Heiko Weyd

Resistance of Short Term Activated T Cells to CD95-Mediated Apoptosis Correlates with De Novo Protein Synthesis of c-FLIPshort

In the early phase of an immune response, T cells are activated and acquire effector functions. Whereas these short term activated T cells are resistant to CD95-mediated apoptosis, activated T cells in prolonged culture are readily sensitive, leading to activation-induced cell death and termination of the immune response. The translation inhibitor, cycloheximide, partially overcomes the apoptosis resistance of short term activated primary human T cells. Using this model we show in this study that sensitization of T cells to apoptosis occurs upstream of mitochondria. Neither death-inducing signaling complex formation nor expression of Bcl-2 proteins is altered in sensitized T cells. Although the caspase-8 inhibitor c-FLIPlong was only slightly down-regulated in sensitized T cells, c-FLIPshort became almost undetectable. This correlated with caspase-8 activation and apoptosis. These data suggest that c-FLIPshort, rather than c-FLIPlong, confers resistance of T cells to CD95-mediated apoptosis in the context of immune responses.

The active caspase-8 heterotetramer is formed at the CD95 DISC.

Caspase-8 plays an important role in the CD95 (APO-1/ Fas) signalling pathway. It is activated at the CD95 DISC. The mechanism of caspase-8 activation at the DISC is in the focus of intensive studies. It is well established that binding of procaspases-8/a and -8/b to the DISC results in autocatalytic cleavage via a two-step mechanism (Figure 1a). The initial cleavage at Asp generates two cleavage intermediates p43/p41 and p12. Subsequently, p12 is rapidly converted to p10. Afterwards, an additional cleavage occurs at Asp, producing the active enzyme subunit p18 and the inactive p26/p24 prodomain. The DED-containing cleavage products p43/p41 and p26/p24 were reported to be present in the DISC in previous biochemical studies. We analysed in more detail all cleavage products of procaspase-8 in the DISC formed upon stimulation of the Blymphoblastoid cell line SKW6.4. Cells were treated with LZCD95L for different time points, followed by immunoprecipitation of the DISC components with the agonistic anti-APO-1 and by immunoblotting with a panel of mAbs. The epitopes recognised by these antibodies are shown in Figure 1a. In addition to the established components of the DISC, including full-length procaspase-8 (p55/p53) and its cleavage product p43/p41, we clearly detected the p18 and p10 subunits of procaspase-8 in the DISC (Figure 1b). The presence of p10 and p18 at the DISC was also observed using other T-cell lines Hut78, CEM, H9 as well as primary T cells (data not shown). The detection of these products became possible because we used different conditions for electrophoresis, focusing on lower molecular weight products in comparison to previous studies. In addition, the presence of the p10 subunit was detected using the C5 antibody that had not been used in the analysis of the lower molecular weight procaspase-8 subunits in the CD95 DISC before. The kinetic analysis in Figure 1b demonstrated that p10 and p18 are detectable at the CD95 DISC within 30 s after stimulation. The amount of both p10 and p18 at the DISC reached its maximum 1–5 min after receptor engagement. After 10 min, the intensity of the bands corresponding to p10 and p18 started to decrease and after 20 min, the intensity of bands was markedly reduced. The kinetic analysis performed with lysates of SKW6.4 cells (Figure 1b) showed the same main features as reported earlier. P10 and p18 were detected in the cytosol 10 min after the receptor stimulation, and their presence reached a maximum after 20 min, exactly at the time when their amount in the DISC was found to be decreased. To ensure that the p10 and p18 subunits are components of the DISC under different stimulation conditions, SKW6.4 were stimulated in parallel with LZ-CD95L and anti-APO-1 antibody. We observed the presence of p10 and p18 in the DISC in both cases (data not shown). To exclude the possibility that p10 and p18 subunits are generated during the washing steps, DISC formation and consecutive immunoprecipitation experiments were performed in the presence of the caspase inhibitor zVAD-fmk. Under these conditions, we could also detect active caspase-8 subunits at the DISC (data not shown). Additionally, we addressed the question of whether the caspase-8 subunits generated at the DISC were functionally active and did not require additional cytosolic processing and can cleave caspase-8 substrates. Thus, in vitro translated, [S]-labelled procaspase-3 was added to protein-A sepharose containing CD95 immunoprecipitates from unstimulated and stimulated cells. Incubation of protein-A sepharose beads with caspase-3 resulted in the cleavage of procaspase-3 to p20, p19, p17 and p12 subunits only in the stimulated case (Figure 1c, lane 3). Cleavage was blocked in the presence of the specific inhibitor of caspase-8 IETD-fmk (Figure 1c, lane 4). To ensure that autocatalytic processing of procaspase-3 does not blur our results in these experiments, we performed a similar assay with procaspase-3 carrying a mutation in the catalytic site (C163S). This mutant was supposed to be a substrate of caspase-8, but it excludes the formation of any cleavage product as a result of genuine caspase-3 proteolytic activity. In the presence of immunoprecipitates from the CD95 DISC, we observed only the first cleavage step with the formation of the p20 subunit (Figure 1c, lane 7). No further processing to the p17 subunit was detected. This observation is in accordance with published data indicating that the generation of the mature p17–p12 enzyme of caspase-3 only occurs autocatalytically. Thus, the caspase-3 mutant C163S is catalytically inactive in our in vitro system. Cleavage was not observed in the presence of IETD-fmk (Figure 1c, lane 8), as well as in the case of immunoprecipitates from unstimulated cells (Figure 1c, lane 6). These data point out that the cleavage of caspase-3 results from the catalytic activity of the caspase-8 heterotetramer formed at the DISC. In previous studies, it has been suggested that formation of the tetramer takes place in the cytosol. In our view, this is a less likely event than the interaction between subunits remaining bound to the DISC. Thus, the DISC not only brings the molecules of procaspase-8 into close proximity to each other, but it also places them in a spatial orientation favourable for their activation. A number of biochemical studies provide evidence that the activation of procaspase-8 occurs via its dimerisation or oligomerisation. The conditions for the dimer formation and the following cleavage steps must be provided by the spatial structure of the DISC. However, further Cell Death and Differentiation (2003) 10, 144–145 & 2003 Nature Publishing Group All rights reserved 1350-9047/03

