Cyclic adenosine monophosphate is a key component of regulatory T cell-mediated suppression.

Naturally occurring regulatory T cells (T reg cells) are a thymus-derived subset of T cells, which are crucial for the maintenance of peripheral tolerance by controlling potentially autoreactive T cells. However, the underlying molecular mechanisms of this strictly cell contact-dependent process are still elusive. Here we show that naturally occurring T reg cells harbor high levels of cyclic adenosine monophosphate (cAMP). This second messenger is known to be a potent inhibitor of proliferation and interleukin 2 synthesis in T cells. Upon coactivation with naturally occurring T reg cells the cAMP content of responder T cells is also strongly increased. Furthermore, we demonstrate that naturally occurring T reg cells and conventional T cells communicate via cell contact-dependent gap junction formation. The suppressive activity of naturally occurring T reg cells is abolished by a cAMP antagonist as well as by a gap junction inhibitor, which blocks the cell contact-dependent transfer of cAMP to responder T cells. Accordingly, our results suggest that cAMP is crucial for naturally occurring T reg cell-mediated suppression and traverses membranes via gap junctions. Hence, naturally occurring T reg cells unexpectedly may control the immune regulatory network by a well-known mechanism based on the intercellular transport of cAMP via gap junctions.

NFATc2 and NFATc3 transcription factors play a crucial role in suppression of CD4+ T lymphocytes by CD4+ CD25+ regulatory T cells.

The phenotype of NFATc2(-/-) c3(-/-) (double knockout [DKO]) mice implies a disturbed regulation of T cell responses, evidenced by massive lymphadenopathy, splenomegaly, and autoaggressive phenomena. The population of CD4(+) CD25(+) T cells from DKO mice lacks regulatory capacity, except a small subpopulation that highly expresses glucocorticoid-induced tumor necrosis factor receptor family-related gene (GITR) and CD25. However, neither wild-type nor DKO CD4(+) CD25(+) regulatory T cells (T reg cells) are able to suppress proliferation of DKO CD4(+) CD25(-) T helper cells. Therefore, combined NFATc2/c3 deficiency is compatible with the development of CD4(+) CD25(+) T reg cells but renders conventional CD4(+) T cells unresponsive to suppression, underlining the importance of NFAT proteins for sustaining T cell homeostasis.

Induction of NFATc2 expression by interleukin 6 promotes T helper type 2 differentiation.

Interleukin (IL)-6 is produced by professional antigen-presenting cells (APCs) such as B cells, macrophages, and dendritic cells. It has been previously shown that APC-derived IL-6 promotes the differentiation of naive CD4+ T cells into effector T helper type 2 (Th2) cells. Here, we have studied the molecular mechanism for IL-6-mediated Th2 differentiation. During the activation of CD4+ T cells, IL-6 induces the production of IL-4, which promotes the differentiation of these cells into effector Th2 cells. Regulation of IL-4 gene expression by IL-6 is mediated by nuclear factor of activated T cells (NFAT), as inhibition of NFAT prevents IL-6-driven IL-4 production and Th2 differentiation. IL-6 upregulates NFAT transcriptional activity by increasing the levels of NFATc2. The ability of IL-6 to promote Th2 differentiation is impaired in CD4+ T cells that lack NFATc2, demonstrating that NFATc2 is required for regulation of IL-4 gene expression by IL-6. Regulation of NFATc2 expression and NFAT transcriptional activity represents a novel pathway by which IL-6 can modulate gene expression.

Pim-1 kinase enhances NFATc activity and neuroendocrine functions in PC12 cells.

The activity of NFATc family transcription factors is tightly regulated in T cells via signaling pathways initiated by stimulation of the T cell receptor or its downstream effectors such as the Pim-1 serine/threonine kinase. Here, we demonstrate that NFATc-dependent transcription is inducible also in NGF-differentiated rat PC12 pheochromocytoma cells treated with phorbol esthers, calcium ionophores and/or forskolin and that the Pim-1 kinase can further potentiate the effects of these agents. PC12 cells share many characteristics with sympathetic neurons and can be induced to produce and release catecholamines, such as dopamine and noradrenaline, and inflammatory cytokines, such as interleukin 6. Interestingly, Pim-1 can synergize with forskolin-induced signaling pathways to stimulate also neuroendocrine functions of PC12 cells.

