Phospholipids and reactive nitrogen intermediates collaborate in expression of the T cell mitogenesis-inhibitory activity of immunosuppressive macrophages induced in mycobacterial infection

We studied the role of phospholipids and nitric oxide in expression of the suppressor activity of splenic macrophages induced by Mycobacterium avium-intracellulare complex infection (MAIC-induced macrophages) in mice against mitogenic response of concanavalin A (Con A)-stimulated splenocytes (SPC) as follows. First, phosphatidylserine (PS) and phosphatidylinositol were found to suppress Con A-induced mitogenesis of SPC via inhibition of IL-2 production and acquisition of IL-2 reactivity in Con A-stimulated T cells. The mitogenesis-inhibitory activity of PS was increased when SPC were cultured under mildly acidic condition (pH 6·3). When SPC were pretreated with PS for 24 h prior to Con A blastogenesis, their mitogenic response was irreversibly abrogated. Second, N^G-monomethyl-l-arginine, an inhibitor of nitric oxide (NO) synthase, was found to attenuate in part the expression of the suppressor activity of MAIC-induced macrophages. Third, reactive nitrogen intermediates (RNI) including NO generated from acidified NO2− exerted potent inhibitory activity against SPC mitogenic response, and the suppressive activity of RNI was significantly augmented by the combination with PS. These findings indicate that phospholipids and RNI play an important role in the expression of suppressor activity of MAIC-induced macrophages as the effector molecules.     

Comparative roles of free fatty acids with reactive nitrogen intermediates and reactive oxygen intermediates in expression of the anti-microbial activity of macrophages against Mycobacterium tuberculosis

We assessed the role of free fatty acids (FFA) in the expression of the activity of macrophages against Mycobacterium tuberculosis in relation to the roles of two major anti-microbial effectors, reactive nitrogen intermediates (RNI) and reactive oxygen intermediates (ROI). Intracellular growth of M. tuberculosis residing inside macrophages was accelerated by treatments of macrophages with either quinacrine (phospholipase A2 (PLA2) inhibitor), arachidonyl trifuloromethylketone (type IV cytosolic PLA2 inhibitor), NG-monomethyl-L-arginine (nitric oxide synthase inhibitor), and superoxide dismutase plus catalase (ROI scavengers). In addition, M. tuberculosis-infected macrophages produced and/or secreted these effectors sequentially in the order ROI (0-3 h), FFA (0-48 h), and RNI (3 to at least 72 h). Notably, membranous FFA (arachidonic acid) of macrophages translocated to M. tuberculosis residing in the phagosomes of macrophages in phagocytic ability- and PLA2-dependent fashions during cultivation after M. tuberculosis infection. FFA, RNI and H2O2-mediated halogenation system (H2O2-halogenation system) displayed strong activity against M. tuberculosis in cell-free systems, while ROI alone exerted no such effects. Combinations of 'FFA + RNI' and 'RNI + H2O2-halogenation system' exhibited synergistic and additive effects against M. tuberculosis, respectively, while 'FFA + H2O2-halogenation system' had an antagonistic effect. Moreover, a sequential attack of FFA followed by RNI exerted synergistic activity against M. tuberculosis. Since M. tuberculosis-infected macrophages showed simultaneous production of RNI with FFA secretion for relatively long periods (approx. 45 h) and prolonged RNI production was seen thereafter, RNI in combination with FFA appear to play critical roles in the manifestation of the activity of macrophages against M. tuberculosis.     

Suppression of Heme Oxygenase-1 by Prostaglandin E2-Protein Kinase A-A-Kinase Anchoring Protein Signaling Is Central for Augmented Cyclooxygenase-2 Expression in Lipopolysaccharide-Stimulated RAW 264.7 Macrophages

Purpose

Prostaglandin (PG) E₂ is an immunomodulatory lipid mediator generated mainly via the cyclooxygenase-2 (COX-2) pathway from arachidonic acid at sites of infection and inflammation. A positive feedback loop of PGE₂ on COX-2 expression is critical for homeostasis during toll-like receptor (TLR)-mediated inflammatory processes. The mechanism of PGE₂-regulated COX-2 expression remains poorly understood. The low-molecular-weight stress protein heme oxygenase-1 (HO-1) contributes to the anti-inflammatory, anti-oxidant and anti-apoptotic response against environmental stress.

Methods

We explored the involvement of HO-1 on PGE₂ regulation of LPS-induced COX-2 expression in RAW 264.7 macrophages.

