Distinct PKC isoforms mediate the activation of cPLA2 and adenylyl cyclase by phorbol ester in RAW264.7 macrophages

The modulatory effects of protein kinase C (PKC) on the activation of cytosolic phospholipase A2 (cPLA2) and adenylyl cyclase (AC) have recently been described. Since the signalling cascades associated with these events play critical roles in various functions of macrophages, we set out to investigate the crosstalk between PKC and the cPLA2 and AC pathways in mouse RAW 264.7 macrophages and to determine the involvement of individual PKC isoforms. The cPLA2 and AC pathways were studied by measuring the potentiation by the phorbol ester PMA of ionomycin-induced arachidonic acid (AA) release and prostagladin E1 (PGE1)-stimulated cyclic AMP production, respectively. PMA at 1 microM caused a significant increase in AA release both in the presence (371%) and absence (67%) of ionomycin induction, while exposure of RAW 264.7 cells to PMA increased PGE1 stimulation of cyclic AMP levels by 208%. Treatment of cells with staurosporine and Ro 31-8220 inhibited the PMA-induced potentiation of both AA release and cyclic AMP accumulation, while Go 6976 (an inhibitor of classical PKC isoforms) and LY 379196 (a specific inhibitor of PKCbeta) inhibited the AA response but failed to affect the enhancement of the cyclic AMP response by PMA. Long term pretreatment of cells with PMA abolished the subsequent effect of PMA in potentiating AA release, but only inhibited the cyclic AMP response by 42%. Neither PD 98059, an inhibitor of MEK, nor genistein, an inhibitor of tyrosine kinases, had any effect on the ability of PMA to potentiate AA or cyclic AMP production. The potentiation of AA release, but not of cyclic AMP formation, by PMA was sensitive to inhibition by wortmannin. This effect was unrelated to the inhibition of PKC activation as deduced from the translocation of PKC activity to the cell membrane. Western blot analysis revealed the presence of eight PKC isoforms (alpha, betaI, betaII, delta, epsilon, mu, lambda and xi) in RAW 264.7 cells and PMA was shown to induce the translocation of the alpha, betaI, betaII, delta, epsilon and mu isoforms from the cytosol to the cell membrane within 2 min. Pretreatment of cells with PMA for 2-24 h resulted in a time-dependent down-regulation of PKCalpha, betaI, betaII, and delta expression, while the levels of the other four PKC isozymes were unchanged after PMA treatment for 24 h. A decrease in the potentiation of AA release by PMA was observed, concomitant with the time-dependent down-regulation of PKC. These results indicate that PKCbeta has a crucial role in the mediation of cPLA2 activation by the phorbol ester PMA, whereas PMA utilizes PKC epsilon and/or mu to up-regulate AC activity.     

Phosphorylation of cytosolic phospholipase A2 and the release of arachidonic acid in human neutrophils

Kinases mediating phosphorylation and activation of cytosolic phospholipase A2 (cPLA2) in intact cells remain to be fully characterized. Platelet-activating factor stimulation of human neutrophils increases cPLA2 phosphorylation. This increase is inhibited by PD 98059, a mitogen-activated protein (MAP)/extracellular signal-regulating kinase (erk) 1 inhibitor, but not by SB 203580, a p38 MAP kinase inhibitor, indicating that this action is mediated through activation of the p42 MAP kinase (erk2). However, platelet-activating factor-induced arachidonic acid release is inhibited by both PD 98059 and SB 203580. Stimulation by TNF-alpha increases cPLA2 phosphorylation, which is inhibited by SB 203580, but not PD 98059, suggesting a role for p38 MAP kinase. LPS increases cPLA2 phosphorylation and arachidonic acid release. However, neither of these actions is inhibited by either PD 98059 or SB 203580. PMA increases cPLA2 phosphorylation. This action is inhibited by PD 98059 but not SB 203580. Finally, FMLP increases cPLA2 phosphorylation and arachidonic acid release. Interestingly, while the FMLP-induced phosphorylation of cPLA2 is not affected by the inhibitors of the p38 MAP kinase or erk cascades, both inhibitors significantly decrease arachidonic acid release stimulated by FMLP. SB 203580 or PD 98059 has no inhibitory effects on the activity of coenzyme A-independent transacylase.     

