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Metabolic reprogramming plays a central role in T cell activation and differentiation, and the inhibition of key metabolic pathways in activated T cells represents a logical approach for the development of new therapeutic agents for treating autoimmune diseases. The widely prescribed antidiabetic drug metformin and the glycolytic inhibitor 2-deoxyglucose (2-DG) have been used to study the inhibition of oxidative phosphorylation and glycolysis, respectively, in murine immune cells. Published studies have demonstrated that combination treatment with metformin and 2-DG was efficacious in dampening mouse T cell activation–induced effector processes, relative to treatments with either metformin or 2-DG alone. In this study, we report that metformin + 2-DG treatment more potently suppressed IFN-γ production and cell proliferation in activated primary human CD4+ T cells than either metformin or 2-DG treatment alone. The effects of metformin + 2-DG on human T cells were accompanied by significant remodeling of activation-induced metabolic transcriptional programs, in part because of suppression of key transcriptional regulators MYC and HIF-1A. Accordingly, metformin + 2-DG treatment significantly suppressed MYC-dependent metabolic genes and processes, but this effect was found to be independent of mTORC1 signaling. These findings reveal significant insights into the effects of metabolic inhibition by metformin + 2-DG treatment on primary human T cells and provide a basis for future work aimed at developing new combination therapy regimens that target multiple pathways within the metabolic networks of activated human T cells.

Metabolic reprogramming plays a central role in T cell activation and differentiation, and the inhibition of key metabolic pathways in activated T cells represents a logical approach for the development of new therapeutic agents for treating autoimmune diseases. The widely prescribed antidiabetic drug metformin and the glycolytic inhibitor 2-deoxyglucose (2-DG) have been used to study the inhibition of oxidative phosphorylation and glycolysis, respectively, in murine immune cells. Published studies have demonstrated that combination treatment with metformin and 2-DG was efficacious in dampening mouse T cell activation-induced effector processes, relative to treatments with either metformin or 2-DG alone. In this study, we report that metformin + 2-DG treatment more potently suppressed IFN-γ production and cell proliferation in activated primary human CD4

The NLRP3 inflammasome is a complex multimeric signaling apparatus that regulates production of the pro-inflammatory cytokine IL-1β. To overcome both the variability among primary immune cells and the limitations of genetic manipulation of differentiated human or murine macrophages, we developed a simplified, reliable and relevant cell-based model for studying the NLRP3 inflammasome using the undifferentiated human myelomonocytic cell line THP1. Undifferentiated THP1 cells constitutively express NLRP3, and NLRP3 inflammasome activation occurred in response to canonical NLRP3 activation stimuli including nigericin, ATP, and urea crystals, culminating in pro-IL-1β cleavage, extracellular release of mature IL-1β, and pyroptosis. We used this THP1 cell system to investigate potential targets of the potent, NLRP3 inflammasome selective inhibitor CP-456,773. We optimized a viral shRNA transduction method for gene expression knockdown (KD), and the KD of NLRP3 itself eliminated inflammasome activation and IL-1β production. NLRP3 inflammasome activation and CP-453,773 pharmacology were not altered in ABCb7- or ABCb10-deficient THP1 cells, eliminating these gene products as candidate pharmacological targets of CP-453,773. For ABCb10, we confirmed our results using CRISPR/CAS9-mediated ABCb10 knockout (KO) THP1 sub-lines. In summary, undifferentiated THP1 cells are fully competent for activation of the NLRP3 inflammasome and production of IL-1β, without differentiation into macrophages, and we describe optimized KD and KO methodologies to manipulate gene expression in these cells. As an example of the utility of undifferentiated THP1 cells for investigations into the biology of the NLRP3 inflammasome, we have used this cell system to rule out ABCb7 and ABCb10 as potential targets of the NLRP3 inflammasome inhibitor CP-453,773.