Results

LPS-induced COX-2 expression in RAW 264.7 macrophages was enhanced by exogenous PGE₂ or cyclic AMP (cAMP) analogue and was suppressed by a COX inhibitor (indomethacin), a protein kinase A (PKA) inhibitor (KT5720), and A kinase anchoring protein (AKAP) disruptors (Ht31 and RIAD). This result suggests that the stimulatory effects of endogenous and exogenous PGE₂ on COX-2 expression are mediated by a cAMP-PKA-AKAP-dependent pathway. The induction of HO-1 was observed in LPS-stimulated RAW 264.7 macrophages. This induction was suppressed by exogenous PGE₂ and enhanced by blockage of the endogenous PGE₂ effect by the PKA inhibitor or AKAP disruptors. In addition, HO-1 induction by the HO activator copper protoporphyrin suppressed LPS-induced COX-2 expression, which was restored by the addition of exogenous PGE₂. The induction of HO-1 inhibited LPS-induced NF-κB p-65 nuclear expression and translocation.

Conclusions

AKAP plays an important role in PGE₂ regulation of COX-2 expression, and the suppression of HO-1 by PGE₂-cAMP-PKA-AKAP signaling helps potentiate the LPS-induced COX-2 expression through a positive feedback loop in RAW 264.7 macrophages.     

Enhancing effect of oxygen radical scavengers on murine macrophage anticryptococcal activity through production of nitric oxide

We examined the roles of reactive nitrogen intermediates (RNI) and reactive oxygen intermediates (ROI) in interferon-gamma (IFN-γ)-induced cryptococcostatic activity of murine peritoneal macrophages using N^G-monomethyl-L-arginine (L-NMMA), a competitive inhibitor of RNI synthesis, and superoxide dismutase (SOD) and catalase, oxygen radical scavengers. IFN-γ-activated macrophages produced nitric oxide (NO) in a dose-dependent manner, as measured by increased nitrite concentration in the culture supernatant. IFN-γ also enhanced the suppressive effect on cryptococcal growth in a similar dose-dependent manner. The induction of killing activity and NO production by an optimal dose of IFN-γ (100 U/ml) was virtually suppressed by 500 μM L-NMMA. These results confirmed the importance of the RNI-mediated effector mechanism in anticryptococcal activity of macrophages. SOD and catalase significantly enhanced the cryptococcostatic activity of macrophages induced by a suboptimal dose of IFN-γ (20 U/ml). The augmenting effect of these reagents was mediated by NO, since they potentiated the production of NO by macrophages and their effects were totally blocked by L-NMMA. Our results indicate that the IFN-γ-induced anticryptococcal activity of macrophages is dependent mostly on RNI, and suggest that the ROI system down-regulates the effector mechanism for cryptococcostasis by suppressing the RNI system.     

Contribution of group II phospholipase A2 to arachidonic acid release and prostaglandin synthesis by mesangial cells

Interleukin 1 (IL-1) and tumour necrosis factor (TNF)α have been found to increase group II phospholipase A₂ (PLA₂) synthesis and secretion by mesangial cells. In all cases 85%–90% of the enzyme is secreted from the cells and a parallel increase in prostaglandin (PGE₂) synthesis is observed. We report here that co-incubation with a monoclonal antibody that specifically binds and neutralizes rat group II PLA₂ attenuates IL-1β and TNFα-stimulated PGE₂ production by 45% and 52%, respectively. CGP43182, a specific inhibitor of group II PLA₂, potently blocks IL-1β- and TNFα-stimulated PGE₂ synthesis in intact mesangial cells with 1C₅₀s of 1.3 and 1.0 μM, respectively.     

Antiinflammatory action of thielocin A1β, a group II phospholipase A2 specific inhibitor,in rat carrageenan-induced pleurisy

Extracellular phospholipase A₂ (PLA₂) activity was detected in exudate from rat carrageenan-induced pleurisy using [³H]oleic acid-labeledEscherichia coli as substrate. Both exudate volume and PLA₂ activity increased up to 24 h after carrageenin injection. Specific absorption of this activity by anti-group II PLA₂ (PLA₂-II) antibody indicated that the PLA₂ activity in the pleural exudate was PLA₂-II. Thielocin Al, a novel type of PLA₂ inhibitor from fungi, inhibited this PLA₂-II activity in a dose-dependent manner (IC₅₀=0.32M). Thielocin A1 correspondingly reduced both exudate volume and PLA₂-II activity in the exudate in a dose-dependent manner when coinjected with carrageenan. The exudate volume was also significantly decreased when indomethacin, a cyclooxygenase inhibitor, or dexamethasone, a steroidal antiinflammatory drug, was coinjected with carrageenan. However, neither indomethacin nor dexamethasone could significantly attenuate the PLA₂-II activity in exudate In addition, indomethacin and dexamethasone significantly reduced the levels of PGE₂ in the exudate. However, thielocin A1 had no effect on the PGE₂ content in the exudate. These results suggest that thielocin A1 shows antiinflammatory activity due to inhibition of PLA₂-II and offer evidence for the significance of PLA₂1I in the propagation of inflammatory processes.     

