TGF-β1 prevents up-regulation of the P2X7 receptor by IFN-γ and LPS in leukemic THP-1 monocytes

The P2X7 receptor is an extracellular ATP-gated cation channel critical in inflammation and immunity, and can be up-regulated by IFN-γ and LPS. This study aimed to examine the effect of TGF-β1 on the up-regulation of P2X7 function and expression in leukemic THP-1 monocytes differentiated with IFN-γ and LPS. Cell-surface molecules including P2X7 were examined by immunofluorescence staining. Total P2X7 protein and mRNA was assessed by immunoblotting and RT-PCR respectively. P2X7 function was evaluated by ATP-induced cation dye uptake measurements. Cell-surface P2X7 was present on THP-1 cells differentiated for 3 days with IFN-γ and LPS but not on undifferentiated THP-1 cells. ATP induced ethidium⁺ uptake into differentiated but not undifferentiated THP-1 cells, and the P2X7 antagonist, KN-62, impaired ATP-induced ethidium⁺ uptake. Co-incubation of cells with TGF-β1 plus IFN-γ and LPS prevented the up-regulation of P2X7 expression and ATP-induced ethidium⁺ uptake in a concentration-dependent fashion with a maximum effect at 5 ng/ml and with an IC₅₀ of ~ 0.4 ng/ml. Moreover, ATP-induced YO-PRO-1²⁺ uptake and IL-1β release were abrogated in cells co-incubated with TGF-β1. TGF-β1 also abrogated the amount of total P2X7 protein and mRNA induced by IFN-γ and LPS. Finally, TGF-β1 prevented the up-regulation of cell-surface CD86, but not CD14 and MHC class II, by IFN-γ and LPS. These results indicate that TGF-β1 prevents the up-regulation of P2X7 function and expression by IFN-γ and LPS in THP-1 monocytes. This suggests that TGF-β1 may limit P2X7-mediated processes in inflammation and immunity.     

Binding of rat α1-inhibitor-3-methylamine to the α2-macroglobulin signaling receptor induces second messengers

Binding of receptor-recognized forms of tetrameric human α₂-macroglobulin (α₂M*) to a macrophage signaling receptor induces cAMP synthesis, increases in inositol 1,4,5-triphosphate (IP₃) synthesis, and a concomitant rise in cytosolic free calcium ([Ca²⁺]_i). The α₂M* signaling receptor is coupled to a pertussis-toxin insensitive G protein. Binding of α₂M* also occurs to the low density lipoprotein receptor-related protein/α₂M receptor (LRP/α₂MR), but this binding does not induce signal transduction. Rat α₁-inhibitor-3 (α₁I₃) is a monomeric member of the α-macroglobulin/complement superfamily. Like α₂M, it can react with proteinases or methylamine which induces a conformational change causing activated α₁I₃ to bind to LRP/α₂MR. We now report that α₁I₃-methylamine binds to the macrophage α₂M* signaling receptor inducing a rapid rise in the synthesis of IP₃ with a subsequent 1.5- to 3-fold rise in [Ca²⁺]_i. α₁I₃-methylamine binding to macrophages also caused a statistically significant elevation in cAMP. Native α₁I₃, like α₂M, was unable to induce signal transduction. α₁I₃ forms a complex with α₁-microglobulin, which has a distinct conformation from α₁I₃ and is recognized by LRP/α₂MR. This complex also induces an increase in [Ca²⁺]_i comparable to the effect of α₁I₃-methylamine on macrophages. It is concluded that activation of α₁I₃ by methylamine or binding of α₁-microglobulin causes similar conformational changes in the inhibitor, exposing the receptor recognition site for the α₂M* signaling receptor, as well as for LRP/α₂MR. © 1996 Wiley-Liss, Inc.     