TLR4 interactor with leucine-rich repeats (TRIL) is a brain-enriched accessory protein that is important in TLR3 and TLR4 signaling. In this study, we generated Tril−/− mice and examined TLR responses in vitro and in vivo. We found a role for TRIL in both TLR4 and TLR3 signaling in mixed glial cells, consistent with the high level of expression of TRIL in these cells. We also found that TRIL is a modulator of the innate immune response to LPS challenge and Escherichia coli infection in vivo. Tril−/− mice produce lower levels of multiple proinflammatory cytokines and chemokines specifically within the brain after E. coli and LPS challenge. Collectively, these data uncover TRIL as a mediator of innate immune responses within the brain, where it enhances neuronal cytokine responses to infection.

Intrarenal crystal formation activates the Nlrp3 inflammasome in myeloid cells and triggers a profound inflammatory response. Here, we studied whether a specific inhibitor of the Nlrp3 inflammasome, CP-456,773, can prevent kidney fibrosis in a murine model of crystal nephropathy induced by diets rich in oxalate or adenine. Inflammasome activation in renal dendritic cells and the resulting interleukin (IL)-1β and IL-18 production were markedly reduced by CP-456,773 treatment both ex vivo and in vivo. We directly visualized intrarenal inflammasome activation and its inhibition by CP-456,773 in vivo by adoptive transfer of bone marrow cells transduced with interleukin-1β-Gaussia luciferase, a proteolytic luciferase-based reporter for inflammasome activation, into irradiated mice. CP-456,773 treatment strongly attenuated kidney fibrosis when given early in the genesis of crystal nephropathy, but was unable to reverse established crystal-induced fibrosis. The urinary IL-18 concentration appeared to be a useful noninvasive biomarker for renal inflammasome activation. Finally, NLRP3 inhibition did not compromise adaptive immune responses as previously reported for the global inhibition of IL-1 signaling. Thus, early NLRP3 inhibition by CP-456,773 may be an effective treatment for crystal nephropathy. Use of iGLuc transfected cells introduces a novel imaging technique for inflammasome activation in mice.

A critical component of innate immune response to infection and tissue damage is the NACHT, LRR, and PYD domains–containing protein 3 (NLRP3) inflammasome, and this pathway and its activation products have been implicated in the pathophysiology of a variety of diseases. NLRP3 inflammasome activation leads to the cleavage of pro–IL-1β and pro–IL-18, as well as the subsequent release of biologically active IL-1β, IL-18, and other soluble mediators of inflammation. In this study, we further define the pharmacology of the previously reported NLRP3 inflammasome–selective, IL-1β processing inhibitor CP-456,773 (also known as MCC950), and we demonstrate its efficacy in two in vivo models of inflammation. Specifically, we show that in human and mouse innate immune cells CP-456,773 is an inhibitor of the cellular release of IL-1β, IL-1α, and IL-18, that CP-456,773 prevents inflammasome activation induced by disease-relevant soluble and crystalline NLRP3 stimuli, and that CP-456,773 inhibits R848- and imiquimod-induced IL-1β release. In mice, CP-456,773 demonstrates potent inhibition of the release of proinflammatory cytokines following acute i.p. challenge with LPS plus ATP in a manner that is proportional to the free/unbound concentrations of the drug, thereby establishing an in vivo pharmacokinetic/pharmacodynamic model for CP-456,773. Furthermore, CP-456,773 reduces ear swelling in an imiquimod cream–induced mouse model of skin inflammation, and it reduces airway inflammation in mice following acute challenge with house dust mite extract. These data implicate the NLRP3 inflammasome in the pathogenesis of dermal and airway inflammation, and they highlight the utility of CP-456,773 for interrogating the contribution of the NLRP3 inflammasome and its outputs in preclinical models of inflammation and disease.

