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M Gadina

Publications and source records attributed to M Gadina.

At least 19 recordsLinked to original sources
PubMed2001

Inducible expression of Stat4 in dendritic cells and macrophages and its critical role in innate and adaptive immune responses.

Autocrine activation of APC by IL-12 has recently been revealed; we demonstrate here that inducible expression of Stat4 in APC is central to this process. Stat4 is induced in dendritic cells (DC) in a maturation-dependent manner and in macrophages in an activation-dependent manner. Stat4 levels directly correlate with IL-12-dependent IFN-gamma production by APC as well as IFN-gamma production by DC during Ag presentation. The Th2 cytokines IL-4 and IL-10 suppress Stat4 induction in DC and macrophages when present during maturation and activation, respectively, diminishing IFN-gamma production. In contrast, IL-4 has no effect on Stat4 levels in mature DC and actually augments IFN-gamma production by DC during Ag presentation, indicating that IL-4 acts differently in a spatiotemporal manner. The functional importance of Stat4 is evident in Stat4(-/-) DC and macrophages, which fail to produce IFN-gamma. Furthermore, Stat4(-/-) macrophages are defective in NO production in response to IL-12 and are susceptible to TOXOPLASMA: Autocrine IL-12 signaling is required for high-level IFN-gamma production by APC at critical stages in both innate and adaptive immunity, and the control of Stat4 expression is likely an important regulator of this process.

Animals↗
PubMed2001

Signaling by type I and II cytokine receptors: ten years after.

Discovered during the past ten years, Janus kinases and signal transducers and activators of transcription have emerged as critical elements in cytokine signaling and immunoregulation. Recently, knockout mice for all the members of these families have been generated, with remarkably specific outcomes. Equally exciting is the discovery of a new class of inhibitors, the suppressor of cytokine signaling family. The phenotypes of mice deficient in these molecules are also striking, underscoring the importance of negative regulation in cytokine signaling.

Animals↗
PubMed2000

IL-12 receptor beta 2 (IL-12R beta 2)-deficient mice are defective in IL-12-mediated signaling despite the presence of high affinity IL-12 binding sites.

Two subunits of the IL-12 receptor (IL-12R), IL-12R beta 1 and IL-12R beta 2, have been identified and cloned. Previous studies demonstrated that the IL-12R beta 1 subunit was required for mouse T and NK cells to respond to IL-12 in vivo. To investigate the role of IL-12R beta 2 in IL-12 signaling, we have generated IL-12R beta 2-deficient (IL-12R beta 2(-/-)) mice by targeted mutation in embryonic stem (ES) cells. Although Con A-activated splenocytes from IL-12R beta 2(-/-) mice still bind IL-12 with both high and low affinity, no IL-12-induced biological functions can be detected. Con A-activated splenocytes of IL-12R beta 2(-/-) mice failed to produce IFN-gamma or proliferate in response to IL-12 stimulation. NK lytic activity of IL-12R beta 2(-/-) splenocytes was not induced when incubated with IL-12. IL-12R beta 2(-/-) splenocytes were deficient in IFN-gamma secretion when stimulated with either Con A or anti-CD3 mAb in vitro. Furthermore, IL-12R beta 2(-/-) mice were deficient in vivo in their ability to produce IFN-gamma following endotoxin administration and to generate a type 1 cytokine response. IL-12-mediated signal transduction was also defective as measured by phosphorylation of STAT4. These results demonstrate that although mouse IL-12R beta 1 is the subunit primarily responsible for binding IL-12, IL-12R beta 2 plays an essential role in mediating the biological functions of IL-12 in mice.

Animals↗
PubMed2000

The docking molecule gab2 is induced by lymphocyte activation and is involved in signaling by interleukin-2 and interleukin-15 but not other common gamma chain-using cytokines.