Intracellular free zinc up-regulates IFN-γ and T-bet essential for Th1 differentiation in Con-A stimulated HUT-78 cells

Zinc deficiency impairs cellular immunity. Up-regulation of mRNA levels of IFN-γ, IL-12Rβ2, and T-bet are essential for Th₁ differentiation. We hypothesized that zinc increases Th₁ differentiation via up-regulation of IFN-γ and T-bet expression. To test this hypothesis, we used zinc-deficient and zinc-sufficient HUT-78 cells (a Th₀ cell line) under different condition of stimulation in this study. We also used TPEN, a zinc-specific chelator, to decrease the bioavailability of zinc in the cells. We measured intracellular free zinc, cytokines, and the mRNAs of T-bet, IFN-γ, and IL-12Rβ2. In this study, we show that in zinc-sufficient HUT-78 cells, mRNA levels of IFN-γ, IL-12Rβ2, and T-bet in PMA/PHA-stimulated cells were increased in comparison to zinc-deficient cells. Although intracellular free zinc was increased slightly in PMA/PHA-stimulated cells, Con-A-stimulated cells in 5 μM zinc medium showed a greater sustained increase in intracellular free zinc in comparison to cells incubated in 1 μM zinc. The cells pre-incubated with TPEN showed decreased mRNA levels of IFN-γ and T-bet mRNAs in comparison to cells without TPEN incubation. We conclude that stimulation of cells by Con-A via TCR, release intracellular free zinc which functions as a signal molecule for generation of IFN-γ and T-bet, and IL-12Rβ2 mRNAs required for Th₁ cell differentiation. These results suggest that zinc increase Th₁ cell differentiation by up-regulation of IFN-γ and T-bet, and IL-12Rbβ2 mRNAs.     

And THP-1 cells do not release LTB4, LTC4, or LTD4 in response to A23187

U937 and THP-1 cells possess some characteristics of human mononuclear phagocytes, cells which synthesize and release LTB₄, LTC₄, and LTD₄. Incubation of these cells with recombinant human interferongamma (IFN-gamma) or Phorbol Myristate Acetate (PMA) induces a more differentiated cell state. We hypothesized that U937 and THP-1 cells would release LTB₄, LTC₄, and LTD₄ in response to stimulation with the non-physiologic agonist, calcium ionophore A23187 and that preincubation with IFN-gamma or PMA might alter leukotriene release by thes cells. We cultured both cell lines for 48 hours in the presence and absence of IFN-gamma (10000 units/ml)n and for 120 hours in the presence and absence of PMA (160 nM) and then challenged them with A23187 (5uM) for 30 minutes at 37°C. The supernatants were deproteinated and assayed by RIA for LTB₄ and LTC₄ and by RP-HPLC for LTB₄, LTC₄, and LTD₄. Neither U937 nor THP-1 cells released quantities of leukotrienes detectable by RIA, <0.3ng/5 × 10⁶ cells. Peripheral blood mononuclear phagocytes from normal volumteers, cultured and challenged in vitro at under identical conditions, released 11.3 ± 2.9 ng LTB₄ and 2.0 ± 1.5 ng LTC₄/10⁶ viable monocytes. The lack of leukotriene production by U937 and THP-1 cells was not altered by preincubation for 48 hours with IFN-gamma (n=3) nor by preincubation with PMA for 120 hours (n=3). We conclude 1) U937 and THP-1 cells do not appear to be appropriate in vitro models for the examination of leukotriene release from normal mononuclear phagocytes. 2) Pre-incubation of U937 and THP-1 cells with IFN-gamma or PMA under the conditions tested, does not induce the ability of these cell lines to release leukotrienes.     