Time-course of phospholipase A2, eicosanoid release and cellular accumulation in rat immunological air pouch inflammation

Phospholipase A₂ activity in the rat air pouch cavity was determined after induction of a reverse passive Arthus reaction. Time-course of phospholipase A₂ activity appeared to correlate with increased prostaglandin E₂ levels in inflammatory exudate and with the influx of mononuclear inflammatory cells.Local administration of anti-inflammatory drugs such as dexamethasone, indomethacin, or a PLA₂ inhibitor such as p-bromophenacyl bromide significantly inhibited exudate volume, cellular influx, granuloma formation, exudate PGE₂ levels and PLA₂ activity, to varying degrees. Dexamethasone treatment significantly reduced all parameters determined, whereas p-bromophenacyl bromide had a significant inhibitory effect on PLA₂ activity and PGE₂ release, and indomethacin only restored PGE₂ levels.These results show that PLA₂ is neither the only nor the most important factor involved in the development of subchronic inflammation.     

Inhibition of neutral sphingomyelinase decreases arachidonic acid mediated inflammation in liver ischemia-reperfusion injury

This study aimed to determine the role of selective neutral sphingomyelinase (N-SMase) inhibition on arachidonic acid (AA) mediated inflammation following liver ischemia-reperfusion (IR) injury. Selective N-SMase inhibitor was administered via intraperitoneal injections. Liver IR injury was created by clamping blood vessels supplying the median and left lateral hepatic lobes for 60 min, followed by 60 min reperfusion. Levels of AA in liver tissue were determined by multiple reaction monitoring (MRM) using ultra fast-liquid chromatography (UFLC) coupled with tandem mass spectrometry (MS/MS). Phospholipase A₂ (PLA₂), cyclooxygenase (COX) and prostaglandin E₂ (PGE₂) were measured in liver tissue. Arachidonic acid levels, activity of PLA₂, COX and PGE₂ levels were significantly increased in postischemic liver tissue compared to nonischemic controls. N-SMase inhibition significantly decreased COX activity and PGE₂ levels in postischemic liver. Future studies evaluating agents blocking N-SMase activity can facilitate the development of treatment strategies to alleviate inflammation in liver I/R injury.     

Phosphatidylserine-containing liposomes: potential pharmacological interventions against inflammatory and immune diseases through the production of prostaglandin E(2) after uptake by myeloid derived phagocytes

Phosphatidylserine (PS), which is normally located on the inner leaflet of the plasma membrane, translocates to the outer leaflet at the early stage of apoptosis. The PS externalization provides a signal for phagocytes to initiate uptake of apoptotic cells. After phagocytosis of apoptotic cells, phagocytes induce the secretion of anti-inflammatory mediators including prostaglandin E₂ (PGE₂). PS-containing liposomes (PSLs) can mimic the effects of apoptotic cells on phagocytes to induce the secretion of PGE₂. PSLs induce the PGE₂ secretion from microglia without induction of either cyclooxygenase (COX)-2 or microsomal prostaglandin E synthase (mPGES)-1. PSLs are found to rather utilize COX-1/mPGES-2 system to produce PGE₂ secretion and then shift microglia and macrophages from pro- to anti-inflammatory phenotype by an autocrine action of PGE₂. Moreover, PSLs inhibit the maturation of dendritic cells and osteoclast precursors. Therefore, PSLs will be potential pharmacological interventions for inflammatory and immune diseases through feedback mechanism utilizing PGE₂.     

Inhibition of phospholipase A2 (PLA2) activity by nifedipine and nisoldipine is independent of their calcium-channel-blocking activity

The effects of several calcium antagonists on phospholipase A₂ (PLA₂) activity were examined. Nifedipine and nisoldipine inhibited a cell-free preparation of PLA₂ in a dose-dependent manner with maximal inhibition of 71–77% observed at 100M. More potent or equipotent dihydropyridine calcium antagonists such as nitrendipine and felodipine did not inhibit PLA₂ activity. In addition, nondihydropyridine calcium antagonists such as diltiazem, verapamil, and cinnarazine failed to reduce PLA₂ activity markedly. Nifedipine and nisoldipine also reduced PLA₂ activity in intact mouse peritoneal macrophages where PLA₂ activity was monitored by free [¹⁴C]arachidonic acid release from [¹⁴C]arachidonic acid-prelabeled cells. When levels of PGE₂ and LTC₄ were measured by radioimmunoassay, it was found that the synthesis of these two metabolites was concomitantly inhibited by nifedipine and nisoldipine. In vivo, nifedipine and nisoldipine inhibited tetradecanoylphorbol acetate (TPA) induced ear edema. UV irradiation of nifedipine and nisoldipine (which destroys the slow caicium-channel-blocking activity of these compounds) did not result in a loss of PLA₂ inhibitory activity. In fact, in both instances the UV-irradiated forms of nifedipine and nisoldipine were slightly more potent PLA₂ inhibitors than the parent compound alone. We therefore conclude that the ability of nifedipine and nisoldipine to inhibit PLA₂ was direct and unrelated to their actions on slow calcium channels.     