Interleukin (IL)-2, a critical cytokine with indispensable functions in regulating lymphoid homeostasis, induces the activation of several biochemical pathways. Precisely how these pathways are linked and how they relate to the biological action of IL-2 is incompletely understood. We previously identified SHP-2 (Src homology 2 domain containing phosphatase 2) as an important intermediate in IL-2-dependent MAPK activation and showed its association with a 98-kDa phosphoprotein in response to IL-2. Here, we demonstrate that Gab2, a recently identified adapter molecule, is the major SHP-2 and phosphatidylinositol 3'-kinase-associated 98-kDa protein in normal, IL-2-activated lymphocytes. We further demonstrate that phosphorylation of both Gab2 and SHP-2 is largely dependent upon tyrosine 338 of the IL-2 receptor beta chain. Gab2 can be a substrate of all the three major classes of non-receptor tyrosine kinases associated with the IL-2R, but in terms of IL-2 signaling, JAK3 but not Lck or Syk is essential for Gab2 phosphorylation. We also demonstrate that only IL-2 and IL-15, but not other gammac cytokines induce Gab2 phosphorylation; the ability to phosphorylate Gab2 correlates with Shc phosphorylation and ERK1/ERK2 activation. Finally, we also show that Gab2 levels are regulated by T cell activation, and resting T cells express little Gab2. Therefore, up-regulation and activation of Gab2 may be important in linking the IL-2 receptor to activation of MAPK and may be an important means of achieving specificity in cytokine signaling.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Importance of the MKK6/p38 pathway for interleukin-12-induced STAT4 serine phosphorylation and transcriptional activity.

Interleukin-12 (IL-12) is a key immunoregulatory cytokine that promotes Th1 differentiation and cell-mediated immune responses. The transcription factor STAT4 (signal transducer and activator of transcription 4) is an important element in mediating IL-12 signals, as evidenced by the fact that STAT4(-/-) mice display impaired responsiveness to IL-12 and deficient Th1 differentiation. STAT4 is inducibly phosphorylated on tyrosine and serine in response to IL-12, but the kinase(s) responsible for the latter event is unknown. Here we show that IL-12 induces STAT4 phosphorylation on serine 721 and that mutation of serine 721 interferes with STAT4 transcriptional activity. In addition, we show that mutation of tyrosine 693 abrogates IL-12-induced STAT4 tyrosine phosphorylation and transcriptional activity. Although the site surrounding serine 721 is an optimum consensus sequence for mitogen-activated family of protein kinases (MAPKs)-mediated phosphorylation, we demonstrate that IL-12 does not induce extracellular signal-regulated kinase (ERK) or c-Jun N-terminal kinase (JNK) activation in T and natural killer (NK) cells and that IL-12-induced STAT4 transcriptional activity is not affected by these kinases. Rather, we show that IL-12 induces p38 activation. Moreover, we demonstrate that p38alpha and its upstream activator, MKK6, phosphorylate STAT4 on serine 721, and are required for STAT4 full transcriptional activity induced by IL-12, establishing the MKK6/p38alpha/STAT4 pathway as an important mediator of IL-12 actions. (Blood. 2000;96:1844-1852)

3T3 Cells↗
PubMed2000

Inhibition of Th1 immune response by glucocorticoids: dexamethasone selectively inhibits IL-12-induced Stat4 phosphorylation in T lymphocytes.

Glucocorticoids are widely used in the therapy of inflammatory, autoimmune, and allergic diseases. As the end-effectors of the hypothalamic-pituitary-adrenal axis, endogenous glucocorticoids also play an important role in suppressing innate and cellular immune responses. Previous studies have indicated that glucocorticoids inhibit Th1 and enhance Th2 cytokine secretion. IL-12 promotes Th1 cell-mediated immunity, while IL-4 stimulates Th2 humoral-mediated immunity. Here, we examined the regulatory effect of glucocorticoids on key elements of IL-12 and IL-4 signaling. We first investigated the effect of dexamethasone on IL-12-inducible genes and showed that dexamethasone inhibited IL-12-induced IFN-gamma secretion and IFN regulatory factor-1 expression in both NK and T cells. This occurred even though the level of expression of IL-12 receptors and IL-12-induced Janus kinase phosphorylation remained unaltered. However, dexamethasone markedly inhibited IL-12-induced phosphorylation of Stat4 without altering its expression. This was specific, as IL-4-induced Stat6 phosphorylation was not affected, and mediated by the glucocorticoid receptor, as it was antagonized by the glucocorticoid receptor antagonist RU486. Moreover, transfection experiments showed that dexamethasone reduced responsiveness to IL-12 through the inhibition of Stat4-dependent IFN regulatory factor-1 promoter activity. We conclude that blocking IL-12-induced Stat4 phosphorylation, without altering IL-4-induced Stat6 phosphorylation, appears to be a new suppressive action of glucocorticoids on the Th1 cellular immune response and may help explain the glucocorticoid-induced shift toward the Th2 humoral immune response.