Double-stranded RNA-induced inducible nitric-oxide synthase expression and interleukin-1 release by murine macrophages requires NF-kappaB activation

The effects of double-stranded RNA (synthetic polyinosinic-polycytidylic acid; poly(I-C)) on macrophage expression of inducible nitric-oxide synthase (iNOS), production of nitric oxide, and release of interleukin-1 (IL-1) were investigated. Individually, poly(I-C), interferon-gamma (IFN-gamma), and lipopolysaccharide (LPS) stimulate nitrite production and iNOS expression by RAW 264.7 cells. In combination, the effects of poly(I-C) + IFN-gamma are additive, while poly(I-C) does not further potentiate LPS-induced nitrite production. These results suggest that poly(I-C) and LPS may stimulate iNOS expression by similar signaling pathways, which may be independent of pathways activated by IFN-gamma. LPS-induced iNOS expression is associated with the activation of NF-kappaB. We show that inhibition of NF-kappaB by pyrrolidinedithiocarbamate prevents poly(I-C) + IFN-gamma-, poly(I-C) + LPS-, and LPS-induced iNOS expression, nitrite production and IkappaB degradation by RAW 264.7 cells. The effects of poly(I-C) on iNOS expression appear to be cell-type specific. Poly(I-C), alone or in combination with IFN-gamma, does not stimulate, nor does poly(I-C) potentiate, IL-1-induced nitrite production by rat insulinoma RINm5F cells. In addition, we show that the combination of poly(I-C) + IFN-gamma stimulates iNOS expression, nitrite production, IkappaB degradation, and the release of IL-1 by primary mouse macrophages, and these effects are prevented by pyrrolidinedithiocarbamate. These findings indicate that double-stranded RNA, in the presence of IFN-gamma, is a potent activator of macrophages, stimulating iNOS expression, nitrite production, and IL-1 release by a mechanism which requires the activation of NF-kappaB.     

p50 (NF-kappa B1) is upregulated in LPS tolerant P388D1 murine macrophages

Cells of the murine macrophage cell line P388D1 express cell surface CD14 and respond to LPS (lipopolysaccharide) stimulation with the production of TNF (tumor necrosis factor). When the cells are stimulated with LPS a second time then little TNF is produced, i.e. the cells are tolerant. Flow cytometry analysis demonstrates that this tolerance is not due to a downregulation of the CD14 cell surface receptor. Analysis of proteins binding to the -516 NF-kappa B motif of the murine TNF promoter reveals that constitutive p50p50 and LPS stimulation lead to mobilization of a heterodimer consisting of p65/c-rel. In tolerant cells less of the p65/c-rel heterodimer is mobilized but there is a strong upregulation of p50p50. These data show that tolerance to LPS in murine macrophages may involve a predominance of p50 homodimers.     

Anandamide synthesis is induced by arachidonate mobilizing agonists in cells of the immune system

The hypothesis that the capability of agents to mobilize arachidonic acid (AA) could predict increased anandamide (ANA) synthesis in a macrophage cell line has been examined. Lipopolysaccharide (LPS), platelet-activating factor (PAF) and cannabinoids such as Delta9-tetrahydrocannabinol (THC) and anandamide were all found to be agonists for the release of AA and led to increased ANA synthesis in RAW264.7 mouse macrophage cells. Nitric oxide, in contrast, stimulated AA release without raising ANA levels. ANA stimulation of its own synthesis indicates the existence of a positive feedback mechanism. The possible involvement of the CB2 receptor in THC-mediated AA release and ANA synthesis is addressed using the antagonist SR144528. ANA synthesis is also increased by the combination of calcium ionophore and indomethacin, suggesting that ANA is metabolized by a cyclooxygenase in this system. The data imply that ANA could play a role in the response of the immune system to cannabinoids and bacterial endotoxins and that AA mobilization is a predictor for increased ANA synthesis.     