Mice lacking Tbk1 activity exhibit immune cell infiltrates in multiple tissues, altered circulating immune cell compartments, and increased susceptibility to LPS-induced lethality. TBK1 is critical for immunity against microbial pathogens that activate TLR4- and TLR3-dependent signaling pathways. To address the role of TBK1 in inflammation, mice were generated that harbor two copies of a mutant Tbk1 allele. This Tbk1Δ allele encodes a truncated Tbk1Δ protein that is catalytically inactive and expressed at very low levels. Upon LPS stimulation, macrophages from Tbk1Δ/Δ mice produce normal levels of proinflammatory cytokines (e.g., TNF-α), but IFN-β and RANTES expression and IRF3 DNA-binding activity are ablated. Three-month-old Tbk1Δ/Δ mice exhibit mononuclear and granulomatous cell infiltrates in multiple organs and inflammatory cell infiltrates in their skin, and they harbor a 2-fold greater amount of circulating monocytes than their Tbk1+/+ and Tbk1+/Δ littermates. Skin from 2-week-old Tbk1Δ/Δ mice is characterized by reactive changes, including hyperkeratosis, hyperplasia, necrosis, inflammatory cell infiltrates, and edema. In response to LPS challenge, 3-month-old Tbk1Δ/Δ mice die more quickly and in greater numbers than their Tbk1+/+ and Tbk1+/Δ counterparts. This lethality is accompanied by an overproduction of several proinflammatory cytokines in the serum of Tbk1Δ/Δ mice, including TNF-α, GM-CSF, IL-6, and KC. This overproduction of serum cytokines in Tbk1Δ/Δ mice following LPS challenge and their increased susceptibility to LPS-induced lethality may result from the reactions of their larger circulating monocyte compartment and their greater numbers of extravasated immune cells.

Tpl2 (cot/MAP3K8) is an upstream kinase of MEK in the ERK pathway. It plays an important role in Tumor Necrosis Factor-alpha (TNF-alpha) production and signaling. We have discovered that 8-halo-4-(3-chloro-4-fluoro-phenylamino)-6-[(1H-[1,2,3]triazol-4-ylmethyl)-amino]-quinoline-3-carbonitriles (4) are potent inhibitors of this enzyme. In order to improve the inhibition of TNF-alpha production in LPS-stimulated human blood, a series of analogs with a variety of substitutions around the triazole moiety were studied. We found that a cyclic amine group appended to the triazole ring could considerably enhance potency, aqueous solubility, and cell membrane permeability. Optimization of these cyclic amine groups led to the identification of 8-chloro-4-(3-chloro-4-fluorophenylamino)-6-((1-(1-ethylpiperidin-4-yl)-1H-1,2,3-triazol-4-yl)methylamino)quinoline-3-carbonitrile (34). In a LPS-stimulated rat inflammation model, compound 34 showed good efficacy in inhibiting TNF-alpha production.

We have previously reported the discovery and initial SAR of the [1,7]naphthyridine-3-carbonitriles and quinoline-3-carbonitriles as Tumor Progression Loci-2 (Tpl2) kinase inhibitors. In this paper, we report new SAR efforts which have led to the identification of 4-alkylamino-[1,7]naphthyridine-3-carbonitriles. These compounds show good in vitro and in vivo activity against Tpl2 and improved pharmacokinetic properties. In addition they are highly selective for Tpl2 kinase over other kinases, for example, EGFR, MEK, MK2, and p38. Lead compound 4-cycloheptylamino-6-[(pyridin-3-ylmethyl)-amino]-[1,7]naphthyridine-3-carbonitrile (30) was efficacious in a rat model of LPS-induced TNF-alpha production.