3T3 Cells↗
PubMed2000

IFN consensus sequence binding protein potentiates STAT1-dependent activation of IFNgamma-responsive promoters in macrophages.

IFNgamma, once called the macrophage-activating factor, stimulates many genes in macrophages, ultimately leading to the elicitation of innate immunity. IFNgamma's functions depend on the activation of STAT1, which stimulates transcription of IFNgamma-inducible genes through the GAS element. The IFN consensus sequence binding protein (icsbgamma or IFN regulatory factor 8), encoding a transcription factor of the IFN regulatory factor family, is one of such IFNgamma-inducible genes in macrophages. We found that macrophages from ICSBP-/- mice were defective in inducing some IFNgamma-responsive genes, even though they were capable of activating STAT1 in response to IFNgamma. Accordingly, IFNgamma activation of luciferase reporters fused to the GAS element was severely impaired in ICSBP-/- macrophages, but transfection of ICSBP resulted in marked stimulation of these reporters. Consistent with its role in activating IFNgamma-responsive promoters, ICSBP stimulated reporter activity in a GAS-specific manner, even in the absence of IFNgamma treatment, and in STAT1 negative cells. Indicative of a mechanism for this stimulation, DNA affinity binding assays revealed that endogenous ICSBP was recruited to a multiprotein complex that bound to GAS. These results suggest that ICSBP, when induced by IFNgamma through STAT1, in turn generates a second wave of transcription from GAS-containing promoters, thereby contributing to the elicitation of IFNgamma's unique activities in immune cells.

Animals↗
PubMed2000

Cytokine regulation of IL-12 receptor beta2 expression: differential effects on human T and NK cells.

The biological activities of IL-12 are mediated through a specific, high-affinity receptor composed of IL-12 receptor(R)beta1 and IL-12Rbeta2 subunits that exist primarily on T and NK cells. Remarkably, the expression of IL-12Rbeta2 on CD4(+) T cells in mouse and humans appears to be differentially regulated by IFN-gamma and IFN-alpha, respectively. Using an antibody specific for the human IL-12Rbeta2 subunit, the effect of IFN-gamma, IFN-alpha, IL-12 and IL-2 on the regulation of IL-12R expression and IL-12 responsiveness of human T and NK cells was assessed. The presence of IFN-alpha or IFN-gamma in cultures enhanced IL-12Rbeta2 expression of CD4(+) and CD8(+) T cells. The enhancing effect of IFN-alpha and IFN-gamma was independent of endogenous IL-12. Furthermore, the clearest effects of IFN-alpha and IFN-gamma on IL-12Rbeta2 expression on T cells were seen by abrograting the inhibition induced by the presence of IL-4 in cultures. In contrast to T cells, IFN-alpha and IFN-gamma had little effect on regulating IL-12Rbeta2 expression on human NK cells. Taken together, these data show that there is differential regulation of IL-12Rbeta2 expression by IFN-alpha and IFN-gamma on human T and NK cells.

Animals↗
PubMed2000

Hierarchy of protein tyrosine kinases in interleukin-2 (IL-2) signaling: activation of syk depends on Jak3; however, neither Syk nor Lck is required for IL-2-mediated STAT activation.

Interleukin-2 (IL-2) activates several different families of tyrosine kinases, but precisely how these kinases interact is not completely understood. We therefore investigated the functional relationships among Jak3, Lck, and Syk in IL-2 signaling. We first observed that in the absence of Jak3, both Lck and Syk had the capacity to phosphorylate Stat3 and Stat5a. However, neither supported IL-2-induced STAT activation, nor did dominant negative alleles of these kinases inhibit. Moreover, pharmacological abrogation of Lck activity did not inhibit IL-2-mediated phosphorylation of Jak3 and Stat5a. Importantly, ligand-dependent Syk activation was dependent on the presence of catalytically active Jak3, whereas Lck activation was not. Interestingly, Syk functioned as a direct substrate of Jak1 but not Jak3. Additionally, Jak3 phosphorylated Jak1, whereas the reverse was not the case. Taken together, our data support a model in which Lck functions in parallel with Jak3, while Syk functions as a downstream element of Jaks in IL-2 signaling. Jak3 may regulate Syk catalytic activity indirectly via Jak1. However, IL-2-mediated Jak3/Stat activation is not dependent on Lck or Syk. While the essential roles of Jak1 and Jak3 in signaling by gammac-utilizing cytokines are clear, it will be important to dissect the exact contributions of Lck and Syk in mediating the effects of IL-2 and related cytokines.