NFAT and NF-kappaB factors-the distant relatives.

NFAT and NF-kappaB proteins are members of a superfamily of transcription factors whose activity plays a crucial role in the activation, proliferation and apoptosis of lymphocytes. Both types of factors share a number of properties, including similar DNA binding domains and rapid nuclear translocation upon antigenic stimulation. While NF-kappaBs control both innate and adaptive immune responses, NFATs control the adaptive immune system which emerged-in parallel with the appearance of the NFAT family-in jawed fish. However, NFATs and NF-kappaBs differ remarkably in their function. Whereas NFATs support activation-induced cell death (AICD) of T and B cells, NF-kappaB proteins frequently exert a strong anti-apoptotic effect on lymphocytes and other cells. While the anti-apoptotic activity of NF-kappaBs contributes to their oncogenic capacity, the pro-apoptotic activity favors NFATs as tumor suppressors in lymphoid cells.

Microtubule associated tumor suppressor 1 deficient mice develop spontaneous heart hypertrophy and SLE-like lymphoproliferative disease.

The microtubule associated tumor suppressor gene 1 (MTUS1) is a recently published tumor suppressor gene, which has also been shown to act as an early component in the growth inhibitory signaling cascade of the angiotensin II type 2 receptor (AT2R). In this study we report the generation of MTUS1 knock-out (KO) mice, which develop normally but reveal higher body weights and slightly decreased blood pressure levels. Twenty-eight percent of the studied MTUS1 KO mice also developed heart hypertrophy and 12% developed nephritis, independent of blood pressure levels. Forty-three percent of the MTUS1 KO mice revealed lymphoid hyperplasia affecting spleen (20%), kidney (37%), lung (23%), lymph nodes (17%), and liver (17%) accompanied with leukocytosis, lymphocytosis, and mild anemia. One animal (3%) developed a marginal zone B-cell lymphoma affecting submandibular salivary gland and regional lymph nodes. The symptoms of all mentioned animals are consistent with a B-cell lymphoproliferative disease with features of systemic lupus erythematosus. In addition, body weight of the MTUS1 KO mice was significantly increased and isolated skin fibroblasts showed increased cell proliferation and decreased cell size, compared to wild-type (WT) fibroblasts in response to depleted FCS concentration and lack of growth factors. In conclusion we herein report the first generation of a MTUS1 KO mouse, developing spontaneous heart hypertrophy and increased cell proliferation, confirming once more the anti-proliferative effect of MTUS1, and a SLE-like lymphoproliferative disease suggesting crucial role in regulation of inflammation. These MTUS1 KO mice can therefore serve as a model for further investigations in cardiovascular disease, autoimmune disease and carcinogenesis.

Epigenetic changes and suppression of the nuclear factor of activated T cell 1 (NFATC1) promoter in human lymphomas with defects in immunoreceptor signaling.

The nuclear factor of activated T cell 1 (Nfatc1) locus is a common insertion site for murine tumorigenic retroviruses, suggesting a role of transcription factor NFATc1 in lymphomagenesis. Although NFATc1 is expressed in most human primary lymphocytes and mature human T- and B-cell neoplasms, we show by histochemical stainings that NFATc1 expression is suppressed in anaplastic large cell lymphomas and classical Hodgkin's lymphomas (HLs). In HL cell lines, NFATc1 silencing correlated with a decrease in histone H3 acetylation, H3-K4 trimethylation, and Sp1 factor binding but with an increase in HP1 binding to the NFATC1 P1 promoter. Together with DNA hypermethylation of the NFATC1 P1 promoter, which we detected in all anaplastic large cell lymphoma and many HL lines, these observations reflect typical signs of transcriptional silencing. In several lymphoma lines, methylation of NFATC1 promoter DNA resulted in a "window of hypomethylation," which is flanked by Sp1-binding sites. Together with the under-representation of Sp1 at the NFATC1 P1 promoter in HL cells, this suggests that Sp1 factors can protect P1 DNA methylation in a directional manner. Blocking immunoreceptor signaling led to NFATC1 P1 promoter silencing and to a decrease in H3 acetylation and H3-K4 methylation but not DNA methylation. This shows that histone modifications precede the DNA methylation in NFATC1 promoter silencing.