Tumor progression loci-2 (Tpl2) (Cot/MAP3K8) is a serine/threonine kinase in the MAP3K family directly upstream of MEK. Recent studies using Tpl2 knockout mice have indicated an important role for Tpl2 in the lipopolysaccharide (LPS) induced production of tumor necrosis factor alpha (TNF-alpha) and other proinflammatory cytokines involved in diseases such as rheumatoid arthritis. Initial 4-anilino-6-aminoquinoline-3-carbonitrile leads showed poor selectivity for Tpl2 over epidermal growth factor receptor (EGFR) kinase. Using molecular modeling and crystallographic data of the EGFR kinase domain with and without an EGFR kinase-specific 4-anilinoquinazoline inhibitor (erlotinib, Tarceva), we hypothesized that we could diminish the inhibition of EGFR kinase by substitution at the C-8 position of our 4-anilino-6-aminoquinoline-3-carbonitrile leads. The 8-substituted-4-anilino-6-aminoquinoline-3-carbonitriles were prepared from the appropriate 2-substituted 4-nitroanilines. Modifications to the C-6 and C-8 positions led to the identification of compounds with increased inhibition of TNF-alpha release from LPS-stimulated rat and human blood, and these analogues were also highly selective for Tpl2 kinase over EGFR kinase. Further structure-activity based modifications led to the identification of 8-bromo-4-(3-chloro-4-fluorophenylamino)-6-[(1-methyl-1H-imidazol-4-yl)methylamino]quinoline-3-carbonitrile, which demonstrated in vitro as well as in vivo efficacy in inhibition of LPS-induced TNF-alpha production.

The synthesis and structure-activity studies of a series of quinoline-3-carbonitriles as inhibitors of Tpl2 kinase are described. Potent inhibitors of Tpl2 kinase with selectivity against a panel of selected kinases in enzymatic assays and specificity in cell-based phosphorylation assays in LPS-treated human monocytes were identified. Selected inhibitors with moderate activity in human whole blood assay effectively inhibited LPS/D-Gal induced TNFalpha release when administered intraperitoneally in mice.

The synthesis and structure-activity studies of a series of 6-substituted-4-anilino-[1,7]-naphthyridine-3-carbonitriles as inhibitors of Tpl2 kinase are described. The early exploratory work described here may lead to the discovery of compounds with significant therapeutic potential for treating rheumatoid arthritis and other inflammatory diseases.

Tumor necrosis factor α (TNFα) is a proinflammatory cytokine with pleiotropic immunological and biological activities. TNFα signaling is triggered by the engagement of soluble TNFα to two types of cell surface receptors, TNFR1 and TNFR2. This recruits cytosolic proteins to the intracellular domains of the receptors and initiates signaling to downstream effectors. In this study, we used a proteomic approach to identify these cytosolic proteins from affinity-purified, endogenous TNFα·TNFR complexes in human myelomonocytic U937 cells. Seven proteins were identified, including TRADD, TRAP2, and TRAF2, which are three proteins known to be recruited to TNFα receptors. NAK, RasGAP3, TRCP1, and TRCP2 were also identified. We further showed that NAK is recruited to TNFR1 in a temporally regulated and TNFα-dependent manner and that it mediates the TNFα-induced production of the chemokine RANTES (regulated on activation normal T cell expressed and secreted). These data demonstrate that NAK is a component of the TNFα·TNFR1 signaling complex and confirm the physiological role of NAK in the TNFα-mediated response.

The c-Jun NH 2 -terminal kinase (JNK) group of mitogen-activated protein kinases is activated in response to a wide array of cellular stresses and proinflammatory cytokines. Roles for JNK in the developing nervous system and T-cell-mediated immunity have been established by detailed studies of mice with compound mutations in the Jnk genes. However, little is known concerning the roles of JNK in other mammalian tissues. Mice lacking both of the ubiquitously expressed isoforms (JNK1 and -2) die during midgestation with neural tube closure defects and brain abnormalities. Here we show that JNK-deficient mice exhibit delayed epithelial development in the epidermis, intestines, and lungs. In addition, JNK-deficient mice exhibit an eyelid closure defect associated with markedly reduced epidermal growth factor (EGF) receptor function, and loss of expression of the ligand EGF. We further demonstrate that adult mice lacking either JNK1 or -2 display striking differences in epidermal proliferation and differentiation, indicative of distinct roles for these kinases in the skin. We conclude that JNK is necessary for epithelial morphogenesis and is an essential regulator of signal transduction by the EGF receptor in the epidermis.