Animals↗
PubMed2000

Jaks and stats as therapeutic targets.

Cytokines have critical functions in regulating immune responses. A large number of these factors bind related receptors termed the Type I and Type II families of cytokine receptors. These receptors activate Janus kinases (Jaks) and Stat family of transcription factors. The essential and specific function of Jaks and Stats is particularly well illustrated by human and mouse mutations. The possibility that these molecules could be targeted to produce novel immunosuppressive compounds is considered in this review.

Animals↗
PubMed1999

Germline mutations in the extracellular domains of the 55 kDa TNF receptor, TNFR1, define a family of dominantly inherited autoinflammatory syndromes.

Autosomal dominant periodic fever syndromes are characterized by unexplained episodes of fever and severe localized inflammation. In seven affected families, we found six different missense mutations of the 55 kDa tumor necrosis factor receptor (TNFR1), five of which disrupt conserved extracellular disulfide bonds. Soluble plasma TNFR1 levels in patients were approximately half normal. Leukocytes bearing a C52F mutation showed increased membrane TNFR1 and reduced receptor cleavage following stimulation. We propose that the autoinflammatory phenotype results from impaired downregulation of membrane TNFR1 and diminished shedding of potentially antagonistic soluble receptor. TNFR1-associated periodic syndromes (TRAPS) establish an important class of mutations in TNF receptors. Detailed analysis of one such mutation suggests impaired cytokine receptor clearance as a novel mechanism of disease.

Amino Acid Sequence↗
PubMed1999

IL-2, but not IL-4 and other cytokines, induces phosphorylation of a 98-kDa protein associated with SHP-2, phosphatidylinositol 3'-kinase, and Grb2.

Binding of IL-2 to its receptor activates several biochemical pathways, including JAK-STAT, Ras-mitogen-activated protein kinase, and phosphatidylinositol 3'-kinase (PI 3'-kinase) pathways. Recently, it has been shown that the SH2-containing phosphatase, SHP-2, becomes phosphorylated in response to IL-2 stimulation, associates with PI3'-kinase and Grb2, and can exert a positive regulatory role in IL-2 signaling. We now report the identification of a prominent 98-kDa protein (p98) found to be phosphorylated in response to IL-2 stimulation and coprecipitated with SHP-2, the p85 subunit of PI 3'-kinase and Grb2. Interestingly, whereas IL-4 is known to activate PI 3'-kinase, we did not observe any p98 phosphorylation in response to IL-4 stimulation. p98 can form a multipartite complex with all these proteins as immunodepleting with anti-p85 antiserum substantially reduced the amount of p98 immunoprecipitated by SHP-2 and Grb2; the converse was also true. Furthermore, phosphorylation of p98 did not occur in cells lacking JAK3, suggesting that it may be a JAK substrate. Finally, deglycosylation of p98 did not alter its migration, suggesting p98 is not a member of the recently described SHP substrate/signal-regulatory proteins family of transmembrane glycoproteins. Thus p98 is a prominent IL-2-dependent substrate that associates with multiple proteins involved in IL-2 signaling and may play an important role in coupling the different signal transduction pathways activated by IL-2.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Involvement of SHP-2 in multiple aspects of IL-2 signaling: evidence for a positive regulatory role.

Binding of IL-2 to its receptor activates several biochemical pathways, but precisely how these pathways are linked is incompletely understood. Here, we report that SHP-2, an SH2-domain containing tyrosine phosphatase, associates with different molecules of the IL-2 signaling cascade. Upon IL-2 stimulation, SHP-2 was coimmunoprecipitated with Grb2 and the p85 subunit of phosphatidylinositol 3-kinase. In contrast, SHP-2 was constitutively associated with JAK1 and JAK3. Finally, SHP-2 expression amplified STAT-dependent transcriptional activation whereas a dominant negative allele inhibited transactivation and the IL-2-induced activation of MAPK (mitogen-activated protein kinase). These results demonstrate the involvement of SHP-2 in multiple pathways of the IL-2 signaling cascade and provide evidence for its positive regulatory role.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Shc mediates IL-6 signaling by interacting with gp130 and Jak2 kinase.

