n-Butyrate inhibits Jun NH(2)-terminal kinase activation and cytokine transcription in mast cells.

Mast cells are well known to contribute to type I allergic conditions but only recently have been brought in association with chronic relapsing/remitting autoimmune diseases such as celiac disease and ulcerative colitis. Since the bacterial metabolite n-butyrate is considered to counteract intestinal inflammation we investigated the effects of this short chain fatty acid on mast cell activation. Using RNAse protection assays and reporter gene technology we show that n-butyrate downregulates TNF-alpha transcription. This correlates with an impaired activation of the Jun NH(2)-terminal kinase (JNK) but not other MAP kinases such as ERK and p38 that are largely unaffected by n-butyrate. As a consequence, we observed a decreased nuclear activity of AP-1 and NF-AT transcription factors. These results indicate that n-butyrate inhibits critical inflammatory mediators in mast cells by relatively selectively targeting the JNK signalling.

Activation of mast cells by incorporation of cholesterol into rafts.

IgE plus antigen-stimulated mast cells degranulate, synthesize leukotrienes and secrete cytokines. According to the coalescence model this process is initiated in specific membrane compartments termed rafts. There, enhanced levels of glycosphingolipids and cholesterol stabilize the interaction of FcepsilonRI and Lyn, and thus facilitate the first steps of signal transduction. Enforced changes in raft architecture by cholesterol deprivation and exogenous application of glycosphingolipids influence these early events by loss of tyrosine kinase activity or receptor-independent signal initiation respectively. Here we show that exogenously added cholesterol accumulates in rafts and activates mast cells. An investigation of the signaling events reveals that in contrast to IgE plus antigen-mediated stimulation, cholesterol triggers p38 mitogen-activated protein kinase and preferentially induces expression of FosB. Consequently, a comparative large-scale microarray analysis demonstrates that a number of IgE plus antigen-induced immediate early genes (peak expression at 30 min after induction) are repressed by cholesterol. These changes further translate into altered expression levels and time kinetics of a number of early genes (peak expression at 2 h after stimulation). As the most prominent example for cholesterol-dependent genes, we identified PAI1 (plasminogen activator inhibitor 1), a protein regarded as a risk factor for atherosclerosis.

Suppression of early T-cell-receptor-triggered cellular activation by the Janus kinase 3 inhibitor WHI-P-154.

BACKGROUND: Therapeutic targeting of Janus kinase 3 (JAK3) has received particular attention, because it is associated with the common gamma signaling of cytokine receptors and thus vitally influences T-cell growth and survival. Recent evidence, however, indicates a critical role for JAK3 in signaling linked to the T-cell antigen receptor. METHODS: In this study we investigated whether targeting JAK3 with a rationally designed inhibitor affects early T-cell activation events. T cells were stimulated by CD3 and CD28 cross-linking, and interleukin (IL)-2 production, activation marker expression, increase of free intracellular Ca2+ concentration, activation of the extracellular-related kinase, and nuclear translocation of transcription factors were evaluated. RESULTS: We found that JAK3 inhibitor treatment dramatically impaired T-cell-receptor (TCR)-induced IL-2 production, surface activation marker expression (CD69, CD154), and homotypic T-cell aggregation. Accordingly, mRNA production of IL-2, interferon-gamma, and IL-10 was profoundly inhibited. Molecular analysis revealed that TCR-triggered phosphorylation of phospholipase C-gamma1, increase in cytoplasmic Ca2+ concentration, and activation of extracellular-related kinase were markedly reduced by the JAK3 inhibitor, resulting in substantially decreased DNA binding of nuclear factor of activated T cells and alkaline phosphatase-1 and subsequent IL-2 promoter activation. Remarkably, on TCR-independent stimulation, IL-2 production, CD69 expression, and blast formation were completely insensitive to JAK3 inhibitor treatment. CONCLUSION: These data indicate that pharmacologic targeting of JAK3 uncouples early TCR-triggered signaling from essential downstream events, which may have important implications for the use of such compounds in T-cell-mediated disorders such as allograft rejection or graft-versus-host disease.

Novel mode of interference with nuclear factor of activated T-cells regulation in T-cells by the bacterial metabolite n-butyrate.

The transcription factor nuclear factor of activated T-cells (NF-AT) plays an essential role in the activation of many early immune response genes. A dynamic equilibrium between calcineurin and cellular kinases controls its phosphorylation and thus regulates its activity by determining its subcellular localization. Here, we demonstrate that T-cell activation in the presence of the bacterial metabolite n-butyrate, which leads to inhibition of interleukin-2 transcription, is characterized by the maintenance of the activity of counter-regulatory kinases glycogen synthase kinase 3 and protein kinase A as well as persistence of intracellular cAMP levels, whereas calcium response and mitogen-activated protein kinase activation were indistinguishable from cells stimulated in the absence of n-butyrate. Nuclear binding of NF-AT was decreased but other transcription factors implicated in interleukin-2 expression such as AP1 and nuclear factor kappaB were unaffected. The effect on NF-AT binding appeared to be the result of increased nuclear export because the export inhibitor leptomycin B completely restored nuclear binding of NF-AT. We, therefore, provide first evidence for interference with NF-AT regulation alternative to the currently understood inhibition of nuclear import. This mechanism might represent a bacterial strategy to subvert host defense, which could be of particular clinical importance in the gastrointestinal tract where high amounts of n-butyrate are physiologically present.

The immune modulator FTY720 targets sphingosine 1-phosphate receptors.

Immunosuppressant drugs such as cyclosporin have allowed widespread organ transplantation, but their utility remains limited by toxicities, and they are ineffective in chronic management of autoimmune diseases such as multiple sclerosis. In contrast, the immune modulating drug FTY720 is efficacious in a variety of transplant and autoimmune models without inducing a generalized immunosuppressed state and is effective in human kidney transplantation. FTY720 elicits a lymphopenia resulting from a reversible redistribution of lymphocytes from circulation to secondary lymphoid tissues by unknown mechanisms. Using FTY720 and several analogs, we show now that FTY720 is phosphorylated by sphingosine kinase; the phosphorylated compound is a potent agonist at four sphingosine 1-phosphate receptors and represents the therapeutic principle in a rodent model of multiple sclerosis. Our results suggest that FTY720, after phosphorylation, acts through sphingosine 1-phosphate signaling pathways to modulate chemotactic responses and lymphocyte trafficking.

Sphingolipids and the regulation of the immune response.

Over the last decade evidence has accumulated that sphingolipids are important and specific signalling molecules for cell-to-cell communication (mediator function) as well as for intracellular signalling processes (second messenger function). In addition, glycosylated sphingolipids are essential building blocks of rafts thereby participating in the initiation of receptor mediated signalling events. In immunology, processes such as T cell apoptosis, Th1 versus Th2 T cell differentiation, phagocytosis, and allergic excitability are either influenced or directly regulated by this class of lipids. Models such as the 'dual function concept' (differentiation of structural components versus signalling molecules) and the 'rheostat concept' (the balance of two or more sphingolipids is essential for the biological function) describe the multiple properties of these signalling molecules.

The synthesis and biological characterization of a ceramide library.

A facile synthesis of a combinatorial ceramide library and their activities in the NF-kappaB pathway and in apoptosis induction/prevention were demonstrated. A novel NF-kappaB activating molecule was discovered among ceramide containing beta-galactose, and the structural requirements of ceramides for apoptosis induction was elucidated.