Ligation of the α2-macroglobulin signaling receptor on macrophages induces synthesis of platelet activating factor

The binding of receptor-recognized forms of α₂-macroglobulin (α₂M) to macrophage α₂M signaling receptors increases inositol-1,4,5-triphosphate synthesis and induces Ca²⁺ mobilization. In this report, we demonstrate that ligation of the macrophage α₂M signaling receptor is also associated with synthesis of platelet activating factor (PAF) by both the de novo and remodeling pathways. Both α₂M-methylamine and a cloned and expressed 20-kDa receptor binding fragment (RBF) from rat α₂M+, stimulated macrophage synthesis of PAF from [³H]acetate, [³H]methylcholine, and 1-O-[³H]alkyl lyso-PAF by two- to threefold. PAF levels reached a peak in 20 min after the cells were exposed to α₂M-methylamine or RBF; they remained elevated for about 1 h after ligand addition to the cells. When [³H]methylcholine was the substrate, pertussis toxin did not block PAF synthesis, but the protein kinase C inhibitor staurosporin reduced synthesis by 65–70%. Cycloheximide completely abolished the increase in synthesis of PAF by macrophages exposed to α₂M-methylamine. By contrast, when [³H]acetate was employed as a precursor, staurosporin or cycloheximide did not abolish the increase in PAF synthesis. These studies suggest that protein kinase C is necessary for the induction of the de novo pathway by α₂M-methylamine. Both α₂M-methylamine and RBF stimulated the activity of lyso-PAF acetyltransferase by about fourfold. Both ligands also stimulated the activity of PAF acetylhydrolase by about six- to sevenfold, indicating that ligation of the α₂M signaling receptor also regulates the degradation of PAF. The ability of receptor-recognized forms of α₂M to regulate levels of PAF suggests that α₂M-proteinase complexes not only regulate macrophage function by activating intracellular signaling but also may indirectly regulate the function of other cells that cannot bind α₂M-proteinase complexes. © 1996 Wiley-Liss, Inc.     

MODULATION OF INTERFERON γ INDUCED INCREASES IN CATHEPSIN B IN THP-1 CELLS BY ADRENERGIC AGONISTS AND ANTAGONISTS

In order to investigate the possible modulation of macrophage function by the autonomic nervous system, the effect of adrenergic agonists and antagonists on interferon (IFN)-γ-induced increases in cathepsin B (CB) in a macrophage-like cell line was studied. It has been shown previously that IFN-γ induces increased CB activity in phorbol myristate acetate (PMA)-primed THP-1 cells. Isoproterenol (ISO) (10μm ), a mixed β-receptor agonist, increased the induction of CB activity in the cells but norepinephrine (10μm ) and epinephrine (10μm ), the α and β receptor agonists, had little effect. The addition of the mixed α-receptor antagonist phentolamine (10μm ) had no effect on ISO induced increases but the mixed β-receptor antagonist propranolol (10μm ) and the selective β₁-receptor antagonist atenolol produced significant inhibition. These results suggest that the activation of β-receptors could be involved in the induction of CB activity in macrophages and provide a possible mechanism for the regulation of macrophage effector function by the autonomic nervous system. Dibutyryl cAMP (1mm ) alone also induced increases in CB in THP-1 cells, and H-89 or HA1004 abrogated the effect of dibutyryl cAMP, suggesting that the effect of ISO on CB could be through the elevation of cAMP and the activation of cAMP-dependent protein kinases.     

Determining conditions for nitric oxide synthesis in Caco-2 cells using Taguchi and factorial experimental designs

Intestinal inflammation correlates well with the increased synthesis of nitric oxide (NO), which is attributed mainly to the up-regulation of inducible nitric oxide synthase (iNOS). We optimized the use of interferon γ (IFN-γ), tumour necrosis factor α (TNF-α), interleukin 1β (IL-1β), lipopolysaccharide (LPS), and phorbol myristate acetate (PMA) as inducers to stimulate NO synthesis in Caco-2 cells using a Taguchi design. The results indicated that IFN-γ was the most important inducer of iNOS in Caco-2 cells. Treating Caco-2 cells with both IFN-γ and PMA using an optimal mixture of 8000 U/ml IFN-γ and 0.1 μg/ml of PMA resulted in a synergistic induction of NO synthesis. Further experiments using a 5-factor/2-level factorial design including Caco-2 growth conditions such as cell passage, culture medium composition, cell seeding time and density, and stimulation time were also performed. Cell seeding and stimulation times significantly (P < 0.05) affected NO synthesis, whereas culture medium and seeding density did not appreciably affect NO synthesis in Caco-2 cells. Western blotting and RT-PCR findings confirmed that the optimal mixture of IFN-γ and PMA effectively up-regulated iNOS mRNA and protein. The induced NO, iNOS mRNA, and protein were all inhibited by the iNOS selective inhibitor, aminoguanidine (AG).     

