Thomas Heinemann

Prevention of Acute Allograft Rejection by Antibody Targeting of TIRC7, a Novel T Cell Membrane Protein

A novel 75 kDa membrane protein, TIRC7, is described that exhibits a central role in T cell activation in vitro and in vivo. Modulation of TIRC7-mediated signals with specific anti-TIRC7 antibodies in vitro efficiently prevents human T cell proliferation and IL-2 secretion. Moreover, anti-TIRC7 antibodies specifically inhibit type 1 subset specific IFN-gamma expression but spare the type 2 cytokine IL-4. Diminished proliferation but not IFN-gamma secretion is reversible by exogenous rIL-2. An anti-TIRC7 antibody that cross-reacts with the 75 kDa rat homolog exhibits inhibition of rat alloimmune response in vitro and significantly prolongs kidney allograft survival in vivo. Targeting of TIRC7 may provide a novel therapeutic approach for modulation of the immune response.

cis-4-Methylsphingosine Decreases Sphingolipid Biosynthesis by Specifically Interfering with Serine Palmitoyltransferase Activity in Primary Cultured Neurons

The effect of six different structurally modified sphingosine analogues on biosynthesis of sphingolipids was studied in primary cultured murine cerebellar neurons. Treatment of cells withcis-4-methylsphingosine at micromolar levels resulted in a markedly decreased sphingolipid biosynthesis, whereas the other compounds examined, trans-4-methylsphingosine,cis-5-methylsphingosine,trans-5-methylsphingosine, cis-sphingosine, and 1-deoxysphingosine, inhibited sphingolipid biosynthesis less efficiently. The inhibition of sphingolipid biosynthesis by the various compounds was paralleled by a decrease of serine palmitoyltransferase activity in situ. For cis-4-methylsphingosine the inhibitory effect on serine palmitoyltransferase activity was shown to be concentration- and time-dependent. Half-maximal reduction of enzyme activity occurred after 24 h of treatment with 10 μm of the compound. The activity of other enzymes of sphingolipid biosynthesis as well as phospholipid and protein biosynthesis was not affected. Analysis of the sphingoid moiety of cellular sphingolipids suggests that the sphingosine analogues listed above were subject to degradation rather than being utilized as precursors for sphingolipid biosynthesis by cultured neurons. Except of 1-deoxysphingosine, the other five sphingosine analogues were shown to be substrates for sphingosine kinase in vitro. After 24 h of treatment of primary cerebellar neurons with the various sphingosine analogues the relative percentage of the respective intracellular 1-phosphate derivatives paralleled exactly the inhibitory effect on serine palmitoyltransferase activity observed when cells were treated with the unphosphorylated compounds. In contrast to the respective 1-phosphate derivatives of the other methyl-branched sphingosine analogues examined,cis-4-methylsphingosine 1-phosphate showed an intracellular accumulation suggesting a delayed turnover rate in cultured murine neurons for this compound. These results suggest that the inhibitory effect of the sphingosine analogues on serine palmitoyltransferase is mediated by their respective 1-phosphate derivatives and that the pronounced effect of cis-4-methylsphingosine is caused by a high intracellular concentration ofcis-4-methylsphingosine 1-phosphate.cis-4-Methylsphingosine, in addition, caused drastic changes in cell morphology of primary cerebellar neurons, which were not observed when these cells were treated with one of the other sphingosine analogues examined.

Principles of Ethical Decision Making Regarding Embrionic Stem Cell Research in Germany

The availability of embryonic stem (ES) cells isolated from human blastocysts may open novel avenues for medical treatment of otherwise incurable diseases. Yet the generation of human ES cells requires the destruction of early human embryos. This confronts us with the moral problem of whether it is justifiable to sacrifice human life in order to treat other human life. This article outlines the development of the German debate about research with ES cells and explicates the arguments that are central to that debate with respect to the aims and means of research with ES cells. With regard to the means, the isolation of ES cells from human embryos raises the question of the moral status of the human embryo. A restrictive position acknowledges the human dignity of the embryo in its very early stage of development and claims that the embryos life must be protected accordingly. In contrast, a gradualist position acknowledges human dignity, and therefore the full level of protection, only when the embryo has reached a certain stage of development. In addition, the intentions behind the generation of human embryos, i.e. exclusively for research purposes, and the mode of generating them, i.e. by nuclear transfer technology, have strong ethical relevance in the German debate. Based on these results, the ethical reasoning underlying the draft of a Stem Cell Act recently passed by the German Parliament is outlined.