Modulation of nitric oxide, hydrogen peroxide and cytokine production in a clonal macrophage model by the trichothecene vomitoxin (deoxynivalenol)

Characterization of how vomitoxin (VT) and other trichothecenes affect macrophage regulatory and effector function may contribute to improved understanding of mechanisms by which these mycotoxins impact the immune system. The RAW 264.7 murine cell line was used as a macrophage model to assess effects of the VT on proliferation and the production of nitric oxide (NO), hydrogen peroxide (H2O2) and cytokines. Using the MTT cleavage assay, VT at concentrations of 50 ng/ml or higher was found to significantly decrease proliferation and viability of RAW 264.7 cells without stimulation or with stimulation by lipopolysaccharide (LPS) or interferon (IFN)-gamma. In the absence of an activation agent, VT (25-250 ng/ml) had negligible effects on the production of NO, H2O2, and cytokines. Upon activation with LPS at concentrations of 10 to 100 ng/ml, VT at 25-100 ng/ml markedly enhanced production of H2O2 but was inhibitory at 250 ng/ml. VT enhancement of H2O2 production was observed as early as 12 h after LPS stimulation. When IFN-gamma was used as the stimulant, VT (25-250 ng/ml) delayed peak H2O2 production. VT (25-250 ng/ml) also markedly decreased NO production in cells activated with LPS or IFN-gamma. Interestingly, VT superinduced TNF-alpha and IL-6 production in LPS-stimulated cells and also elevated TNF-alpha in IFN-gamma stimulated cells. These results suggest that VT can selectively and concurrently upregulate or downregulate critical functions associated with activated macrophages.     

Hyperglycemic levels of glucose inhibit interleukin 1 release from RAW 264.7 murine macrophages by activation of protein kinase C

Diabetic patients with hyperglycemia (high blood glucose) have frequent and persistent bacterial infections linked to significantly diminished bactericidal activity and macrophage function. Interleukin-1 (IL-1), released primarily from activated macrophages, is a key mediator of effective host defense against microorganisms. We observe that hyperglycemic levels of D-glucose (8-20 mM) inhibit the release of IL-1 by lipopolysaccharide-stimulated RAW 264.7 murine macrophage cells. An inhibitor of glucose transport and metabolism, 2-deoxyglucose, prevents this inhibition of IL-1 release. High levels (8-20 mM) of fructose and mannose (but not galactose or L-glucose) also inhibit the release of IL-1 activity, suggesting that metabolism is required for IL-1 inhibition. Immunoprecipitation and activity measurements demonstrate that high glucose levels block the release of IL-1 but do not inhibit IL-1 production. High glucose levels (20 mM) increase protein kinase C (PKC) activity, and inhibitors of PKC block the inhibitory effects of glucose. Phorbol 12-myristate 13-acetate, an agonist of PKC, mimics glucose-induced inhibition of IL-1 release. These results demonstrate that high glucose levels inhibit IL-1 release (but not production) by RAW 264. 7 murine macrophages, and this inhibition is mediated by PKC activation. These studies suggest that persistent infections in hyperglycemic patients may be due to an inhibition of IL-1 release from macrophages.     

LPS tolerance in monocytes/macrophages: three 3' cytosins are required in the DNA binding motif for detection of upregulated NF-kappa B p50 homodimers

When monocytes are stimulated with LPS (lipopolysaccharide) repeatedly then the initially high expression of the TNF (tumor necrosis factor) gene is only very low, i.e. the cells are tolerant to LPS. Tolerant cells still express the CD14 receptor and they can still be activated to mobilize NF-kappa B into nucleus. Analysis of the binding proteins employing the -605 motif of the human TNF promoter (GGGGCTGTCCC) revealed that in tolerant cells of the human monocytic cell line Mono Mac 6 there is a predominance of p50p50 of NF-kappa B. We now show that a mutant motif that exchanges the terminal 3' C for a G fails to bind the p50 homodimer that is upregulated in LPS toler ant human Mono Mac 6 cells. The same is true for nuclear extracts taken from the murine P388D1 macrophage cell line when tested with the -516 motif of the murine TNF promoter (GGGGGCTTTCCC). Here the wild type motif gives efficient binding of p50p50 that again is upregulated in tolerant cells whereas a mutant with a 3' G shows hardly any binding of p50p50. Conversely, the murine kappa light chain enhancer motif (GGGGACTTTCCG) does not efficiently bind the nuclear p50p50 from tolerant murine P388 macrophages. Binding is, however, readily detected when the 3' G is replaced by a C. These data show that the detection of upregulated p50 homodimers in LPS tolerant cells is dependent on subtle differences in the sequence of the DNA binding motif.     

