Differential susceptibility of activated macrophage cytotoxic effector reactions to the suppressive effects of transforming growth factor-beta 1

We examined the effects of TGF-beta 1 on induction of several activated macrophage antimicrobial activities against the protozoan parasite Leishmania, and on induction of tumoricidal activity against the fibrosarcoma tumor target 1023. TGF-beta by itself did not affect the viability of either the intracellular or extracellular target in concentrations up to 200 ng/ml. As little as 1 ng/ml TGF-beta, however, suppressed more than 70% of the intracellular killing activity of macrophages treated with lymphokines. In contrast, more than 100 ng/ml TGF-beta was required to suppress intracellular killing by cells activated with an equivalent amount of recombinant IFN-gamma. Addition of TGF-beta for up to 30 min after exposure to activation factors significantly reduced macrophage killing of intracellular parasites. Pretreatment of macrophages with TGF-beta was even more effective: treatment of cells with TGF-beta for 4 h before addition of activation factors abolished all macrophage intracellular killing activity. Regardless of treatment sequence, however, TGF-beta had absolutely no effect, at any concentration tested, on activated macrophage resistance to infection induced by lymphokines or by the cooperative interaction of IFN-gamma and IL-4. Effects of TGF-beta on tumoricidal activity of activated macrophages was intermediate to that of its effects on intracellular killing or resistance to infection. Lymphokine-induced tumor cytotoxicity was marginally (25%) affected by TGF-beta; 200 ng/ml was able to suppress IFN-gamma-induced tumoricidal activity by 40%. Thus, TGF-beta dramatically suppressed certain activated macrophage cytotoxic effector reactions, but was only partially or not at all effective against others, even when the same activation agent (IFN-gamma) was used. The biochemical target for TGF-beta suppressive activity in these reactions may be the pathway for nitric oxide production from L-arginine, because TGF-beta also inhibited the generation of nitric oxide by cytokine-activated macrophages.     

Mycobacterium avium inhibition of IFN-gamma signaling in mouse macrophages: Toll-like receptor 2 stimulation increases expression of dominant-negative STAT1 beta by mRNA stabilization

Mycobacterial infections of macrophages have been shown to inhibit the ability of the macrophage to respond to IFN-gamma. We previously reported that Mycobacterium avium infection of mouse macrophages decreases IFN-gamma-induced STAT1 tyrosine phosphorylation and STAT1 DNA binding. Because macrophages respond to M. avium through Toll-like receptor 2 (TLR2), we determined whether TLR2 stimulation inhibits the response to IFN-gamma. Treatment of mouse RAW264.7 macrophages with TLR2 agonists inhibited the induction of IFN-gamma-inducible genes by IFN-gamma. In contrast to M. avium infection, TLR2 agonists did not inhibit the IFN-gamma induction of DNA-binding activity of STAT1 and the tyrosine phosphorylation of STAT1alpha. Instead, IFN-gamma induction of RAW264.7 cells treated with TLR2 agonists resulted in an increase in the tyrosine phosphorylation of the dominant-negative STAT1beta. TLR2 stimulation of RAW264.7 cells increased both STAT1beta protein and mRNA expression, suggesting that the increased STAT1beta phosphorylation results from increased STAT1beta expression. Because STAT1alpha and STAT1beta mRNA have different 3' untranslated regions, and 3' untranslated regions can regulate mRNA stability, we examined the effects of TLR2 stimulation on mRNA stability. TLR2 stimulation of RAW264.7 cells increased the stability of STAT1beta mRNA, while not affecting the stability of STAT1alpha mRNA. The ability of STAT1beta to function as a dominant negative was confirmed by overexpression of STAT1beta in RAW264.7 macrophages by transient transfection, which inhibited IFN-gamma-induced gene expression. These findings suggest that M. avium infection of mouse macrophages inhibits IFN-gamma signaling through a TLR2-dependent increase in STAT1beta expression by mRNA stablization and a TLR2-independent inhibition of STAT1 tyrosine phosphorylation.     

