Sabine Jakob

Intestinal Epithelial Cells Synthesize Glucocorticoids and Regulate T Cell Activation

Glucocorticoids (GCs) are important steroid hormones with widespread activities in metabolism, development, and immune regulation. The adrenal glands are the major source of GCs and release these hormones in response to psychological and immunological stress. However, there is increasing evidence that GCs may also be synthesized by nonadrenal tissues. Here, we report that the intestinal mucosa expresses steroidogenic enzymes and releases the GC corticosterone in response to T cell activation. T cell activation causes an increase in the intestinal expression of the steroidogenic enzymes required for GC synthesis. In situ hybridization analysis revealed that these enzymes are confined to the crypt region of the intestinal epithelial layer. Surprisingly, in situ–produced GCs exhibit both an inhibitory and a costimulatory role on intestinal T cell activation. In the absence of intestinal GCs in vivo, activation by anti-CD3 injection resulted in reduced CD69 expression and interferon-γ production by intestinal T cells, whereas activation by viral infection led to increased T cell activation. We conclude that the intestinal mucosa is a potent source of immunoregulatory GCs.

Fas (CD95/Apo-1) ligand regulation in T cell homeostasis, cell-mediated cytotoxicity and immune pathology

Members of the tumor necrosis factor (TNF) superfamily are crucially involved in the regulation of T cell activation, homeostasis and cytotoxicity. In particular, Fas ligand (FasL), expressed by activated T lymphocytes, induces cell-mediated cytotoxicity and may also be responsible for apoptotic suicide. Tight regulation of this death-inducing ligand is a prerequisite for proper immune defense and homeostasis. In this review, we will discuss various aspects of FasL regulation in cell-mediated cytotoxicity, immune homeostasis and the immunopathology of diseases.

The nuclear receptor LRH-1 critically regulates extra-adrenal glucocorticoid synthesis in the intestine

The nuclear receptor liver receptor homologue-1 (LRH-1, NR5A2) is a crucial transcriptional regulator of many metabolic pathways. In addition, LRH-1 is expressed in intestinal crypt cells where it regulates the epithelial cell renewal and contributes to tumorigenesis through the induction of cell cycle proteins. We have recently identified the intestinal epithelium as an important extra-adrenal source of immunoregulatory glucocorticoids. We show here that LRH-1 promotes the expression of the steroidogenic enzymes and the synthesis of corticosterone in murine intestinal epithelial cells in vitro. Interestingly, LRH-1 is also essential for intestinal glucocorticoid synthesis in vivo, as LRH-1 haplo-insufficiency strongly reduces the intestinal expression of steroidogenic enzymes and glucocorticoid synthesis upon immunological stress. These results demonstrate for the first time a novel role for LRH-1 in the regulation of intestinal glucocorticoid synthesis and propose LRH-1 as an important regulator of intestinal tissue integrity and immune homeostasis.

TRAIL-induced apoptosis : between tumor therapy and immunopathology

The death ligand members of the tumor necrosis factor (TNF) family are potent inducers of apoptosis in a variety of cell types. In particular, TNF‐related apoptosis‐inducing ligand (TRAIL) has recently received much scientific and commercial attention because of its potent tumor cell‐killing activity while leaving normal untransformed cells mostly unaffected. Furthermore, TRAIL strongly synergizes with conventional chemotherapeutic drugs in inducing tumor cell apoptosis, making it a most promising candidate for future cancer therapy. Increasing evidence indicates, however, that TRAIL may also induce or modulate apoptosis in primary cells. A particular concern is the potential side effect of TRAIL‐based tumor therapies in the liver. In this review we summarize some of the recent findings on the role of TRAIL in tumor cell and hepatocyte apoptosis.

Detection of apoptosis in vivo using antibodies against caspase-induced neo-epitopes

Cell death induction by apoptosis is an important process in the maintenance of tissue homeostasis as well as tissue destruction during various pathological processes. Consequently, detection of apoptotic cells in situ represents an important technique to assess the extent and impact of cell death in the respective tissue. While scoring of apoptosis by histological assessment of apoptotic cells is still a widely used method, it is likely biased by sensitivity problems and observed-based variations. The availability of caspase-mediated neo-epitope-specific antibodies offers new tools for the detection of apoptosis in situ. Here, we discuss the use of immunohistochemical detection of cleaved caspase 3 and lamin A for the assessment of apoptotic cells in paraffin-embedded liver tissue. Furthermore, we evaluate the effect of tissue pretreatment and antigen retrieval on the sensitivity of apoptosis detection, background staining and maintenance of tissue morphology.