Prostaglandin E2 synthesis is differentially affected by reactive nitrogen intermediates in cultured rat microglia and RAW 264.7 cells

We studied the effects of nitric oxide (NO) on prostanoid production, cyclooxygenase (COX-2) expression and [3H]arachidonic acid (AA) release in RAW 264.7 macrophagic cells and rat microglial primary cultures. Inhibition of NO synthesis enhanced microglial prostanoid production without affecting that of RAW 264.7 cells. Both 3-morpholinosydnonimine (SIN-1), (which, by releasing NO and superoxide, leads to the formation of peroxynitrite), and S-nitroso-N-acetylpenicillamine (SNAP), (which releases only NO), inhibited microglial prostanoid production, by preventing COX-2 expression. In contrast, in RAW 264.7 cells, SIN-1 enhanced both basal and LPS-stimulated prostanoid production by upregulating COX-2, while SNAP stimulated basal production and slightly inhibited the LPS-induced production, as a cumulative result of enhanced AA release and depressed COX-2 expression. Thus, reactive nitrogen intermediates can influence prostanoid production at distinct levels and in different way in the two cell types, and results obtained with RAW 264.7 cells can not be extrapolated to microglia.     

Adenosine and its receptor agonists potentiate nitric oxide synthase expression induced by lipopolysaccharide in RAW 264.7 murine macrophages

The effect of adenosine and its agonists on nitric oxide synthase (NOS) activity and the production of nitrite induced by lipopolysaccharide (LPS) in RAW 264.7 cells were investigated. Nitrite production and NOS activity in the RAW 264.7 cells were increased up to 2.5 fold after co-exposure of the cells to LPS and adenosine or its agonists, as compared to LPS alone. Adenosine and its agonists had no effect on NOS activity when incubated alone with RAW 264.7 cells. Enhancement caused by adenosine or its agonists was dose-dependent but the effect was neither A1 nor A2 receptor specific. These findings suggest that during pathological conditions such as inflammation or trauma, the significant amounts of cellular adenosine which are released may increase the production of NO by macrophages.     

Identification of an inhibitor targeting macrophage production of monocyte chemoattractant protein-1 as TGF-beta 1

Monocyte chemoattractant protein-1 (MCP-1) is expressed in a diverse range of cells in response to various pathologic stimuli, whereas little is known about endogenous inhibitors of MCP-1 expression. I sought negative regulators of MCP-1 in culture medium conditioned by several cell lines and found that glomerular mesangial cells exclusively secrete a factor that inhibits expression of MCP-1 by activated macrophages. Treatment of J774.2 macrophages with conditioned medium from mesangial cells blunted the induction of MCP-1 by LPS. Even after the induction, subsequent treatment of macrophages with the conditioned medium down-regulated the MCP-1 expression. Medium conditioned by normal rat glomeruli contained a similar inhibitory activity that was enhanced in regenerating glomeruli, where mesangial cells are activated. The activity of the conditioned medium was not diminished, but enhanced by heat treatment, which was consistent with the unique property of TGF-beta family of molecules. Indeed, the mesangial cell-derived medium contained active TGF-beta 1. An anti-TGF-beta 1 neutralizing Ab abolished the inhibitory effect exerted by the mesangial cell medium, and externally added TGF-beta 1 suppressed the MCP-1 expression by macrophages at both mRNA and protein levels. The inhibitory effect of TGF-beta 1 on MCP-1 was observed in other macrophage cell lines, RAW264.7 and NR8383, and peritoneal macrophages, but not in fibroblastic cell line NRK49F. Treatment of J774.2 macrophages with TGF-beta 1 inhibited LPS induction of c-jun that was found to be crucial for the MCP-1 expression. These data demonstrate that TGF-beta 1 functions as an inhibitor of MCP-1 expression in macrophages possibly via down-regulation of c-Jun/activator protein-1.     

