Emphasis Type="Italic">Yersinia</Emphasis> outer protein P induces cleavage of RIP and caspase-8 at the level of the DISC independently of death receptors in dendritic cells

Yersinia outer protein P (YopP) is injected by Y. enterocolitica into host cells thereby inducing apoptotic and necrosis-like cell death in dendritic cells (DC). Here we show the pathways involved in DC death caused by the catalytic activity of YopP. Infection with Yersinia enterocolitica, translocating catalytically active YopP into DC, triggered procaspase-8 cleavage and c-FLIP_L degradation. YopP-dependent caspase-8 activation was, however, not mediated by tumor necrosis factor (TNF) receptor family members since the expression of both CD95/Fas/APO-1 and TRAIL-R2 on DC was low, and DC were resistant to apoptosis induced by agonistic anti-CD95 antibodies or TNF-related apoptosis-inducing ligand (TRAIL). Moreover, DC from TNF-Rp55^−/− mice were not protected against YopP-induced cell death demonstrating that TNF-R1 is also not involved in this process. Activation of caspase-8 was further investigated by coimmunoprecitation of FADD from Yersinia-infected DC. We found that both cleaved caspase-8 and receptor interacting protein 1 (RIP1) were associated with the Fas-associated death domain (FADD) indicating the formation of an atypical death-inducing signaling complex (DISC). Furthermore, degradation of RIP mediated by the Hsp90 inhibitor geldanamycin significantly impaired YopP-induced cell death. Altogether our findings indicate that Yersinia-induced DC death is independent of death domain containing receptors, but mediated by RIP and caspase-8 at the level of DISC.     

The CD85j+ NK cell subset potently controls HIV-1 replication in autologous dendritic cells

Natural killer (NK) cells and dendritic cells (DC) are thought to play critical roles in the first phases of HIV infection. In this study, we examined changes in the NK cell repertoire and functions occurring in response to early interaction with HIV-infected DC, using an autologous in vitro NK/DC coculture system. We show that NK cell interaction with HIV-1-infected autologous monocyte-derived DC (MDDC) modulates NK receptor expression. In particular, expression of the CD85j receptor on NK cells was strongly down-regulated upon coculture with HIV-1-infected MDDC. We demonstrate that CD85j(+) NK cells exert potent control of HIV-1 replication in single-round and productively HIV-1-infected MDDC, whereas CD85j(-) NK cells induce a modest and transient decrease of HIV-1 replication. HIV-1 suppression in MDCC by CD85j(+) NK cells required cell-to-cell contact and did not appear mediated by cytotoxicity or by soluble factors. HIV-1 inhibition was abolished when NK-MDDC interaction through the CD85j receptor was blocked with a recombinant CD85j molecule, whereas inhibition was only slightly counteracted by blocking HLA class I molecules, which are known CD85j ligands. After masking HLA class I molecules with specific antibodies, a fraction of HIV-1 infected MDDC was still strongly stained by a recombinant CD85j protein. These results suggest that CD85j(+) NK cell inhibition of HIV-1 replication in MDDC is mainly mediated by CD85j interaction with an unknown ligand (distinct from HLA class I molecules) preferentially expressed on HIV-1-infected MDDC.     

Dendritic cells are resistant to apoptosis through the Fas (CD95/APO-1) pathway.

Immunoregulation of lymphocytes and macrophages in the peripheral immune system is achieved in part by activation-induced cell death. Members of the TNF receptor family including Fas (CD95) are involved in the regulation of activation-induced cell death. To determine whether activation-induced cell death plays a role in regulation of dendritic cells (DCs), we examined interactions between Ag-presenting murine DCs and Ag-specific Th1 CD4+ T cells. Whereas mature bone marrow- or spleen-derived DCs expressed high levels of Fas, these DCs were relatively insensitive to Fas-mediated killing by the agonist mAb, Jo-2, as well as authentic Fas ligand expressed on the CD4+ T cell line, A.E7. The insensitivity to Fas-mediated apoptosis was not affected by priming with IFN-gamma and/or TNF-alpha or by blocking the DC survival signals TNF-related activation-induced cytokine and CD40L. However, apoptosis could be induced with C2-ceramide, suggesting that signals proximal to the generation of ceramide might mediate resistance to Fas. Analysis of protein expression of several anti-apoptotic mediators revealed that expression of the intracellular inhibitor of apoptosis Fas-associated death domain-like IL-1-converting enzyme-inhibitory protein was significantly higher in Fas-resistant DCs than in Fas-sensitive macrophages, suggesting a possible role for Fas-associated death domain-like IL-1-converting enzyme-inhibitory protein in DC resistance to Fas-mediated apoptosis. Our results demonstrate that murine DCs differ significantly from other APC populations in susceptibility to Fas-mediated apoptosis during cognate presentation of Ag. Because DCs are most notable for initiation of an immune response, resistance to apoptosis may contribute to this function.     