TGF-beta utilizes SMAD3 to inhibit CD16-mediated IFN-gamma production and antibody-dependent cellular cytotoxicity in human NK cells

TGF-beta can be a potent suppressor of lymphocyte effector cell functions and can mediate these effects via distinct molecular pathways. The role of TGF-beta in regulating CD16-mediated NK cell IFN-gamma production and antibody-dependent cellular cytotoxicity (ADCC) is unclear, as are the signaling pathways that may be utilized. Treatment of primary human NK cells with TGF-beta inhibited IFN-gamma production induced by CD16 activation with or without IL-12 or IL-2, and it did so without affecting the phosphorylation/activation of MAP kinases ERK and p38, as well as STAT4. TGF-beta treatment induced SMAD3 phosphorylation, and ectopic overexpression of SMAD3 resulted in a significant decrease in IFN-gamma gene expression following CD16 activation with or without IL-12 or IL-2. Likewise, NK cells obtained from smad3(-/-) mice produced more IFN-gamma in response to CD16 activation plus IL-12 when compared with NK cells obtained from wild-type mice. Coactivation of human NK cells via CD16 and IL-12 induced expression of T-BET, the positive regulator of IFN-gamma, and T-BET was suppressed by TGF-beta and by SMAD3 overexpression. An extended treatment of primary NK cells with TGF-beta was required to inhibit ADCC, and it did so by inhibiting granzyme A and granzyme B expression. This effect was accentuated in cells overexpressing SMAD3. Collectively, our results indicate that TGF-beta inhibits CD16-mediated human NK cell IFN-gamma production and ADCC, and these effects are mediated via SMAD3.     

Essential role of STAT1 in caspase-independent cell death of activated macrophages through the p38 mitogen-activated protein kinase/STAT1/reactive oxygen species pathway

Unlike other immune cells, activation of macrophages by stimulating agents, such as lipopolysaccharide (LPS), confers significant resistance to many apoptotic stimuli, but the underlying mechanism of this phenomenon remains largely unknown. Here, we demonstrate that LPS-induced early caspase activation is essential for macrophage survival because blocking caspase activation with a pancaspase inhibitor (zVAD [benzyloxycarbonyl-Val-Ala-Asp]) rapidly induced death of activated macrophages. This type of death process by zVAD/LPS was principally mediated by intracellular generation of superoxide. STAT1 knockout macrophages demonstrated profoundly decreased superoxide production and were resistant to treatment with zVAD/LPS, indicating the crucial involvement of STAT1 in macrophage death by zVAD/LPS. STAT1 level and activity were reciprocally regulated by caspase activation and were associated with cell death. Activation of STAT1 was critically dependent upon serine phosphorylation induced by p38 mitogen-activated protein kinase (MAPK) because a p38 MAPK inhibitor nullified STAT1 serine phosphorylation, reactive oxygen species (ROS) production, and macrophage death by zVAD/LPS. Conversely, p38 MAPK activation was dependent upon superoxide and was also nullified in STAT1 knockout macrophages, probably due to impaired generation of superoxide. Our findings collectively indicate that STAT1 signaling modulates intracellular oxidative stress in activated macrophages through a positive-feedback mechanism involving the p38 MAPK/STAT1/ROS pathway, which is interrupted by caspase activation. Furthermore, our study may provide significant insights in regards to the unanticipated critical role of STAT1 in the caspase-independent death pathway.     