NFATc1 autoregulation: a crucial step for cell-fate determination.

Nuclear factor of activated T cell c (NFATc) transcription factors appeared in evolution with the emergence of lymphocytes in jawed fish. They have decisive roles in the development of the immune system and adaptive immune responses. Following immunoreceptor stimulation, NFAT factors control the expression of a large set of genes and thereby the fate of peripheral lymphocytes. NFATc1 and NFATc2 are the most prominent NFAT factors in peripheral T cells; they overlap in their function but differ remarkably in the mode of expression. NFATc2 is constitutively synthesized in T cells, whereas the expression of NFATc1/alphaA, the most prominent of six NFATc1 isoforms in peripheral T cells, is strongly induced following T-cell receptor and co-receptor stimulation and maintained by positive autoregulation. Findings concerning NFATc1 autoregulation in peripheral T lymphocytes and other cells suggest that positive autoregulation of NFATc1 is a crucial step in cell-fate determination.

NFAT transcription factors in control of peripheral T cell tolerance.

The Ca++-regulated calcineurin/NFAT cascade is one of the crucial signalling pathways that controls adaptive immunity. However, a number of novel experimental data suggest that, in addition to their role in T cell activation, NFATc transcription factors play also a decisive role in the generation of peripheral tolerance against self-antigens. This function of NFATc factors is mediated by controlling activation-induced cell death and clonal anergy of T helper cells and the activity of regulatory T cells. The multi-functional role of NFATc proteins characterize these transcription factors as key regulators of immunological tolerance and, if dysregulated, of development of autoimmune diseases.

Specific and redundant roles for NFAT transcription factors in the expression of mast cell-derived cytokines.

By virtue of their ability to express a plethora of biologically highly active mediators, mast cells (MC) are involved in both adaptive and innate immune responses. MC-derived Th2-type cytokines are thought to act as local amplifiers of Th2 reactions, including chronic inflammatory disorders such as allergic asthma, whereas MC-derived TNF-alpha is a critical initiator of antimicrobial defense. In this study, we demonstrate that the transcription factors NFATc1 and NFATc2 are part of a MC-specific signaling network that regulates the expression of TNF-alpha and IL-13, whereas NFATc3 is dispensable. Primary murine bone marrow-derived MC from NFATc2(-/-) mice, activated by either ionomycin or IgE/Ag cross-link, display a strong reduction in the production of these cytokines, compared with bone marrow-derived MC from wild-type mice. Detailed analyses of TNF-alpha and IL-13 expression using small interfering RNA-mediated knockdown reveals that both NFATc2 and NFATc1 are able to drive the expression of these cytokines, whereas neither degranulation nor the expression of IL-6 depends on NFAT activity. These results support the view that high NFAT activity is necessary for TNF-alpha and IL-13 promoter induction in MC, irrespective of whether NFATc2 or NFATc1 or a combination of both is present.

B cell hyperresponsiveness and expansion of mature follicular B cells but not of marginal zone B cells in NFATc2/c3 double-deficient mice.

Marginal zone (MZ) B cells and peritoneal B-1 cells provide a first defense system of thymus-independent Ab responses against foreign pathogens and therefore share a number of functional properties. Recently, development of B-1a cells was shown to be controlled by the transcription factor NFATc1. We show here that mice deficient for NFATc2 and c3 display a distinct lower representation of MZ B cells, which is correlated with a reduced capturing of trinitrophenyl-Ficoll. In contrast, mature follicular B cells from NFATc2/c3-/- mice are strongly increased in number. NFATc2/c3-/- B cells exhibit a marked increase in BCR-induced intracellular Ca2+ mobilization and proliferation. However, trinitrophenyl-Ficoll-specific IgM and IgG3 responses of NFATc2/c3-deficient mice are intact, and chimeric mice reconstituted with NFATc2/3-deficient B cells show a normal number of MZ B cells and normal BCR responses. These observations suggest that the strongly elevated Th2 cytokine milieu in NFATc2/c3-deficient mice leads to a hyperactivation of mature, follicular B cells, whereas MZ B cells are less responsive to these signals.