Sequential Involvement of NK Cells and CD8+ T Cells in Granuloma Formation of Rhodococcus aurantiacus-Infected Mice

We investigated the effect of in vivo administration of antibodies against T-cell subsets and natural killer (NK) cells on endogenous gamma interferon (IFN-γ) production and granuloma formation in Rhodococcus aurantiacus-infected mice. High titers of endogenous IFN-γ were detected in the extracts of the livers and spleens during 24 hr of the infection, reaching the peak at 8 hr, and the IFN-γ production was reduced by in vivo administration of anti-NK 1.1 monoclonal antibody (MAb) or antibody against asialo GM1⁺ cells. Endogenous IFN-γ declined until 2 days of the infection, then reappeared from 1 week and peaked at 3 weeks. Endogenous IFN-γ at 1 and 3 weeks was reduced by in vivo administration of anti-CD8 MAb, but not by anti-CD4 MAb or anti-NK 1.1 MAb. Granulomatous lesions in the livers and spleens began to appear from 1 week of the infection and developed in 3 weeks. In vivo administration of rat anti-IFN-γ MAb reduced the development of granulomas. In addition, granuloma formation was reduced by depletion of NK cells prior to the infection or depletion of CD8⁺ T cells at 1 week of the infection. Based on these findings, it is presumed that the biphasic production of IFN-γ is attributable to NK cells in the early phase of the infection and CD8⁺ T cells in the phase of granuloma formation, and that granuloma formation is regulated by NK cells and CD8⁺ T cells through the secretion of endogenous IFN-γ.     

Thyroid hormone analogues potentiate the antiviral action of interferon-γ by two mechanisms

L-thyroxine (L-T₄) potentiates the antiviral activity of human interferon-γ (IFN-γ) in HeLa cells. We have added thyroid hormone and analogues to cells either 1) for 24 h pretreatment prior to 24 h of IFN-γ (1.0 IU/ml), 2) for 24 h cotreatment with IFN-γ, 3) for 4 h, after 20 h cell incubation with IFN-γ, alone, or 4) for 24 h pretreatment and 24 h cotreatment with IFN-γ. The antiviral effect of IFN-γ was then assayed. L-T₄ potentiated the antiviral action of IFN-γ by a reduction in virus yield of more than two logs, the equivalent of a more than 100-fold potentiation of the IFN's antiviral effect. 3,3′,5-L-triiodothyronine (L-T₃) was as effective as L-T₄ when coincubated for 24 h with IFN-γ but was less effective than L-T₄ when coincubated for only 4 h. D-T₄, D-T₃, 3,3′,5-triiodothyroacetic acid (triac), tetraiodothyroacetic acid (tetrac), and 3,5-diiodothyronine (T₂) were inactive. When preincubated with L-T₄ for 24 h prior to IFN-γ treatment, tetrac blocked L-T₄ potentiation, but, when coincubated with L-T₄ for 4 h after 20 h IFN-γ, tetrac did not inhibit the L-T₄ effect. 3,3′,5′-L-triiodothyronine (rT₃) also potentiated the antiviral action of IFN-γ, but only in the preincubation model. Furthermore, the effects of rT₃ preincubation and L-T₃ coincubation were additive, resulting in 100-fold potentiation of the IFN-γ effect. When L-T₄, L-T₃, or rT₃, plus cycloheximide (5 μg/ml), was added to cells for 24 h and then removed prior to 24 h IFN-γ exposure, the potentiating effect of the three iodothyronines was completely inhibited. In contrast, IFN-γ potentiation by 4 h of L-T₄ or L-T₃ coincubation was not inhibited by cycloheximide (25 μg/ml). These studies demonstrate two mechanisms by which thyroid hormone can potentiate IFN-γ's effect: 1) a protein synthesis-dependent mechanism evidenced by enhancement of IFN-γ's antiviral action by L-T₄, L-T₃, or rT₃ preincubation, and inhibition of enhancement by tetrac and cycloheximide, and 2) a protein synthesis-independent (posttranslational) mechanism, not inhibited by tetrac or cycloheximide, demonstrated by 4 h coincubation of L-T₄ or L-T₃, but not rT₃, with IFN-γ. The protein synthesis-dependent pathway is responsive to rT₃, a thyroid hormone analogue generally thought to have little effect on protein synthesis. A posttranslational mechanism by which the antiviral action of IFN-γ can be regulated has not previously been described. © 1996 Wiley-Liss, Inc.     