Photosensitivity, Apoptosis, and Cytokines in the Pathogenesis of Lupus Erythematosus: a Critical Review

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Comparative analysis of protocols to induce human CD4+Foxp3+ regulatory T cells by combinations of IL‐2, TGF‐beta, retinoic acid, rapamycin and butyrate

The underlying pathomechanisms of lupus erythematosus (LE), a multifactorial autoimmune disease, remain elusive. Due to the clinical evidence demonstrating a clear relationship between ultraviolet (UV) light exposure and skin lesions of LE, photosensitivity has been proven to be an important factor in the pathogenesis of the disease. Standardised photoprovocation with UVA and UVB irradiation has been shown to be a reliable model for evaluating photosensitivity in patients with cutaneous LE (CLE) and analysing the underlying medical conditions of the disease. In this respect, UV irradiation can cause aberrant induction of apoptosis in keratinocytes and contribute to the appearance of excessive apoptotic cells in the skin of CLE patients. Moreover, apoptotic cells that cannot be cleared by phagocytes may undergo secondary necrosis and release proinflammatory compounds and potential autoantigens, which may contribute to the inflammatory micromilieu that leads to formation of skin lesions in the disease. In addition to UV-mediated induction of apoptosis, the molecular and cellular factors that may cause the abnormal long-lasting photoreactivity in CLE include mediators of inflammation, such as cytokines and chemokines. In particular, interferons (IFNs) are important players in the early activation of the immune system and have a specific role in the immunological interface between the innate and the adaptive immune system. The fact that treatment with recombinant type I IFNs (α and β) can induce not only systemic organ manifestations but also LE-like skin lesions provides additional evidence for a pathogenetic role of these IFNs in the disease.

Annexin A1 on the Surface of Early Apoptotic Cells Suppresses CD8 + T Cell Immunity

Regulatory T cells (Tregs) suppress other immune cells and are critical mediators of peripheral tolerance. Therapeutic manipulation of Tregs is subject to numerous clinical investigations including trials for adoptive Treg transfer. Since the number of naturally occurring Tregs (nTregs) is minute, it is highly desirable to develop a complementary approach of inducing Tregs (iTregs) from naïve T cells. Mouse studies exemplify the importance of peripherally induced Tregs as well as the applicability of iTreg transfer in different disease models. Yet, procedures to generate iTregs are currently controversial, particularly for human cells. Here we therefore comprehensively compare different established and define novel protocols of human iTreg generation using TGF-β in combination with other compounds. We found that human iTregs expressed several Treg signature molecules, such as Foxp3, CTLA-4 and EOS, while exhibiting low expression of the cytokines Interferon-γ, IL-10 and IL-17. Importantly, we identified a novel combination of TGF-β, retinoic acid and rapamycin as a robust protocol to induce human iTregs with superior suppressive activity in vitro compared to currently established induction protocols. However, iTregs generated by these protocols did not stably retain Foxp3 expression and did not suppress in vivo in a humanized graft-versus-host-disease mouse model, highlighting the need for further research to attain stable, suppressive iTregs. These results advance our understanding of the conditions enabling human iTreg generation and may have important implications for the development of adoptive transfer strategies targeting autoimmune and inflammatory diseases.