Leishmania inhibits STAT1-mediated IFN-gamma signaling in macrophages: increased tyrosine phosphorylation of dominant negative STAT1beta by Leishmania mexicana

Previous studies have demonstrated that Leishmania donovani attenuates STAT1-mediated signaling in macrophages; however it is not clear whether other species of Leishmania, which cause cutaneous disease, also interfere with macrophage IFN-gamma signaling. Therefore, we determined the effect of Leishmania major and Leishmania mexicana infection on STAT1-mediated IFN-gamma signaling pathway in J774A.1 and RAW264.7 macrophages. We found that both L. major and L. mexicana suppressed IFNgammaRalpha (alpha subunit of interferon gamma receptor) and IFN-gammaRbeta (beta subunit of interferon gamma receptor) expression, reduced levels of total Jak1 and Jak2, and down-regulated IFN-gamma-induced Jak1, Jak2 and STAT1 activation. The effect of L. mexicana infection on Jak1, Jak2 and STAT1 activation was more profound when compared with L. major. Although tyrosine phosphorylation of STAT1alpha was decreased in IFN-gamma stimulated macrophages infected with L. major or L. mexicana, those infected with L. mexicana showed a significant increase in phosphorylation of the dominant negative STAT1beta. These findings indicate that L. major and L. mexicana attenuate STAT1-mediated IFN-gamma signaling in macrophages. Furthermore, they also demonstrate that L. mexicana preferentially enhances tyrosine phosphorylation of dominant negative STAT1beta, which may be one of the several survival mechanisms used by this parasite to evade the host defense mechanisms.     

TGF-beta does not inhibit IL-12- and IL-2-induced activation of Janus kinases and STATs

The immune system is an important target for the cytokine TGF-beta1, whose actions on lymphocytes are largely inhibitory. TGF-beta has been reported to inhibit IL-12- and IL-2-induced cell proliferation and IFN-gamma production by T cells and NK cells; however, the mechanisms of inhibition have not been clearly defined. It has been suggested by some studies that TGF-beta blocks cytokine-induced Janus kinase (JAK) and STAT activation, as in the case of IL-2. In contrast, other studies with cytokines like IFN-gamma have not found such an inhibition. The effect of TGF-beta on the IL-12-signaling pathway has not been addressed. We examined this and found that TGF-beta1 did not have any effect on IL-12-induced phosphorylation of JAK2, TYK2, and STAT4 although TGF-beta1 inhibited IL-2- and IL-12-induced IFN-gamma production. Similarly, but in contrast to previous reports, we found that TGF-beta1 did not inhibit IL-2-induced phosphorylation of JAK1, JAK3, and STAT5A. Furthermore, gel shift analysis showed that TGF-beta1 did not prevent activated STAT4 and STAT5A from binding to DNA. Our results demonstrate that the inhibitory effects of TGF-beta on IL-2- and IL-12-induced biological activities are not attributable to inhibition of activation of JAKs and STATs. Rather, our data suggest the existence of alternative mechanisms of inhibition by TGF-beta.     

Interleukin-1 inhibits the induction of insulin-like growth factor-I by growth hormone in CWSV-1 hepatocytes

Sepsis results in hepatic "growth hormone (GH) resistance" with reductions in plasma IGF-I despite a two- to fourfold increase in circulating GH. In this study, we examine the effects of IL-1 on GH receptor (GHR) expression, GH signaling (via the JAK/STAT and MAPK pathways), and the induction of gene expression [IGF-I mRNA and serine protease inhibitor (Spi) 2.1] by GH in CWSV-1 hepatocytes. Incubation of cells with IL-1beta (10 ng/ml, 24 h) had no effect on the relative abundance of GHR or signaling proteins JAK2, STAT5b, and ERK1/2 in cell lysates. Baseline phosphorylation of GHR, JAK2, STAT5b, and ERK1/2 was minimal. After GH stimulation, tyrosine phosphorylation of GHR, JAK2, STAT5b, and ERK1/2 increased 2- to 10-fold. However, neither the time course nor the magnitude of GHR, JAK2, and ERK1/2 phosphorylation by GH were significantly altered by IL-1. The GH-induced translocation of STAT5b to the nucleus was not prevented by IL-1. Although phosphorylated STAT5 in nuclear extracts from GH + IL-1 cells was decreased by 24% (vs. controls) 15 min after GH stimulation, this did not result in reduced STAT5-DNA binding activity. Pretreatment with IL-1 did not significantly decrease IGF-I mRNA stability. We conclude that IL-1 only minimally affects the time course of JAK2/STAT5 and MAPK signaling by GH. Therefore, an inhibitory effect of IL-1 on IGF-I and Spi 2.1 mRNA synthesis by GH represents the most likely mechanism for IL-1-mediated GH resistance.     