The c-Jun NH2-terminal kinase (JNK) group of mitogen-activated protein kinases is stimulated in response to a wide array of cellular stresses and proinflammatory cytokines. Mice lacking individual members of theJnkfamily (Jnk1,Jnk2, andJnk3) are viable and survive without overt structural abnormalities. Here we show that mice with a compound deficiency inJnkexpression can survive to birth, but fail to close the optic fissure (retinal coloboma). We demonstrate that JNK initiates a cytokine cascade of bone morphogenetic protein-4 (BMP4) and sonic hedgehog (Shh) that induces the expression of the paired-like homeobox transcription factor Pax2 and closure of the optic fissure. Interestingly, the role of JNK to regulate BMP4 expression during optic fissure closure is conserved inDrosophiladuring dorsal closure, a related morphogenetic process that requires JNK-regulated expression of the BMP4 ortholog Decapentaplegic (Dpp).

Mitogen-activated protein (MAP) kinases have been implicated as important mediators of the inflammatory response. Here we report that c-Jun NH(2)-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 MAP kinase activities are reprogrammed during the IL-6 induced macrophage-like differentiation of the murine myeloid M1 cell line. Moreover, p38 inhibition upregulates JNK and ERK activity in M1 cells and in thioglycollate-elicited peritoneal exudate macrophages. IL-6-induced M1 differentiation also induces expression of the anti-inflammatory cytokine IL-10, and p38 inhibition potentiates this increase in IL-10 expression in an ERK-dependent manner. Thus, we speculate that during inflammatory conditions in vivo macrophage p38 may regulate JNK and ERK activity and inhibit IL-10 expression. These data highlight the importance of p38 in the molecular mechanisms of macrophage function.

Background Toll-like receptor 4 (TLR4) is activated by bacterial endotoxin, a pathogen-associated molecular pattern (PAMP). It has been suggested that TLR4 can also be activated by damage-associated molecular pattern (DAMP) proteins such as HSP70. It remains a challenge to provide unequivocal evidence that DAMP proteins themselves play a role in TLR4 activation, as the DAMP proteins used are often contaminated with endotoxin and other TLR ligands introduced during protein expression and/or purification. Results Here we report that the activation of TLR4 on primary human macrophage cultures by recombinant HSP70 is not solely due to contaminating endotoxin. Polymyxin B pretreatment of HSP70 preparations to neutralize contaminating endotoxin caused significant reductions in the amount of TNF-α induced by the recombinant protein. However, digestion of HSP70 with Proteinase K-agarose beads also dramatically reduced the TNF-α response of macrophages to HSP70, while leaving levels of contaminating endotoxin largely unchanged relative to controls. Conclusions These results indicate that the stimulatory effect of recombinant HSP70 requires both the presence of endotoxin and structural integrity of the heat shock protein itself.

We report here the synthesis and SAR of a new series of thieno[3,2-d]pyrimidines as potent Tpl2 kinase inhibitors. The proposed binding mode suggests the potential flipped binding mode depending on the substitution. Biacore studies show evidence of binding of these molecules to the protein kinase. The kinome inhibition profile of these molecules suggests good selectivity.

Synthesis, modeling and structure–activity relationship of indazoles as inhibitors of Tpl2 kinase are described. From a high throughput screening effort, we identified an indazole hit compound 5 that has a single digit micromolar Tpl2 activity. Through SAR modifications at the C3 and C5 positions of the indazole, we discovered compound 31 with good potency in LANCE assay and cell-based p -Erk assay.