IL-6 is a multifunctional cytokine involved in hemopoiesis, immune regulation, inflammation, neural development, and infection. IL-6 belongs to a family of related cytokines that includes leukemia inhibitory factor, oncostatin M, IL-11, ciliary neurotropic factor, and cardiotropin-1, all of which initiate signaling through a receptor-associated gp130. IL-6 induces homodimerization of gp130 and activates the Jak/STAT pathway of signal transduction. In addition, IL-6 stimulates the mitogen-activated protein kinases designated ERK (extracellular signal-regulated kinase)-1 and -2. Activation of ERK-1 and -2 may involve the Src homology-2 containing proteins Shc and Grb2. Here we provide evidence that Shc could function as signaling molecules for IL-6 in DeFew-IL-6R/gp130 cells, a human B lymphoma cell line engineered to express high levels of both the IL-6R (p80) and the gp130 subunit. IL-6 was shown to promote the rapid tyrosine phosphorylation of gp130, Jak2, and Shc proteins. Moreover, Shc associated both in vivo and in vitro with phosphorylated gp130 through the Shc-Src homology-2 domain. We also report that Shc bound to activated Jak2 by using the Shc amino terminal phosphotyrosine interaction domain. Following IL-6 stimulation, Shc physically associated with Grb2. Thus, the data point to Shc proteins as a functional link between the Jak2 and Ras pathways of IL-6 signal transduction.

Adaptor Proteins, Signal Transducing↗
PubMed1995

A study of the intracellular routing of cytotoxic ribonucleases.

Several ribonucleases serve as cytotoxic agents in host defense and in physiological cell death pathways. Although certain members of the pancreatic ribonuclease A superfamily can be toxic when applied to the outside of cells, they become thousands of times more toxic when artificially introduced into the cytosol, indicating that internalization is the rate-limiting step for cytotoxicity. We have used three agents that disrupt the Golgi apparatus by distinct mechanisms, retinoic acid, brefeldin A, and monensin, to probe the intracellular pathways ribonucleases take to reach the cytosol. Retinoic acid and monensin potentiate the cytotoxicity of bovine seminal RNase, Onconase, angiogenin, and human ribonuclease A 100 times or more. Retinoic acid-mediated potentiation of ribonucleases is completely blocked by brefeldin A. Ribonucleases appear to route more efficiently into the cytosol through the Golgi apparatus disrupted by monensin or retinoic acid. Intracellular RNA degradation by BS-RNase increased more than 100 times in the presence of retinoic acid confirming that the RNase reaches the cytosol and indicating that degradation of RNA is the intracellular lesion causing toxicity. As retinoic acid alone and Onconase are in clinical trials for cancer therapy, combinations of RNases and retinoic acid in vivo may offer new clinical utility.

Amino Acid Sequence↗
PubMed1995

Preclinical evaluation of the ribosome-inactivating proteins PAP-1, PAP-S and RTA in mice.

In a preclinical mouse model the plant ribosome-inactivating proteins (RIPs) pokeweed antiviral proteins PAP-1, and PAP-S and ricin A-chain (RTA) induced a pathological elevation of serum concentrations of glutamate pyruvate transaminase (GPT) and blood urea nitrogen (BUN) and had a significant immunosuppressive effect on B- and T-lymphocytes. The present analysis and comparison of the biodistribution and systemic/organ toxicity associated with RIP injection suggest a possible in vivo mechanism of action of PAP-1 and PAP-S and identify several limitations in the clinical use of these two toxins and RTA. When administered intravenously, PAP-1 and PAP-S consistently accumulated in kidneys and induced histologically documented damage to kidney and liver, with a LD50 of 3.3 mg/kg and 1.6 mg/kg for PAP-1 and PAP-S, respectively. In mice injected with PAP-S after chlorpromazine (CPZ) administration, GPT levels returned to normal between 24 and 72 h after toxin injection, while the BUN levels remained elevated. Mortality of the animals was delayed but all mice eventually succumbed. All the three toxins inhibited the expansion of anti-sheep red blood cells (SRBC) antibody-forming cells and the production of anti-SRBC antibody levels, although PAP-S showed the most potent activity. Despite the immunosuppressive activity, all toxins were highly immunogenic.

Alanine Transaminase↗