Rho plays a key role in TGF-beta1-induced cytoskeletal rearrangement in human retinal pigment epithelium

Proliferative vitroretinopathy (PVR) is caused by retinal pigment epithelial (RPE) cell proliferation and transformation into fibrotic cells that produce extracellular matrix (ECM) components. Transforming growth factor beta1 (TGF-beta1) is known to play an important role in PVR pathogenesis. To determine how TGF-beta1 mediates the pathogenic changes in RPE cells, we characterized the effects of TGF-beta1 on the morphology, ECM accumulation, and stress fiber formation of ARPE-19 cells, a human RPE cell line. We then elucidated the signaling pathways that mediate these effects. Serum-starved ARPE-19 cells were incubated with 10 ng/ml TGF-beta1 and their morphological changes were examined by phase-contrast microscopy. Actin reorganization was examined by immunochemistry and confocal microscopy. Protein phosphorylation was analyzed by Western blot analysis. TGF-beta1 treatment induced cytoskeleton reorganization, alpha-SMA expression, increased the phosphorylation of ERK, Smad2/3, and AKT, and activated RhoA and Rac1. Cytoskeletal rearrangement was prevented by pretreatment with a Rho inhibitor and by expression of a dominant negative form of Rho. TGF-beta1 also increased LIM kinase and cofilin phosphorylation and the Rho inhibitor blocked this effect. We propose that TGF-beta1 induces human RPE cells to undergo cytoskeletal actin rearrangement via Rho GTPase-dependent pathways that modulate LIM kinase and cofilin activity. This inhibits actin depolymerization and induces the cytoskeletal rearrangements in RPE cells that result in the characteristic features of PVR.     

Both the suppressor of cytokine signaling 1 (SOCS-1) kinase inhibitory region and SOCS-1 mimetic bind to JAK2 autophosphorylation site: implications for the development of a SOCS-1 antagonist

Suppressor of cytokine signaling (SOCS)-1 protein modulates signaling by IFN-gamma by binding to the autophosphorylation site of JAK2 and by targeting bound JAK2 to the proteosome for degradation. We have developed a small tyrosine kinase inhibitor peptide (Tkip) that is a SOCS-1 mimetic. Tkip is compared in this study with the kinase inhibitory region (KIR) of SOCS-1 for JAK2 recognition, inhibition of kinase activity, and regulation of IFN-gamma-induced biological activity. Tkip and a peptide corresponding to the KIR of SOCS-1, ((53))DTHFRTFRSHSDYRRI((68)) (SOCS1-KIR), both bound similarly to the autophosphorylation site of JAK2, JAK2(1001-1013). The peptides also bound to JAK2 peptide phosphorylated at Tyr(1007), pJAK2(1001-1013). Dose-response competitions suggest that Tkip and SOCS1-KIR similarly recognize the autophosphorylation site of JAK2, but probably not precisely the same way. Although Tkip inhibited JAK2 autophosphorylation as well as IFN-gamma-induced STAT1-alpha phosphorylation, SOCS1-KIR, like SOCS-1, did not inhibit JAK2 autophosphorylation but inhibited STAT1-alpha activation. Both Tkip and SOCS1-KIR inhibited IFN-gamma activation of Raw 264.7 murine macrophages and inhibited Ag-specific splenocyte proliferation. The fact that SOCS1-KIR binds to pJAK2(1001-1013) suggests that the JAK2 peptide could function as an antagonist of SOCS-1. Thus, pJAK2(1001-1013) enhanced suboptimal IFN-gamma activity, blocked SOCS-1-induced inhibition of STAT3 phosphorylation in IL-6-treated cells, enhanced IFN-gamma activation site promoter activity, and enhanced Ag-specific proliferation. Furthermore, SOCS-1 competed with SOCS1-KIR for pJAK2(1001-1013). Thus, the KIR region of SOCS-1 binds directly to the autophosphorylation site of JAK2 and a peptide corresponding to this site can function as an antagonist of SOCS-1.     