Mice vaccinated with allergen-pulsed myeloid dendritic cells are not protected from developing allergen-induced Th2 responses.

BACKGROUND: Dendritic cells (DC) play a decisive role in the induction of allergen-induced Th1 and Th2 responses. Since the induction of allergen-specific Th1 responses has shown to inhibit allergen-induced Th2-type inflammation, in this study we investigated whether manipulated myeloid-derived DC pulsed with the specific allergen would predominantly induce allergen-specific Th1 responses thereby reducing the development of Th2 responses. METHODS: Murine bone marrow (BM)-DC were generated and pulsed with ovalbumin (OVA) and CpG oligodeoxynucleotides (CpG-ODN). Langerhans cells (LC) were also isolated and pulsed in vitro with OVA. Subsequently, mice were vaccinated intravenously with either CpG/OVA-pulsed BM-DC or OVA-pulsed LC, and the protocol to induce OVA-specific Th2 responses using OVA/alum sensitization was initiated. Airway inflammation and OVA-specific serum antibody levels were evaluated 6 days after the intranasal challenge with OVA. RESULTS: The application ofCpG/OVA-pulsed BM-DC was unable to reduce airway eosinophilia and inflammation in OVA/alum-immunized mice. OVA-specific IgG1 or IgE serum levels were also not reduced. The experiments using LC pulsed with OVA yielded similar results. However, mice vaccinated with CpG/OVA-pulsed BM-DC had greatly enhanced levels of serum OVA-specific IgG2a, suggesting the induction of allergen-specific Th1 responses in vivo. Moreover, allergen-induced mast cell degranulation was decreased using this approach. CONCLUSIONS: Taken together, our results demonstrated that the vaccination with OVA-pulsed BM-DC matured with CpG-ODN or OVA-pulsed LC did not result in a reduction in allergen-specific Th2 responses in a murine model of severe atopic asthma. Other DC-based vaccination strategies should be evaluated in order to prevent the development of allergic disorders.

A tumor-suppressor function for NFATc3 in T-cell lymphomagenesis by murine leukemia virus.

Nuclear factor of activated T cell (NFAT) transcription factors play a central role in differentiation, activation, and elimination of lymphocytes. We here report on the finding of provirus integration into the Nfatc3 locus in T-cell lymphomas induced by the murine lymphomagenic retrovirus SL3-3 and show that NFATc3 expression is repressed in these lymphomas. The provirus insertions are positioned close to the Nfatc3 promoter or a putative polyadenylated RNA (polyA) region. Furthermore, we demonstrate that NFATc3-deficient mice infected with SL3-3 develop T-cell lymphomas faster and with higher frequencies than wild-type mice or NFATc2-deficient mice. These results identify NFATc3 as a tumor suppressor for the development of murine T-cell lymphomas induced by the retrovirus SL3-3.

cGMP-dependent protein kinase type II regulates basal level of aldosterone production by zona glomerulosa cells without increasing expression of the steroidogenic acute regulatory protein gene.

The renin-angiotensin-aldosterone system plays a pivotal role in the regulation of salt and water homeostasis. Here, we demonstrate the expression and functional role of cGMP-dependent protein kinases (PKGs) in rat adrenal cortex. Expression of PKG II is restricted to adrenal zona glomerulosa (ZG) cells, whereas PKG I is localized to the adrenal capsule and blood vessels. Activation of the aldosterone system by a low sodium diet up-regulated the expression of PKG II, however, it did not change PKG I expression in adrenal cortex. Both, activation of PKG II in isolated ZG cell and adenoviral gene transfer of wild type PKG II into ZG cells enhanced aldosterone production. In contrast, inhibition of PKG II as well as infection with a PKG II catalytically inactive mutant had an inhibitory effect on aldosterone production. Steroidogenic acute regulatory (StAR) protein that regulates the rate-limiting step in steroidogenesis is a new substrate for PKG II and can be phosphorylated by PKG II in vitro at serine 55/56 and serine 99. Stimulation of aldosterone production by PKG II in contrast to stimulation by PKA did not activate StAR gene expression in ZG cells. The results presented indicate that PKG II activity in ZG cells is important for maintaining basal aldosterone production.