The effects of γ-interferon on human peripheral blood monocyte/macrophage-mediated bone particle degradation

γ-Interferon (IFN-γ) has recently been demonstrated to inhibit the ability of mononuclcar phagocytes to degrade bone particles. We have further addressed the specificity, potency and mechanism of this activity using human recombinant IFN-γ. Adherent peripheral blood mononuclear leukocytes from normal human volunteers were cultured with washed, sieved (⩽75 μm) ⁴⁵Ca-labelled rat bone particles for 3 days, after which bone particle degradation (7.1 ± 1.6%, n = 11) was calculated from the fraction of⁴⁵Ca released into the medium. As little as 5 U/ml IFN-γ significantly suppressed bone particle degradation and 50 U/ml was associated with consistent marked suppression (74.0 ± 3.5% inhibition, P < 0.001, n = 11). IFN-γ was not suppressive if added to cells 24 h or more after exposure to bone particles. Addition of indomethacin (10 μM) did not reverse the effect of IFN-γ, suggesting that it was not prostaglandin-mediated. In addition, 1,25(OH)₂D₃ (10 nM) did not remove the inhibitor, effect of IFN-γ.Contact of mononuclear phagocytes with bone particles and secretion of soluble factors from these cells have both been demonstrated to play a role in their ability to degrade bone particles. IFN-γ (50 U/ml) inhibited monocyte/macrophage interaction with another unopsonized surface, i.e., one μm fluorescent latex particles, decreasing the number of internalized particles from 12.6 ± 2.9 per cell to 5.9 ± 1.4 per cell (P < 0.01, n = 15), as measured using flow cytometry. However, binding of bone particles by the cells was not diminished by IFN-γ. Exogenous α-imerferon and human recombinant IL-1β, TNF-α, and lymphotoxin did not alter bone particle degradation. In addition, endogenous IL-1β release from human monocyte/macrophages exposed to bone particles was negligible and unaffected by IFN-γ.We conclude that IFN-γ is a potent and specific inhibitor of monocyte/macrophage-mediated bone particle degradation, and that this activity does not appear to be due to effects on the ability of monocytes to bind bone particles or to release IL-1 in response to the particles     

Opioid-Induced Increase in [Ca2+]i in ND8-47 Neuroblastoma × Dorsal Root Ganglion Hybrid Cells Is Mediated Through G Protein-Coupled δ-Opioid Receptors and Desensitized by Chronic Exposure to Opioid