The production of IFN-gamma by IL-12/IL-18-activated macrophages requires STAT4 signaling and is inhibited by IL-4

Macrophages release IFN-gamma on combined stimulation with IL-12 and IL-18, but the signaling requirements of this process and its regulation by other cytokines are unknown. Here, we demonstrate that STAT4 is indispensable for IL-12/IL-18-induced production of IFN-gamma by mouse peritoneal macrophages. Type 2 NO synthase (NOS2), which we previously found to be a prerequisite for IL-12-induced IFN-gamma production in NK cells, was not required for IFN-gamma production by these macrophages. IL-12 alone already induced the expression of IFN-gamma mRNA, but nuclear translocation of STAT4, the release of IFN-gamma protein, and the subsequent production of NO was strictly dependent on the simultaneous presence of IL-18. NF-kappa B, which mediates IL-18 effects in T cells, was only weakly activated by IL-12 and/or IL-18 in macrophages. Known inhibitors of macrophage functions (e.g., IL-4 and TGF-beta) also suppressed macrophage IFN-gamma production and the subsequent production of NOS2-derived NO. The inhibitory effect of IL-4 was paralleled by nuclear translocation of STAT6, which in EMSAs was able to bind to the same DNA oligonucleotide as STAT4. These results further define the production of IFN-gamma by macrophages and point to a diversity in the signals required for IFN-gamma production by various cell types.     

PI 3 kinase-dependent Akt kinase and PKCepsilon independently regulate interferon-gamma-induced STAT1alpha serine phosphorylation to induce monocyte chemotactic protein-1 expression

Monocyte chemotactic protein-1 (MCP-1) recruits activated phagocytes to the site of tissue injury. Interferon-gamma (IFN-gamma) present in the microenvironment of glomerulus acts on mesangial cells to induce local production of MCP-1. The mechanism by which IFN-gamma stimulates expression of MCP-1 is not clear. We therefore examined the role of PI 3 kinase signaling in regulating the IFN-gamma-induced MCP-1 expression in mesangial cells. Blocking PI 3 kinase activity with Ly294002 attenuated IFN-gamma-induced MCP-1 protein and mRNA expression. IFN-gamma increased Akt kinase activity in a PI 3 kinase-dependent manner. Expression of dominant negative Akt kinase inhibited serine phosphorylation of STAT1alpha, without any effect on its tyrosine phosphorylation, and decreased IFN-gamma-induced expression of MCP-1. These data for the first time indicate a role for PI 3 kinase-dependent Akt kinase in MCP-1 expression. We have recently shown that along with Akt, PKCepsilon is a downstream target of PI 3 kinase in IFN-gamma signaling. Similar to dominant negative Akt kinase, dominant negative PKCepsilon also inhibited serine phosphorylation of STAT1alpha without any effect on tyrosine phosphorylation. Dominant negative PKCepsilon also abrogated MAPK activity, resulting in decrease in IFN-gamma-induced MCP-1 expression. Furthermore, Akt and PKCepsilon are present together in a signaling complex. IFN-gamma had no effect on this complex formation, but did increase PKCepsilon-associated Akt kinase activity. PKCepsilon did not regulate IFN-gamma-induced Akt kinase. Finally, expression of dominant negative Akt kinase blocked IFN-gamma-stimulated MAPK activation. These data provide the first evidence that PI 3 kinase-dependent Akt and PKCepsilon activation independently regulate MAPK activity and serine phosphorylation of STAT1alpha to increase expression of MCP-1.