Maturation-dependent expression and function of the CD49d integrin on monocyte-derived human dendritic cells

Dendritic cells (DC) are highly specialized APC that are critical for the initiation of T cell-dependent immune responses. DC exert a sentinel function while immature and, after activation by inflammatory stimuli or infectious agents, mature and migrate into lymphoid organs to prime T cells. We have analyzed integrin expression on monocyte-derived DC (MDDC) and found that expression of CD49d integrins (CD49d/CD29 and CD49d/beta7) was induced/up-regulated during TNF-alpha- or LPS-initiated MDDC maturation, reflecting the induction/up-regulation of CD49d and beta7 mRNA. CD49d mRNA steady-state level increased more than 10 times during maturation, with the highest levels observed 24 h after TNF-alpha treatment. CD49d integrin expression conferred mature MDDC with an elevated capacity to adhere to the CS-1 fragment of fibronectin, and also mediated transendothelial migration of mature MDDC. Up-regulation of CD49d integrin expression closely paralleled that of the mature DC marker CD83. CD49d integrin expression was dependent on cell maturation, as its induction was abrogated by N:-acetylcysteine, which inhibits NF-kappaB activation and the functional and phenotypic maturation of MDDC. Moreover, CD49d integrin up-regulation and MDDC maturation were prevented by SB203580, a specific inhibitor of p38 mitogen-activated protein kinase, but were almost unaffected by the mitogen-activated protein/extracellular signal-related kinase kinase 1/2 inhibitor PD98059. Our results support the existence of a link between functional and phenotypic maturation of MDDC and CD49d integrin expression, thus establishing CD49d as a maturation marker for MDDC. The differential expression of CD49d on immature and mature MDDC might contribute to their distinct motility capabilities and mediate mature DC migration into lymphoid organs.     

Apoptosis in human embryo development: 2. Cerebellum

We analysed the spatial and temporal distribution of apoptosis in human cerebellum development, during embryonic and fetal periods. Cerebella excised from two human embryos (8 weeks old) and eight fetuses (12-22 weeks old), were paraffin embedded and serially sectioned. Apoptotic cells were identified by propidium iodide staining, and TUNEL. In addition, immunohistochemistry for suicide receptor Fas(APO-1/CD95) was performed. We determined the distribution and percentage of apoptotic cells as well as Fas(APO-1/CD95)-positive cells in different regions and stages of development. Apoptotic cells were seen in both proliferative zones and postmitotic regions along the migratory pathways as well as in the developing cerebellar cortex in all examined stages. The Fas(APO-1/CD95) immunoreactivity was present in all examined stages in a small population of apoptotic cells: either neuroblasts or differentiated cells in postmitotic zones. These findings suggest that apoptosis drives the selection of the cells which are committed to differentiate during the early stages of cerebellar development. The differences between apoptotic cells distribution and Fas receptor expression suggest that cell selection is driven by different apoptotic pathways.     

Activation of CD95 (APO-1/Fas) signaling by ceramide mediates cancer therapy-induced apoptosis.

We report here that anticancer drugs such as doxorubicin lead to induction of the CD95 (APO-1/Fas) system of apoptosis and the cellular stress pathway which includes JNK/SAPKs. Ceramide, which accumulates in response to different types of cellular stress such as chemo- and radiotherapy, strongly induced expression of CD95-L, cleavage of caspases and apoptosis. Antisense CD95-L as well as dominant-negative FADD inhibited ceramide- and cellular stress-induced apoptosis. Fibroblasts from type A Niemann-Pick patients (NPA), genetically deficient in ceramide synthesis, failed to up-regulate CD95-L expression and to undergo apoptosis after gamma-irradiation or doxorubicin treatment. In contrast, JNK/SAPK activity was still inducible by doxorubicin in the NPA cells, suggesting that activation of JNK/SAPKs alone is not sufficient for induction of the CD95 system and apoptosis. CD95-L expression and apoptosis in NPA fibroblasts were restorable by exogenously added ceramide. In addition, NPA fibroblasts undergo apoptosis after triggering of CD95 with an agonistic antibody. These data demonstrate that ceramide links cellular stress responses induced by gamma-irradiation or anticancer drugs to the CD95 pathway of apoptosis.     