Abstract: δ-Receptor agonists induce a concentration-dependent increase in intracellular calcium concentration ([Ca²⁺]_i) in ND8-47 cells by activating dihydropyridine-sensitive Ca²⁺ channels. The role of G proteins in transducing the opioid effect has been studied. Pretreatment of cells with pertussis toxin (100 ng/ml, 24 h) almost completely blocked [d -Ser²,Leu⁵]enkephalin-Thr (DSLET)-induced increase in [Ca²⁺]_i. Cholera toxin (10 nM, 24 h) had no effect on DSLET-induced response. Pretreatment of the cells with 1 µM DSLET for 1 h resulted in a 30% inhibition of DSLET-induced increase in [Ca²⁺]_i and a 78% inhibition after exposure for 24 h. After 1 h of exposure to DSLET, there was a decrease in agonist affinity with no significant changes in receptor density. Cells exposed to 1 µM DSLET for 24 h demonstrate a nearly 90% decrease in [³H]diprenorphine binding, with a decrease in affinity for agonist at the remaining binding sites. G protein subunits α_i2, α_i3, α_s, and α_q were detected in ND8-47 cell membranes by western blot; α_o and α_i1 were not present. Chronic DSLET treatment had no significant effect on the quantity of each of the α-subunits. These results suggest that the DSLET-induced increase in [Ca²⁺]_i is mediated through pertussis toxin-sensitive G proteins (probably G_i2 or G_i3) and the attenuation of this response in chronically treated cells is associated with a relatively rapid reduction in receptor affinity to DSLET and a slow reduction in receptor density.     

Contribution of intercellular adhesion molecule 1 (ICAM-1) to control Mycobacterium avium infection

Mycobacterium avium is a facultative intracellular opportunistic pathogen especially relevant in cases of people living with AIDS. The aim of this study was to evaluate the role of intercellular adhesion molecule 1 (ICAM-1) in the inflammatory response against M. avium infection. Mice deficient for ICAM-1 (ICAM KO) and infected with M. avium presented increased bacterial load in the spleen, liver and lungs compared to C57BL/6. Moreover, ICAM deficient mice presented reduced granuloma area in liver at 30 days post-infection with reduced numbers of lymphocytes and granulocytes. The assessment of in vitro cytokine production by ICAM KO spleen cells showed lower levels of IFN-γ compared to C57BL/6, whereas TNF-α remained unaltered. Additionally, the production of IFN-γ in liver and spleen tissues was also diminished in ICAM-1 KO mice. Interestingly, a persistent reduction in IFN-γ production was observed in CD3⁺NK1.1⁺ cells of ICAM-1 deficient mice compared to wild-type animals. Together, these results demonstrate the importance of ICAM-1 in the efficient control of M. avium infection and granuloma formation and highlights its role on CD3⁺NK1.1⁺ cell population as important for IFN-γ production during infection.     

Activation of Unusual Mac-1+CD4− CD8− T Cells Bearing αβ Antigen Receptor in Murine Lungs during Infection with Mycobacterium bovis BCG: Regulation by Interferon-γ

We have recently (Kawakami et al, Immunol. Lett. 1995;46: 143) demonstrated that unusual Mac-1⁺CD4^?CD8^? T cells bearing αβ antigen receptor (Mac-1⁺ αβ T cells) reside in a considerable proportion in murine lungs. The present study was performed to examine the dynamics of accumulation of these cells in the lungs following intravenous administration of Mycobacterium bovis BCG (BCG). Mac-1⁺ αβ T cells accumulated rapidly 24 hr after infection, followed by a gradual increase over the observation period of 15 days. Furthermore, the expression of Ia, ICAM-1 and FcγR II/III on their surface intensified dramatically after BCG infection. The kinetics of enhancement of Ia expression was slower than that of ICAM-1, with the maximum level attained in one day in the latter molecule but in two weeks in the former. Neutralization of endogenous IFN-γ by specific mAb completely blocked the augmented expression of Ia on Mac-1⁺ αβ T cells after BCG infection, but did not have any significant effect on that of ICAM-1. In contrast, in vivo administration of IFN-γ enhanced the expression of ICAM-1 as well as that of Ia. Our results indicate that accumulation of Mac-1 αβ T cells within the lung is associated with a differential change in the expression of surface antigens, and suggest that these cells may play a role in the host defense against mycobacterial infection.     