Antisense oligonucleotides targeting protein kinase C-alpha, -beta I, or -delta but not -eta inhibit lipopolysaccharide-induced nitric oxide synthase expression in RAW 264.7 macrophages: involvement of a nuclear factor kappa B-dependent mechanism

The signaling pathway for protein kinase C (PKC) activation and the role of PKC isoforms in LPS-induced nitric oxide (NO) release were studied in RAW 264.7 macrophages. The tyrosine kinase inhibitor genestein attenuated LPS-induced NO release and inducible nitric oxide synthase (iNOS) expression, as did the phosphoinositide-specific phospholipase C (PI-PLC) inhibitor U73122 and the phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor D609. LPS stimulated phosphatidylinositol (PI) hydrolysis and PKC activity in RAW cells; both were inhibited by genestein. The PKC inhibitors (staurosporine, calphostin C, Ro 31-8220, or Go 6976) or long-term 12-O-tetradecanoylphorbol 13-acetate (TPA) treatment also resulted in inhibition of LPS-induced NO release and iNOS expression. Western blot analysis showed expression of PKC-alpha, -betaI, -delta, -eta, and -zeta in RAW cells; down-regulation of PKC-alpha, -betaI, and -delta, but not -eta, was seen after long-term TPA treatment, indicating the possible involvement of one or all of PKC-alpha, -betaI, and -delta, but not -eta, in LPS-mediated effects. Treatment with antisense oligonucleotides for these isoforms further demonstrated the involvement of PKC-alpha, -betaI, and delta, but not -eta, in LPS responses. Stimulation of cells with LPS for 1 h caused activation of NF-kappaB in the nuclei by detection of NF-kappaB-specific DNA-protein binding; this was inhibited by genestein, U73122, D609, calphostin C, or antisense oligonucleotides for PKC-alpha, -betaI, and -delta, but not -eta. These data suggest that LPS activates PI-PLC and PC-PLC via an upstream tyrosine kinase to induce PKC activation, resulting in the stimulation of NF-kappaB DNA-protein binding, then initiated the expression of iNOS and NO release. PKC isoforms alpha, betaI, and delta were shown to be involved in the regulation of these LPS-induced events.     

Macrophages expressing a fusion protein derived from bactericidal/permeability-increasing protein and IgG are resistant to endotoxin

OBJECTIVES: To generate a recombinant fusion protein (FP) based on the endotoxin-binding domain of bactericidal/permeability-increasing protein (BPI) and the constant domain of IgG and to test its ability to inhibit lipopolysaccharide (LPS)-induced macrophage tumor necrosis factor alpha (TNF-alpha) secretion. DESIGN: A murine macrophage cell line, RAW 264.7, was transfected with a BPI-IgG FP before incubation with LPS. The amount of LPS-induced TNF-alpha protein secreted was measured and compared with that secreted by cells transfected with a control construct. SETTING: Basic science research laboratory. MAIN OUTCOME MEASURES: Secreted TNF-alpha protein concentration. RESULTS: After transfection, RAW 264.7-cell FP expression was detected in cell lysates and supernatants. At each LPS dose tested, cells transfected with the FP gene secreted less TNF-alpha than did cells transfected with a control construct. CONCLUSIONS: The FP possesses substantial antiendotoxin activity, as delineated by inhibition of LPS-induced TNF-alpha secretion by murine macrophages transfected with the fusion gene construct. In the future, such FP may be used as a clinical reagent to reduce the morbidity and mortality associated with serious gram-negative bacterial infections in surgical patients.     