TIRC7 Deficiency Causes In Vitro and In Vivo Augmentation of T and B Cell Activation and Cytokine Response

The membrane protein T cell immune response cDNA 7 (TIRC7) was recently identified and was shown to play an important role in T cell activation. To characterize the function of TIRC7 in more detail, we generated TIRC7-deficient mice by gene targeting. We observed disturbed T and B cell function both in vitro and in vivo in TIRC7−/− mice. Histologically, primary and secondary lymphoid organs showed a mixture of hypo-, hyper-, and dysplastic changes of multiple lymphohemopoietic compartments. T cells from TIRC7−/− mice exhibited significantly increased proliferation and expression of IL-2, IFN-γ, and IL-4 in response to different stimuli. Resting T cells from TIRC7−/− mice exhibited decreased CD62L, but increased CD11a and CD44 expression, suggesting an in vivo expansion of memory/effector T cells. Remarkably, activated T cells from TIRC7−/− mice expressed lower levels of CTLA-4 in comparison with wild-type cells. B cells from TIRC7-deficient mice exhibited significantly higher in vitro proliferation following stimulation with anti-CD40 Ab or LPS plus IL-4. B cell hyperreactivity was reflected in vivo by elevated serum levels of various Ig classes and higher CD86 expression on B cells. Furthermore, TIRC7 deficiency resulted in an augmented delayed-type hypersensitivity response that was also reflected in increased mononuclear infiltration in the skin obtained from TIRC7-deficient mice food pads. In summary, the data strongly support an important role for TIRC7 in regulating both T and B cell responses.

TIRC7 Inhibits T Cell Proliferation by Modulation of CTLA-4 Expression

Ab targeting of TIRC7 has been shown previously to inhibit T cell proliferation and Th1 lymphocyte-associated cytokine production. In this study, we demonstrate that Ab targeting of TIRC7 induces early cell surface expression of CTLA-4. The majority of stimulated CD4+ and CD8+ human T cells coexpress CTLA-4 and TIRC7. Similar to CTLA-4, TIRC7 rapidly accumulates at the site of Ag adhesion upon T cell activation. TIRC7 seems to colocalize with CTLA-4 in human T cells, and both molecules are associated with clathrin-coated vesicles, indicating they share intracellular transport systems. Moreover, Ab targeting of TIRC7 results in an early activation of CTLA-4 transcription. The inhibition of cell proliferation mediated by TIRC7 is dependent on CTLA-4 expression because the TIRC7-mediated inhibitory effects on cell proliferation and cytokine expression are abolished by Ab blockade of CTLA-4. Splenocytes obtained from CTLA-4-deficient mice are not responsive to TIRC7 Ab targeting. Thus, TIRC7 acts as an upstream regulatory molecule of CTLA-4 expression.

Cis-4-methylsphingosine phosphate induces apoptosis in neuroblastoma cells by opposite effects on p38 and ERK mitogen-activated protein kinases.

Abstract Intracellular phosphorylation of cis-4-methylsphingosine was previously shown to result in a metabolically stable compound that accumulates in Swiss 3T3 fibroblasts and mimics the mitogenic effect induced by the shortlived sphingosine metabolite, sphingosine 1-phosphate. In the present study incubation of neuroblastoma B104 cells with cis-4-methylsphingosine (10 M) also resulted in an intracellular accumulation of its phosphorylated derivative that was, however, associated with the concentrationdependent induction of apoptosis, not observed after treatment with 10 M of sphingosine-1-phosphate or sphingosine, respectively. In B104 cells, cis-4-methylsphingosine stimulated p38 mitogenactivated protein kinase (p38 MAPK) and simultaneously inhibited extracellular signalregulated kinase (ERK), whereas sphingosine and sphingosine 1-phosphate only stimulated p38 MAPK without suppression of ERK. Inhibition of cis-4-methylsphingosine phosphorylation reduced both, apoptosis and concurrent regulation of mitogenactivated protein kinases (MAPKs), suggesting that the unusual accumulation of the phosphorylated sphingoid base was responsible for the biological effects. Furthermore, inhibition of p38 MAPK prevented cis-4-methylsphingosineinduced apoptosis, while suppression of the ERK pathway in the presence of sphingosine or sphingosine-1-phosphate resulted in apoptosis, indicating that the simultaneous opposite regulation of the two MAPKs was required for the induction of apoptosis.

Phosphorylated cis-4-Methylsphingosine Mimics the Mitogenic Effect of Sphingosine-1-phosphate in Swiss 3T3 Fibroblasts