Consequences of Fas-mediated human dendritic cell apoptosis induced by measles virus

Mortality from measles virus (MV) infection is caused mostly by secondary infections associated with a pronounced immunosuppression. Dendritic cells (DCs) represent a major target of MV and could be involved in immunosuppression. In this study, human monocyte-derived DCs were used to demonstrate that DC apoptosis in MV-infected DC-T-cell cocultures is Fas mediated, whereas apoptotic T cells could not be rescued by blocking the Fas pathway. Two novel consequences of DC apoptosis after MV infection were demonstrated. (i) Fas-mediated apoptosis of DCs facilitates MV release, while CD40 activation enhances MV replication in DCs. Indeed, detailed studies of infectious MV release and intracellular MV nucleoprotein (NP) showed that inhibition of CD40-CD40L ligand interaction blocks NP synthesis. We conclude that the CD40 ligand expressed by activated T cells first enhances MV replication in DCs, and then Fas ligand produced by activated T cells induces Fas-mediated apoptosis of DCs, thus facilitating MV release. (ii) Not only MV-infected DCs but also bystander uninfected DCs undergo a maturation process confirmed by CD1a, CD40, CD80, CD86, CD83, and major histocompatibility complex type II labeling. The bystander maturation effect results from contact and/or engulfment of MV-induced apoptotic DCs by uninfected DCs. A model is proposed to explain how both a specific immune response and immunosuppression can simultaneously occur after MV infection through Fas-mediated apoptosis and CD40 activation of DCs.     

Mature dendritic cells are protected from Fas/CD95-mediated apoptosis by upregulation of Bcl-XL

The clinical use of dendritic cells (DC) as tumor vaccines is very much dependent on their survival potential. Members of the tumor necrosis factor (TNF) receptor superfamily and their ligands are involved in the regulation of cell death. Fas (CD95) is a representative protein that promotes apoptosis. The Bcl-2 family of proteins functions as an integrator of diverse pro- and anti-apoptotic signals. It has been found that DC maturation facilitates their survival, and thus has an anti-apoptotic function. However, little is known about the underlying mechanisms. We investigated the effects of TNF-alpha and lipopolysaccharide (LPS) on the expression of apoptotic molecules during differentiation and maturation of DC under serum-free conditions, and correlated this to the sensitivity to apoptosis by the Fas-mediated pathway. Indeed, DC activation effectively inhibited DC apoptosis, which was predominantly accompanied by the upregulation of Bcl-X(L) and to a lesser extent Bcl-2, while Bax and FLICE inhibitory protein (FLIP) remained unchanged. In contrast, in the presence of serum FLIP was also upregulated. We conclude that under serum-free conditions, Bcl-X(L) rather than FLIP plays the main role in protection against DC apoptosis.     

Fas-mediated apoptosis of proliferating, transiently growth-arrested, and senescent normal human fibroblasts

Previous studies suggest that apoptotic signaling may require proteins that are critical to cellular proliferation and cell cycle regulation. To further examine this question, proliferating, transiently growth-arrested, and senescent normal human fibroblasts were induced to undergo apoptosis in response to two distinct mediators of apoptosis-Fas (APO-1/CD95) death receptor and staurosporine. Ligation of the Fas receptor in the presence of cycloheximide or actinomycin D resulted in apoptosis of proliferating cells, cells transiently growth arrested by gamma-irradiation or serum starvation (i.e., G(0) arrest), and permanently growth-arrested senescent fibroblasts. Proliferating and G(0)-arrested cells were also susceptible to staurosporine-mediated apoptosis. Surprisingly, gamma-irradiated cells did not undergo staurosporine-mediated apoptosis, and remained viable for a prolonged time. Fas-mediated apoptosis of senescent fibroblasts was evidenced by chromosome condensation and by activation of caspase-8 and -3, proteases crucial for the execution of the Fas apoptosis pathway. In addition, ligation of the Fas receptor in G(0)-arrested cells did not result in the activation of p34(cdc2) kinase, arguing that activation of this kinase is not essential in this apoptotic process. From these studies we conclude that proliferating, transiently growth-arrested, and senescent normal human fibroblasts are susceptible to apoptotic signals and that apoptosis is not necessarily dependent upon cell cycle or proliferative state of the cell.     

Actin integrity is indispensable for CD95/Fas-induced apoptosis of HIV-specific CD8<Superscript>+</Superscript> T cells

We have recently provided data suggesting a potential role for mitochondria and Bcl-2-family molecules in apoptosis sensitivity of HIV-specific CD8⁺ T cells. Here, we report on the role of filamentous (F) actin in this process. Disruption of actin by cytochalasin D (cytD) or lantrunculin A remarkably reduced CD95/Fas-induced apoptosis of HIV-specific CD8⁺ T cells while their spontaneous apoptosis was unaffected. This inhibition cannot be attributed to changes of CD95/Fas distribution or levels in these cells. Furthermore, cytD treatment reduced CD95/Fas-induced apoptosis of CD8⁺ T cells from HIV⁺ patients independently of their differentiation status. CD95/Fas-induced apoptosis of both CD38⁺ and CD38⁻ HIV-specific CD8⁺ T cells was inhibited by cytD treatment indicating that actin mediates this apoptotic process independently of the activation level of these cells. CytD was found to reduce the activation of caspase-8 induced by short treatment of purified CD8⁺ T cells from HIV⁺ patients with anti-CD95/Fas. Our data reveal actin as a critical mediator of HIV-specific CD8⁺ T cell apoptosis; further analysis of the molecular mechanisms governing this process may potentially contribute to design new therapies targeting the enhancement of the immune system in HIV infection.