Regulation of Activation-induced Fas (CD95/Apo-1) Ligand Expression in T Cells by the Cyclin B1/Cdk1 Complex

Fas (CD95/Apo-1) ligand-mediated apoptosis has been recognized as an important mechanism of cell-mediated cytotoxicity and maintenance of immune homeostasis. Chronically activated T cells undergo activation-induced cell death (AICD), which depends on simultaneous Fas and Fas ligand expression. Previous reports have suggested that AICD might be linked to cell cycle progression of T cells and therefore to the expression of cell cycle-related molecules. In particular, cyclin B1 has been implicated in the induction of AICD in T cells. In this study, we have investigated the role of cyclin B1 in AICD and the expression of effector molecules involved in this form of cell death. Our results show that inhibition of cyclin B1 blocks AICD in T cells through specific inhibition of Fas ligand expression but not Fas-induced apoptosis. This effect of cyclin B1 appears to be mediated through the cyclin B1/cyclin-dependent kinase 1 (Cdk1/Cdc2) complex because overexpression of cyclin B1 enhances FasL promoter activity, whereas a dominant-negative version of Cdk1 blocks Fas ligand promoter induction. We provide further evidence that cyclin B1/Cdk1 regulates FasL transcription through the regulation of NFκB activation because dominant-negative Cdk1 inhibits activation-induced NFκB reporter and Rel A-induced FasL promoter activity. In conclusion, our data support a link between cell cycle progression, activation-induced Fas ligand expression, and apoptosis in T cells.

Sensitizing antigen-specific CD8 + T cells for accelerated suicide causes immune incompetence

Death receptor-mediated activation-induced apoptosis of antigen-specific T cells is a major mechanism of peripheral tolerance induction and immune homeostasis. Failure to undergo activation-induced cell death (AICD) is an important underlying cause of many autoimmune diseases. Thus, enhancing the T cells own suicide mechanism may provide an efficient therapy for the treatment of autoimmune diseases. Bisindolylmaleimide VIII (Bis VIII), a PKC inhibitor, can sensitize T cells for death receptor-induced apoptosis and thus can inhibit the development of T cell-mediated autoimmune disease in vivo. In this study, we have analyzed the functional consequences of accelerated suicide for a protective CD8+ T cell-mediated immune response. Our data indicate that CD8+ T cells are sensitized by Bis VIII to AICD, both in vitro and in vivo. The sensitizing effect of Bis VIII appears to be mediated by specific downmodulation of the antiapoptotic molecule cellular FLICE-like inhibitory protein (cFLIP(L)). Importantly, Bis VIII administration during an acute lymphocytic choriomeningitis virus (LCMV) infection causes the depletion of virus-specific CD8+ T cells and subsequently impaired cytotoxicity and virus clearance. We conclude that resistance to death receptor-induced apoptosis is crucial for the efficient induction of a protective immune response, and that Bis VIII-based immunotherapies have to be applied under well-controlled conditions to avoid the induction of immune incompetence and the inability to respond to pathogen infection.

Active caspase 3 and DNA fragmentation as markers for apoptotic cell death in primary and metastatic liver tumours

Aims: The induction of tumour cell death by apoptosis is a major goal of cancer therapy and the in situ detection of apoptosis in tumour tissue has become an important diagnostic parameter. Different apoptosis detection methods assess distinct biochemical processes in the dying cell. Thus, their direct comparison is mandatory to evaluate their diagnostic value. The aim of this study was to compare the immunohistochemical detection of active caspase 3 and single‐stranded DNA in primary and metastatic liver tumours as markers of apoptotic cell death. Methods: We studied detection of active caspase 3 and single‐stranded DNA in 20 primary hepatocellular carcinomas (HCC) and 20 liver metastases from colorectal carcinomas (CRC) using immunohistochemistry on paraffin sections. Results: Our results reveal that both methods are suitable and sensitive techniques for the in situ detection of apoptosis, however, they also demonstrate that immunohistochemistry for active caspase 3 and single‐stranded DNA have differential sensitivities in HCC and CRC. Conclusion: The sensitivity of apoptosis detection using immunohistochemistry for active caspase 3 and single‐stranded DNA may be tumour cell type dependent.

TRAIL and thymocyte apoptosis: not so deadly?