Although Toll-like receptor 9 (TLR9) has been implicated in regulating cytokine and type I interferon (IFN) production during malaria in humans and mice, the high AT content of the Plasmodium falciparum genome prompted us to examine the possibility that malarial DNA triggered TLR9-independent DNA sensing pathways. Over 6000 ATTTTTAC (“AT-rich”) motifs are present in the genome of P. falciparum, which we show here potently induce type I IFNs. Parasite DNA, parasitized erythrocytes and oligonucleotides containing the AT-r motif induce type I IFNs via a pathway that did not involve previously described sensors including TLR9, DAI, RNA polymerase-III or IFI16/p204. Rather, AT-rich DNA sensing involved an unknown receptor that coupled to STING, TBK1 and IRF3-IRF7 signaling pathway. Mice lacking both IRF3 and IRF7, the kinase TBK1 or the type I IFN receptor were resistant to otherwise lethal cerebral malaria. Collectively, these observations implicate AT-rich DNA sensing via STING, TBK1 and IRF3-IRF7 in P. falciparum malaria.

Objectives. To investigate the expression and function of triggering receptor expressed on myeloid cells-1 (TREM-1) in the synovium of human RA patients as well as the level of soluble TREM-1 in the plasma of RA patients. Methods. Twenty-four RA synovial samples were analysed by gene expression oligonucleotide microarrays. Expression levels of TREM-1 mRNA in murine CIA paws were determined by quantitative PCR (qPCR). TREM-1 protein expression was detected by immunohistochemistry in five RA synovial samples and two OA synovial samples. TREM-1-positive cells from five RA synovial tissues were analysed by FACS staining to determine the cell type. Activation of TREM-1 was tested in five RA synovial samples. Soluble TREM-1 was measured in serum from 32 RA patients. Results. The expression of TREM-1 mRNA was found to increase 6.5-fold in RA synovial samples, whereas it was increased 132-fold in CIA paws. Increased numbers of TREM-1-positive cells were seen in RA synovium sections and these cells co-expressed CD14. Using a TREM-1-activating cross-linking antibody in RA synovial cultures, multiple pro-inflammatory cytokines were induced. The average amount of soluble TREM-1 in plasma from RA patients was found to be higher than that in plasma from healthy volunteers. Conclusions. These findings suggest that the presence of high levels of functionally active TREM-1 in RA synovium may contribute to the development or maintenance of RA, or both. Inhibiting TREM-1 activity may, therefore, have a therapeutic effect on RA. High levels of soluble TREM-1 in the plasma of RA patients compared with healthy volunteers may indicate disease activity.

Tumor necrosis factor alpha (TNFalpha) is a pro-inflammatory cytokine that controls the initiation and progression of inflammatory diseases such as rheumatoid arthritis. Tpl2 is a MAPKKK in the MAPK (i.e. ERK) pathway, and the Tpl2-MEK-ERK signaling pathway is activated by the pro-inflammatory mediators TNFalpha, interleukin (IL)-1beta, and bacterial endotoxin (lipopolysaccharide (LPS)). Moreover, Tpl2 is required for TNFalpha expression. Thus, pharmacologic inhibition of Tpl2 should be a valid approach to therapeutic intervention in the pathogenesis of rheumatoid arthritis and other inflammatory diseases in humans. We have developed a series of highly selective and potent Tpl2 inhibitors, and in the present study we have used these inhibitors to demonstrate that the catalytic activity of Tpl2 is required for the LPS-induced activation of MEK and ERK in primary human monocytes. These inhibitors selectively target Tpl2 in these cells, and they block LPS- and IL-1beta-induced TNFalpha production in both primary human monocytes and human blood. In rheumatoid arthritis fibroblast-like synoviocytes these inhibitors block ERK activation, cyclooxygenase-2 expression, and the production of IL-6, IL-8, and prostaglandin E(2), and the matrix metalloproteinases MMP-1 and MMP-3. Taken together, our results show that inhibition of Tpl2 in primary human cell types can decrease the production of TNFalpha and other pro-inflammatory mediators during inflammatory events, and they further support the notion that Tpl2 is an appropriate therapeutic target for rheumatoid arthritis and other human inflammatory diseases.