Mice deficient in nuclear factor of activated T-cell transcription factor c2 mount increased Th2 responses after infection with Nippostrongylus brasiliensis and decreased Th1 responses after mycobacterial infection.

Infection of nuclear factor of activated T-cell transcription factor c2 (NFATc2)-deficient mice with the helminth Nippostrongylus brasiliensis led to a distinct increase in interleukin-4 (IL-4) and IL-5 protein synthesis by lymph node and spleen cells and to elevated serum immunoglobulin E (IgE) levels in comparison to those seen with infected control mice. While IL-4, IL-5, and IL-13 mRNA expression was also enhanced in lymph node cells from the lungs of infected NFATc2(-/-) mice, the number of T cells secreting Th2-type lymphokines remained the same in mice infected with N. brasiliensis. In contrast, lymphocytes from NFATc2-deficient mice infected with Mycobacterium bovis BCG secreted less gamma interferon than lymphocytes from infected control mice. These findings indicate that NFATc2 is an activator of Th1 responses and a suppressor of Th2 responses in vivo.

Oxidized LDL suppresses NF-kappaB and overcomes protection from apoptosis in activated endothelial cells.

Atherosclerosis is a chronic inflammatory disease associated with enhanced apoptotic cell death in vascular cells, partly induced by oxidized low-density lipoprotein (OxLDL). However, proinflammatory stimuli such as lipopolysaccharide (LPS) or tumor necrosis factor-alpha (TNF-alpha) activate endothelial cells (EC) and inhibit apoptosis through induction of nuclear factor kappaB (NF-kappaB)-dependent genes. This study therefore investigated whether OxLDL or its component, lysophosphatidylcholine (LPC), interacts with the effect of LPS or TNF-alpha on cell survival. Human EC were incubated with LPS, TNF-alpha, OxLDL, or LPC alone or in combinations. OxLDL (100 to 200 microg/ml) and LPC (100 to 300 microM) induced apoptosis dose-dependently. LPS and TNF-alpha had no effect on cell survival in the presence or absence of OxLDL or LPC. LPS and TNF-alpha both induced the antiapoptotic gene A20, whereas OxLDL and LPC suppressed its induction. Expression of A20 is regulated by NF-kappaB. OxLDL and LPC dose-dependently suppressed NF-kappaB activity. For functional analysis, bovine EC were transfected with A20 encoding expression constructs in sense and antisense orientation. Bovine EC that overexpressed A20 were protected against OxLDL-induced apoptosis, whereas expression of antisense A20 rendered cells more sensitive to OxLDL. These results suggest that OxLDL not only induces cell death, as has been shown before, but also compromises antiapoptotic protection of activated EC. OxLDL sensitizes EC to apoptotic triggers by interfering with the induction of A20 during the inflammatory response seen in atherosclerotic lesions. This inhibition is based on repression of NF-kappaB activation. The effect may be caused by the OxLDL component LPC.

Inhibition of cGMP-dependent protein kinase II by its own splice isoform.

cGMP- and cAMP-dependent protein kinases (cGK I, cGK II, and cAK) are important mediators of many signaling pathways that increase cyclic nucleotide concentrations and ultimately phosphorylation of substrates vital to cellular functions. Here we demonstrate a novel mRNA splice isoform of cGK II arising from alternative 5' splicing within exon 11. The novel splice variant encodes a protein (cGK II Delta(441-469)) lacking 29 amino acids of the cGK II Mg-ATP-binding/catalytic domain, including the conserved glycine-rich loop consensus motif Gly-x-Gly-x-x-Gly-x-Val which interacts with ATP in the protein kinase family of enzymes. cGK II Delta(441-469) has no intrinsic enzymatic activity itself, however, it antagonizes cGK II and cGK I, but not cAK. Thus, the activation and cellular functions of cGK II may be determined not only by intracellular cGMP levels but also by alternative splicing which may regulate the balance of expression of cGK II versus its own inhibitor, cGK II Delta(441-469).