Death receptor/death ligand interactions have been recognized as important triggers of cell death in various cell types. So far, best understood are the signaling events that are activated upon binding of the Fas ligand to the Fas (CD95) receptor, and subsequently lead to apoptosis in the target cell. Fas-mediated apoptosis plays a crucial role in many physiological and pathophysiological events, such as immune homeostasis, immune privilege, tissue turnover, and cellmediated cytotoxicity. Similar to Fas ligand, TRAIL/Apo-2 ligand (Tumor Necrosis Factor-related apoptosis-inducing ligand) has been proposed to represent an important cytotoxic effector molecule, by which cytotoxic T cells and natural killer cells eliminate their target cells. TRAIL has particularly received strong scientific attention due to its proposed role in tumor immune surveillance and its potential therapeutic application in cancer patients. Apart from tumor immunity, however, TRAIL has also been implicated in various other aspects of immune cell regulation. For example, neutralization of TRAIL causes an exacerbation of experimental rheumatoid arthritis, autoimmune encephalomyelitis and diabetes, suggesting that TRAIL-induced apoptosis or effector cell modulation represents an important regulatory process in the pathogenesis of autoimmune diseases. Recently, Lamhamedi-Cherradi et al. have proposed TRAIL/TRAIL receptor-mediated cell death as an underlying mechanism of thymic negative selection. Thymocytes from TRAIL-deficient mice were found to be more resistant to antigenor CD3/T cell receptor (TCR)-mediated apoptosis, and soluble TRAIL receptor was reported to block thymic cell death. This finding, however, is quite controversial and has therefore been questioned by other investigators. For example, Simon et al. have not been able to block thymic negative selection by soluble TRAIL receptor, and Cretney et al. have found normal antigen-driven negative selection in the very same TRAIL-deficient mice. Furthermore, CD3/TCRmediated apoptosis in vivo has been reported to require the release of systemic glucocorticoids and may thus not necessarily reflect the biology of negative selection. Particularly incompatible with this proposed role of TRAIL in thymic negative selection is the observation that the adaptor molecule FADD (Fas-associated death domain) is a crucial element in the TRAIL-induced signaling pathways leading to apoptosis, however, overexpression of dominant-negative FADD does not alter thymic negative selection. Thus, the role of TRAIL–TRAIL receptor interaction in the establishment of central tolerance is far from being well accepted. The different publications describing TRAIL or TRAIL-R expression in the thymus, or their involvement in thymic negative selection are summarized in Table 1. They demonstrate that TRAIL and TRAIL receptor are expressed in the thymus and thymocytes, and that thymocytes are sensitive to TRAIL, yet come to different conclusions regarding the role of TRAIL–TRAIL receptor interaction in TCR-induced thymocyte apoptosis. The discrepancy between these different reports is difficult to reconcile and may partially depend on the different experimental systems used. We have thus reinvestigated the role of TRAIL in activation-induced apoptosis in thymocytes using TRAIL-deficient mice, and have extended our experiments to other apoptosis triggers. Both, in vitro and in vivo activation of thymocytes by CD3/ TCR ligation using anti-CD3e-crosslinking antibodies caused massive induction of apoptosis in CD4þCD8þ immature thymocytes (Figure 1a and b). Importantly, double-positive thymocytes from wild type and TRAIL-deficient mice expressed low but detectable levels of TRAIL-R (as assessed by flow cytometry), which became upregulated upon stimulation with antiCD3 (data not shown). In agreement with the finding of Lamhamedi-Cherradi et al., we found a significant inhibition of anti-CD3-induced apoptosis in TRAIL-deficient thymocytes, in vitro as well as in vivo. This would indicate that TRAIL might play an important role in CD3/TCR-mediated thymocyte apoptosis and possibly also negative selection. Death ligand/death receptor interactions are not only involved in activation-induced apoptosis and cell-mediated cytotoxicity, but have also been implicated in apoptosis induction upon DNA damage. DNA damage may induce the expression of death receptors and also death ligands through p53and/orJun kinase/NFkB-dependent mechanisms. Since thymocytes are exquisitely sensitive to DNA damage, we analyzed thymocyte apoptosis in response to UV and girradiation in vitro and in vivo as well as other cell death triggers, that is, the glucocorticoid dexamethasone and Fas ligation in TRAIL-deficient and control mice. Surprisingly, we observed that UVand g-irradation as well as glucocorticoid-induced apoptosis was significantly attenuated in TRAIL-deficient thymocytes. In marked contrasts, however, induction of Fas receptor-mediated apoptosis occurred with similar kinetics in wild type and TRAIL-deficient thymocytes (Figure 1a and b). Apoptosis resistance of TRAIL-deficient thymocytes does thus not appear to be restricted to CD3/TCR-mediated apoptosis, but extends to other apoptosis triggers, with the exception of Fas. While it is conceivable that DNA damage-induced apoptosis in thymocytes may involve TRAIL/TRAIL receptor interaction, there is no evidence so far that glucocorticoids signal via TRAIL. So far, the key mediators of glucocorticoid-induced Cell Death and Differentiation (2004) 11, S213–S215 & 2004 Nature Publishing Group All rights reserved 1350-9047/04

Phenotyping, functional characterization, and developmental changes in canine intestinal intraepithelial lymphocytes

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