The phosphorylated derivative of sphingosine, sphingosine-1-phosphate, is a short-living metabolite of ultimate ceramide degradation and was shown to act as an intracellular signaling molecule, stimulating cell proliferation in quiescent Swiss 3T3 fibroblasts and inducing the release of calcium from intracellular stores (Zhang, H., Desai, N. N., Olivera, A., Seki, T., Brooker, G., and Spiegel, S. (1991) J. Cell. Biol. 114, 155–167). In the present study, 24-h treatment of Swiss 3T3 fibroblasts with the synthetic sphingosine analoguecis-4-methylsphingosine resulted in proliferation of quiescent Swiss 3T3 fibroblasts that was 3-fold stronger than that of equimolar sphingosine-1-phosphate. The phosphorylated derivative ofcis-4-methylsphingosine accumulated drastically in the cells. Simultaneous treatment with the sphingosine kinase inhibitorl-threo-sphinganine reduced both the amount of phosphorylated cis-4-methylsphingosine and cell proliferation induced by this compound by about 50%, indicating that the phosphorylated derivative mediated the proliferative stimulus. The mitogenic effect of cis-4-methylsphingosine was associated with a mobilization of intracellular calcium in Swiss 3T3 fibroblasts that was similar to that induced by sphingosine-1-phosphate. The results demonstrate that the phosphorylated derivative ofcis-4-methylsphingosine mimics the previously reported mitogenic action of sphingosine-1-phosphate in Swiss 3T3 cells, and the stronger effect most likely corresponds to the unusual accumulation of this compound.

Antibody targeting of TIRC7 results in significant therapeutic effects on collagen-induced arthritis in mice

TIRC7 is a cell surface molecule which is expressed in T and B lymphocytes and negatively regulates their function. Anti‐TIRC7 specific monoclonal antibody (mAb) inhibited T cell memory response to recall antigens. Up‐regulation of TIRC7 on lymphocytes from joint tissue of patients with Rheumatoid Arthritis (RA) and mice with collagen induced arthritis (CIA) suggested TIRC7 as a novel target to promote anti‐inflammatory reaction. Anti‐TIRC7 mAb administration significantly inhibited the induction and progression of CIA and the anti‐collagen IgG1 and IgG2a antibody response. Combination therapy of anti‐TIRC7 mAb and soluble TNF‐α receptor demonstrated an increased inhibitory effect over the single compounds on CIA. The results demonstrate the therapeutic potential of TIRC7 targeting with mAb in diseases associated with exaggerated T and B cell responses.

HLA-DR Alpha 2 Mediates Negative Signalling via Binding to Tirc7 Leading to Anti-Inflammatory and Apoptotic Effects in Lymphocytes In Vitro and In Vivo

Classically, HLA-DR expressed on antigen presenting cells (APC) initiates lymphocyte activation via presentation of peptides to TCR bearing CD4+ T-Cells. Here we demonstrate that HLA-DR alpha 2 domain (sHLA-DRα2) also induces negative signals by engaging TIRC7 on lymphocytes. This interaction inhibits proliferation and induces apoptosis in CD4+ and CD8+ T-cells via activation of the intrinsic pathway. Proliferation inhibition is associated with SHP-1 recruitment by TIRC7, decreased phosphorylation of STAT4, TCR-ζ chain & ZAP70, and inhibition of IFN-γ and FasL expression. HLA-DRα2 and TIRC7 co-localize at the APC-T cell interaction site. Triggering HLA-DR - TIRC7 pathway demonstrates that sHLA-DRα2 treatment inhibits proinflammatory-inflammatory cytokine expression in APC & T cells after lipopolysaccaride (LPS) stimulation in vitro and induces apoptosis in vivo. These results suggest a novel antiproliferative role for HLA-DR mediated via TIRC7, revise the notion of an exclusive stimulatory interaction of HLA-DR with CD4+ T cells and highlights a novel physiologically relevant regulatory pathway.

Ethically Appropriate Handling of Incidental Findings in Human Neuroimaging Research

The possibility of discovering incidental findings is a serious ethical and juridical issue in human neuroimaging research. There is consensus among neuroscientists and neuroradiologists that appropriately handling incidental findings requires ethical guidelines. The Bonn Neuroethics Working Group is organizing a comprehensive process among neuroscientists from German-speaking countries with the aim of identifying the points of consent and preparing a proposal for future guidelines. This process began in 2005 and included: a mini-symposium which was held at the 50th Annual Meeting of the German Society for Clinical Neurophysiology and Functional Neuroimaging (DGKN) in Bad Nauheim (March 23, 2006); publishing the first proposal in Deutsches Ärzteblatt (2007, July) [1]; and a closed meeting of neuroscientists which was held in Bonn in August 2008. The feedback received from the researchers was recorded in detail and entered into a revised version of the guideline proposal which was then distributed among the participants and other researchers in January 2009. Once again, we requested detailed feedback. On June 19, 2009, after another substantial revision, the final version was distributed to 190 researchers heading neuroscientific working groups, institutes, and clinics in Germany, Austria, and Switzerland. Professor A. Spickhoff (Department of Law, University of Regensburg, Germany) provided juridical expertise for all versions of the proposal. In their guest editorial, “Response of the German Society of Neuroradiology” which appeared in Clinical Neuroradiology, Hentschel and von Kummer plainly reject the guideline proposal “on behalf of the German Society of Neuroradiology (DGNR)” ([2], p. 110). Contrary to the view outlined in the proposal, they claim