Interferon-α Abrogates the Suppressive Effect of Apoptotic Cells on Dendritic Cells in an In Vitro Model of Systemic Lupus Erythematosus Pathogenesis

Prevention of an immune response against self-antigens derived from apoptotic cells is essential to preclude autoimmune and chronic inflammatory diseases. Here, we describe apoptosis induced externalization of endogenous cytosolic annexin 1 initiating an anti-inflammatory effector mechanism that suppresses the immune response against antigens of apoptotic cells. Cytosolic annexin 1 rapidly translocated to the apoptotic cell surface and inhibited dendritic cell (DC) activation induced by Toll like receptors (TLR). Annexin 1-inhibited DC showed strongly reduced secretion of pro-inflammatory cytokines (e.g. TNF and IL-12) and costimulatory surface molecules (e.g. CD40 and CD86), while anti-inflammatory mediators like PD-L1 remained unchanged. T cells stimulated by such DC lacked secretion of interferon-γ (IFN-γ) and TNF but retained IL-10 secretion. In mice, annexin 1 prevented the development of inflammatory DC and suppressed the cellular immune response against the model antigen ovalbumin (OVA) expressed in apoptotic cells. Furthermore, annexin 1 on apoptotic cells compromised OVA-specific tumor vaccination and impaired rejection of an OVA-expressing tumor. Thus, our results provide a molecular mechanism for the suppressive activity of apoptotic cells on the immune response towards apoptotic cell-derived self-antigens. This process may play an important role in prevention of autoimmune diseases and of the immune response against cancer.

The tolerogenic function of annexins on apoptotic cells is mediated by the annexin core domain

Objective. An increased incidence of apoptotic cells and an increased activation of dendritic cells (DC) may be involved in the pathogenesis of systemic lupus erythematosus (SLE). We investigated the characteristics of apoptotic neutrophils and monocyte-derived DC of patients with SLE, their interaction, and the influence of autoantibodies and inflammatory cytokines on this interaction. Methods. Kinetics of neutrophil apoptosis and DC activation were studied by flow cytometry. To analyze the interaction of apoptotic cells with phagocytes, crossover coculture experiments were performed with DC from patients with SLE and apoptotic Jurkat T cells as well as with apoptotic neutrophils from patients with SLE and the monocytic cell line U937. SLE serum and cytokines were added to this coculture, and activation and suppression of DC were quantified by levels of inflammatory cytokine secretion. Results. Apoptotic neutrophils and DC from patients with SLE showed no inherent defects compared to healthy controls, and the suppressive nature of their interaction was not affected. Autoantibodies as well as the inflammatory cytokines interleukin 17 (IL-17) and IL-1β had no influence on the interaction in this setup. Interferon (IFN)-α, however, substantially reduced the suppressive effect of apoptotic cells on DC. Conclusion. The data suggest that aberrant immune reactivity in SLE is not generally due to an intrinsic defect in apoptotic cells, their processing, or their interaction with DC, but likely arises from the milieu in which this interaction takes place. Our study highlights the importance of IFN-α during early stages of SLE and its potential as a therapeutic target.

Life, death and tolerance

Immunological tolerance is constantly being maintained in the periphery by dendritic cells processing material from apoptotic cells (ACs) in the steady-state. Although research has focused on the uptake of ACs by phagocytes, tolerogenic signals exposed by the ACs are much less well defined. In this article, we show that the annexin (Anx) family members AnxA5 and AnxA13 translocate to the surface of ACs to function as redundant tolerogenic signals in vitro and in vivo. Exposure of bone marrow–derived dendritic cells to AnxA5 or AnxA13 in vitro resulted in the inhibition of both proinflammatory cytokine secretion and the upregulation of costimulatory molecules upon TLR stimulation. The highly conserved Anx core domain was sufficient to mediate these effects, whereas recognition by N-formyl peptide receptor family members was dispensable. In vivo, coinjection of OVA-expressing and Anx-expressing ACs prevented induction of Ag-specific CD8+ T cells. Moreover, mice immunized with Anx-expressing ACs became refractory to an antigenic challenge. These results suggest that several Anxs contribute to AC-induced suppression of dendritic cell activation. Therefore, manipulating Anx-mediated immunosuppression may prove beneficial for patients with cancer or autoimmune diseases and chronic inflammatory disorders.

Antibodies against annexins, use thereof for therapy and diagnosis. use of annexins for therapy and diagnosis.

• CD95 triggers death as well as non-death activities - e.g. proliferation, invasion and inflammation.