Bidirectional signaling between Yersinia and its target cell

Lipopolysaccharide (LPS) exhibits a wide variety of bioactivities. Although it was generally proposed that the lipid A component represented the active center responsible for most of the bioactivities of LPS, a variety of lipid A partial structures and analogues were reported to have different properties. Lipopolysaccharide of the Re595 mutant of Salmonella minnesota is lack of O and part of the core polysaccharide (2 keto-3-deoxyoctanate (KDO) left on lipid A). Re595 lipid A (LA) and Re595 monophosphoryl lipid A (MPLA) differ in structure from Re595 LPS by lacking KDO and KDO plus phosphoryl group respectively. Whether these lipid A-common Re595 LPS preparations differed in activities, we investigated their effects on nitric oxide (NO), TNF-alpha, IL-6, and IL-12 induction from murine macrophage cell line RAW 264.7. RAW 264.7 cells (2 x 10(5) cells ml(-1)) were stimulated with these LPS preparations at 1 microg ml(-1) for 48 h. Re595 LPS, Re595 LA and Re595 MPLA significantly induced NO, TNF-alpha and IL-6 production; NO, TNF-alpha and IL-6 inducing capacities were in the order of LPS = LA > MPLA, LPS = LA = MPLA, and LPS = LA > MPLA respectively. However, these preparations did not induce IL-12 production from RAW cells even when stimulated in combination with IFN-gamma (20 U ml(-1)). IFN-gamma itself also induced NO, TNF-alpha and IL-6 production from RAW 264.7 cells. When RAW 264.7 cells were stimulated with IFN-gamma plus any of these preparations, effects were additive and synergistic for NO and IL-6 responses respectively. But TNF-alpha responses of RAW cells against these preparations were almost equal when cultured alone or in combination with IFN-gamma. Pre-treatment of RAW cells either with LPS, LA or MPLA at low concentration (0.1 microg ml(-1)) for 60 min before pulsing with IFN-gamma (20 IU ml(-1)) plus LPS (1 microg ml(-1)) for an additional 48 h, significantly (P < 0.01) decreased NO response. Although to a lesser extent, TNF-alpha and IL-6 responses were also decreased. Complete inhibition of NO inducing effect of these LPS preparations was achieved with polymyxin B at 40 microg ml(-1). But the concentration of polymyxin B to get a significant (P < 0.05) inhibitory effect on LPS was four times higher than that for LA or MPLA. Unexpectedly, polymyxin B also inhibited INF-gamma-induced NO production from RAW cells in a dose-dependent fashion. These findings suggested that effect of LPS was dependent, at least in part, on both the LPS polysaccharide chain length and the hydrophilic portion of LPS. In addition, not only LPS but also LA and MPLA exert either enhancing or suppressive effects, depending on their concentrations and the timing of their addition with respect to co-stimulators.     

A physiologic role of Bcl-xL induced in activated macrophages

Activated macrophages produce nitric oxide (NO) that is an important effector molecule for their antimicrobial and antitumor activities. Since this NO is also toxic for themselves, they have self-defense mechanisms. To elucidate the mechanisms in a physiologic condition, expression of bcl-2 family genes were examined in peritoneal macrophages and RAW264 macrophage cell line activated with IFN-gamma and LPS. Bcl-xL, but not bcl-2 and bax mRNA, was highly inducible within 3 h after stimulation. The induction required new protein synthesis, but was independent of effects of synthesized NO. Since activated RAW264 were more resistant to NO-induced apoptosis mediated by the exposure to S-nitroso-N-acetyl-penicillamine (SNAP) than nonactivated RAW264, the inducible Bcl-xL may play a role in the protection from NO toxicity. To confirm the protective function, RAW264 were stably transfected with bcl-xL. Those transfectants activated with IFN-gamma and LPS appeared highly resistant to NO-induced cell death detected within 24 h after stimulation, although their NO production was similar to those of parental RAW264 and neomycin control-transfected cells. Furthermore, bcl-xL transfectants displayed substantial protection from SNAP-induced apoptosis. These results establish a link between self-defense to the synthesized NO and the induction of Bcl-xL in activated macrophages.