Introduction Tumor necrosis factor-alpha (TNFα) participates in many inflammatory processes. TNFα modulators show beneficial effects for the treatment of many diseases including rheumatoid arthritis. The purpose of this study was to validate a rat pharmacokinetic/pharmacodynamic (PK/PD) model for rapid assessment of drug candidates that intended to interrupt TNFα synthesis or release. Methods Rats received intravenous (IV) or oral administrations of test article or dose vehicle, followed by LPS challenge. Plasma levels of test article and TNFα were determined. The areas under the concentration–time curves (AUCdrug and AUCTNFα) were calculated. The overall percentage of inhibition on TNFα release in vivo was calculated by comparing AUCTNFα of the test article treated group against that for the vehicle control group. Results The dosing vehicles tested in this study did not increase plasma TNFα level. At IV dose of up to 100 μg/kg, LPS did not alter the pharmacokinetics of the compound tested. Using a selective TNFα converting enzyme (TACE) inhibitor as model compound, this PK/PD model demonstrated its ability to correlate plasma test article concentration with its biological activity of lowering the LPS-induced TNFα plasma levels in vivo. Discussion A rat PK/PD model for evaluation of the effect of drug candidates on LPS-induced TNFα synthesis and/or release has been investigated. This model provides integrated information on pharmacokinetics and in vivo potency of the test articles.

Tumor necrosis factor alpha (TNFalpha) is a proinflammatory cytokine with pleiotropic immunological and biological activities. TNFalpha signaling is triggered by the engagement of soluble TNFalpha to two types of cell surface receptors, TNFR1 and TNFR2. This recruits cytosolic proteins to the intracellular domains of the receptors and initiates signaling to downstream effectors. In this study, we used a proteomic approach to identify these cytosolic proteins from affinity-purified, endogenous TNFalpha.TNFR complexes in human myelomonocytic U937 cells. Seven proteins were identified, including TRADD, TRAP2, and TRAF2, which are three proteins known to be recruited to TNFalpha receptors. NAK, RasGAP3, TRCP1, and TRCP2 were also identified. We further showed that NAK is recruited to TNFR1 in a temporally regulated and TNFalpha-dependent manner and that it mediates the TNFalpha-induced production of the chemokine RANTES (regulated on activation normal T cell expressed and secreted). These data demonstrate that NAK is a component of the TNFalpha.TNFR1 signaling complex and confirm the physiological role of NAK in the TNFalpha-mediated response.

NO, produced from l-arginine in a reaction catalyzed by NO synthase, is an endogenous free radical with multiple functions in mammalian cells. Here, we demonstrate that endogenously produced NO can suppress c-Jun N-terminal kinase (JNK) activation in intact cells. Treatment of BV-2 murine microglial cells with IFN-γ induced endogenous NO production, concomitantly suppressing JNK1 activation. Similarly, IFN-γ induced suppression of JNK1 activation in RAW264.7 murine macrophage cells and rat alveolar macrophages. The IFN-γ-induced suppression of JNK1 activation in BV-2, RAW264.7, or rat alveolar macrophage cells was completely prevented by NG-nitro-l-arginine, a NO synthase inhibitor. Interestingly, the IFN-γ-induced suppression of JNK1 activation was not affected by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of guanylyl cyclase. 8-Bromo-cGMP, a membrane-permeant analogue of cGMP, did not change JNK1 activation in intact cells either. In contrast, S-nitro-N-acetyl-dl-penicillamine (SNAP), a NO donor, inhibited JNK1 activity in vitro. Furthermore, a thiol reducing agent, DTT, reversed not only the in vitro inhibition of JNK1 activity by SNAP but also the in vivo suppression of JNK1 activity by IFN-γ. Substitution of serine for cysteine-116 in JNK1 abolished the inhibitory effect of IFN-γ or SNAP on JNK1 activity in vivo or in vitro, respectively. Moreover, IFN-γ enhanced endogenous S-nitrosylation of JNK1 in RAW264.7 cells. Collectively, our data suggest that endogenous NO mediates the IFN-γ-induced suppression of JNK1 activation in macrophage cells by means of a thiol-redox mechanism.