Pretreatment with interferon-γ protects microglia from oxidative stress via up-regulation of Mn-SOD

Microglial cells, resident macrophage-like immune cells in the brain, are exposed to intense oxidative stress under various pathophysiological conditions. For self-defense against oxidative injuries, microglial cells must be equipped with antioxidative mechanisms. In this study, we investigated the regulation of antioxidant enzyme systems in microglial cells by interferon-γ (IFN-γ) and found that pretreatment with IFN-γ for 20 h protected microglial cells from the toxicity of various reactive species such as hydrogen peroxide (H₂O₂), superoxide anion, 4-hydroxy-2(E)-nonenal, and peroxynitrite. The cytoprotective effect of IFN-γ pretreatment was abolished by the protein synthesis inhibitor cycloheximide. In addition, treatment of microglial cells with both IFN-γ and H₂O₂ together did not protect them from the H₂O₂-evoked toxicity. These results imply that protein synthesis is required for the protection by IFN-γ. Among various antioxidant enzymes such as manganese or copper/zinc superoxide dismutase (Mn-SOD or Cu/Zn-SOD), catalase, and glutathione peroxidase (GPx), only Mn-SOD was up-regulated in IFN-γ-pretreated microglial cells. Transfection with siRNA of Mn-SOD abolished both up-regulation of Mn-SOD expression and protection from H₂O₂ toxicity by IFN-γ pretreatment. Furthermore, whereas the activities of Mn-SOD and catalase were up-regulated by IFN-γ pretreatment, those of Cu/Zn-SOD and GPx were not. These results indicate that IFN-γ pretreatment protects microglial cells from oxidative stress via selective up-regulation of the level of Mn-SOD and activity of Mn-SOD and catalase.     

Transforming growth factor β1 inhibits interleukin-1-induced but enhances ionomycin-induced interferon-γ production in a t cell lymphoma: Comparison with the effects of rapamycin

Transforming growth factor β1 (TGF-β1) is a multifunctional cytokine whose potent immunomodulatory activity is well documented. To explore the mechanisms of this activity we examined the effect of TGF-β1 on the production of IFN-γ measured at the mRNA and protein levels in the YAC-1 cell lymphoma. In previous studies, this model proved useful to characterize the mode of action of the immunosuppressant rapamycin (RAP). Here, we found that when induced by IL-1 or IL-1 + PMA, the production of IFN-γ is suppressed by both TGF-β1 (ED₅₀ = 1.9 pM) and RAP (ED₅₀ = 0.2 nM). In contrast, when induced by the calcium ionophore ionomycin, in the absence or in the presence of PMA, this production is enhanced up to 10-fold by TGF-β (ED₅₀ = 1.8 pM) and 1.5—3-fold by RAP. Therefore, in YAC-1 cells, TGF-β1 exerts opposite effects on IFN-γ production depending on the mode of activiation, and these effects parallel those of RAP. To further analyze the mode of action of TGF-β1 in this system, we used okadaic acid (OA), an inhibitor of serine/threonine protein phosphatases. Treatment with OA rendered the expression of IFN-γ mRNA induced by IL-1 insensitive to TGF-β1 or RAP, indicating that activation of a phosphatase may play a role in the suppressive effect of both agents. However, OA did not prevent the augmentation of ionomycin-mediated induction of IFN-β mRNA by either TGF-β1 or RAP. Hence, the up-regulation of IFN-β production by TGF-β1 and RAP may involve a different biochemical mechanism that that mediating their suppressive action. These observations also favor the hypothesis that the two agents act on the same regulatory pathways. This was further supported by the finding that TGF-β1 and RAP modulate IFN-γ production in an additive rather than synergistic fashion. However, their effects could be dissociated in mutants of YAC-1 cells selected for resistance to the inhibition of IL-1-mediated IFN-γ induction by RAP. Moreover, the IFN-γ modulatory action of RAP in YAC-1 cells was accompanied by an antiproliferative effect, whereas TGF-β1 failed to alter the growth of these cells. Therefore, the immunomodulatory action of TGF-β1 may result from the dis ruption of biochemical processes related to, although distinct from, those affected by RAP. © 1994 Wiley-Liss, Inc.     

DcR3 protects THP-1 macrophages from apoptosis by increasing integrin α4

In order to analyze the function of DcR3 for the regulation of cell adhesion and apoptosis in macrophages, we investigated the expression of decoy receptor 3 (DcR3) in THP-1 monocytes/macrophages.DcR3 was expressed in THP-1 and increased by phorbol 12-myristate 13-acetate (PMA). The formation of macrophage aggregates was observed when THP-1 cells were differentiated by PMA or stimulated with DcR3-Fc. Undifferentiated THP-1 cells were also induced to form aggregates by DcR3-Fc. The expression of integrin α4 was significantly increased by DcR3-Fc. CHX-induced apoptosis in THP-1 was inhibited by DcR3-Fc, of which inhibition against CHX-induced apoptosis and aggregate formation were ameliorated by anti-VLA4 antibody.DcR3 may play a significant role in macrophages not only by a decoy receptor but also by increasing α4 integrin.     

Interferon-inducible immunity-related GTPase Irgm1 regulates IFNγ-dependent host defense,lymphocyte survival and autophagy

IFN-γ is a pleiotropic cytokine that plays a key role in host resistance, yet when not properly regulated can become detrimental to the host. The interferon-inducible Immunity Related GTPase family M member 1 (Irgm1), previously characterized as an effector molecule required for macrophage microbicidal activity, has been shown recently to control IFN-γ-dependent cell survival and host resistance. Irgm1 regulates the expansion/survival of mature effector CD4⁺ T lymphocytes by protecting them from IFN-γ-induced autophagic cell death. Importantly, mice deficient in both IFN-γ and Irgm1 were rescued from the lymphocyte depletion and increased mortality that typically occurs in Irgm1^–/– animals following pathogen exposure. We propose that Irgm1 plays a major role in maintaining T lymphocyte homeostasis during host IFN-γ responses by protecting these cells from autophagy-dependent cell death.     

Activation of Calcium-Phospholipid-Dependent Protein Kinase Enhances Benzodiazepine and Barbiturate Potentiation of the GABAA Receptor

Abstract: The effect of calcium-phospholipid-dependent protein kinase (PKC) on GABA_A receptor function was examined in Xenopus oocytes expressing recombinant human GABA_A receptor using two-electrode voltage-clamp measurements. Phorbol 12-myristate 13-acetate (PMA), a potent activator of PKC, inhibited GABA-gated chloride currents by ～72% in oocytes expressing α_lβ₁γ_2L subunit cDNAs. Phorbol 12-monomyristate (PMM), a negative control analogue of PMA, did not alter GABA_A receptor responses. To investigate whether activation of PKC could alter the modulatory responses of the receptor complex, the effect of PMA on benzodiazepine and barbiturate potentiation of GABA responses was assessed. In oocytes expressing α_lβ₁γ_2s subunit cDNAs, diazepam (300 nM) potentiated GABA responses by ～160%. Following PMA (5-25 nM/) treatment, diazepam potentiation was significantly increased to 333%. No effect of the inactive phorbol ester PMM (25 nM) was observed on diazepam potentiation of GABA responses. PMA enhancement of diazepam potentiation of GABA responses was also observed in oocytes expressing α_lβ₁γ_2Ssubunit cDNAs, indicating that the unique PKC site present in the Tγ_2LL subunit is not required for observing the PMA effect. PMA (5-25 nM) also enhanced pentobarbital potentiation of GABA responses. In oocytes expressing α_lβ₁γ_2L subunit cDNAs, pentobarbital (25 μM) potentiated GABA receptor responses by ～97%. Following treatment with PMA (5-25 nM), pentobarbital potentiation of GABA responses increased to ～ 156%. The present results suggest that protein phosphorylation may alter the coupling between the allosteric modulatory sites within the GABA_A receptor complex.