Further studies on the mechanism of suppression of human lymphocyte transformation by human alpha fetoprotein

We investigated the possibility that noncovalent binding of negatively charged molecules such as prostaglandins (PGs) might be partly responsible for human alpha fetoprotein (HAFP) heterogeneity with respect to charge and the suppression of human lymphocyte responses, since PGs are potent suppressors of lymphocyte transformation. Indomethacin, an inhibitor of PG synthesis, had no effect upon the transformation of adherent-cell-depleted human lymphocytes, nor did it interfere with the capacity of HAFP to inhibit lymphocyte transformation. When a partially suppressive dose of HAFP was added to mitogen-stimulated lymphocytes together with varying doses of prostaglandin E₁ or E₂ (PGE₁ or PGE₂), no evidence of inhibitory synergy was demonstrable; their combined suppressive action was either less than or approximately equal to the sum of their respective inhibitory effects. Addition of PGE₂ to a nonsuppressive dose of an impotent HAFP preparation resulted in no greater lymphocyte suppression than that achieved by PGE₂ alone. Analysis of the kinetics of suppression of lymphocyte transformation by PG and HAFP yielded unequivocal evidence for their disparate mechanisms of action. Suppression of lymphocyte transformation by PGE₂ is evident by 24 to 40 hr of culture, persists throughout the entire culture period (including peak deoxyribonucleic acid [DNA] synthesis at 72 to 88 hr and beyond), and does not vary in profundity at any time. By contrast, HAFP-induced suppression of lymphocyte DNA synthesis is lacking at 24 to 40 hr, reaches a peak at 72 to 88 hr, and is waning by 96 to 112 hr. Cultures containing HAFP synthesize more DNA than control cultures during the 120 to 136 hr culture period. These observations, together with our earlier demonstration of a subpopulation of human lymphocytes resistant to HAFP inhibition, suggest that, after the first 24 to 40 hr of culture, HAFP may enhance the proliferation of a suppressor population which gradually dampens the proliferative response. HAFP is capable of suppressing the proliferative response of periodate-treated lymphocytes; this confirms that it does not act via competition with the cell membrane for a mitogen-binding site. Immunofluorescence studies indicate that human peripheral blood lymphocytes lack membrane-associated HAFP or HAFP receptors of high affinity. Our results indicate that PG, either present as a putative contaminant in HAFP isolates or endogenously synthesized by lymphocytes (or monocytes), plays no role in lymphocyte suppression by HAFP.     

Implication of Prostaglandin E2 in Soluble Factor-Mediated Immune Suppression by Murine Decidual Cells

ABSTRACT: Cells from the uteri of pregnant mice mediate profound nonantigen-specific and nonmajor histocompatibility complex-restricted immune suppression in vitro. In part, those cells accomplish suppression by releasing soluble suppressor factors. The purpose of the present study was to initiate identification of uterine cell suppressor factors. Immune suppression was assayed by the effect of decidual cells or in vitro generated supernatants of decidual cells on mixed lymphocyte reactions (MLR). The following findings support the designation of prostaglandin E₂ (PGE₂) as a primary suppressor molecule originating with decidual cells: (1) Suppression mediated by supernatants of decidual cells was relieved by removal of lipids but not proteins; (2) indomethacin, an inhibitor of prostaglandin synthesis, produced partial relief of suppression mediated by uterine cells and totally inhibited soluble suppressor factor generation by those cells; (3) decidual cells produced high levels of both PGE₂ and PGF_2a; (4) the addition of exogenous PGE₂ at levels comparable to those found in the decidual cell supernatants restored suppression by decidual cells and their supernatants whereas the addition of PGF_2a had no effect; (5) inhibition of the lipoxygenase pathway of arachidonate metabolism had no effect on cell or supernatant mediated suppression; (6) nonspecific suppressor mechanisms, such as arginine depletion and peroxide generation, were excluded as possible mediators of MLR suppression by decidual cells and their supernatants. Fractionation of decidual cells revealed at least three indomethacin-sensitive cell types: small, lymphocytelike cells, macrophages, and a third population of large decidual cells that was not identified by specific markers. The data strongly suggest that prostaglandins, primarily PGE₂, are involved in immunosuppression by cells from the uteri of pregnant mice.     

IL-1β Stimulates the Expression of Prostaglandin Receptor EP4 in Human Chondrocytes by Increasing Production of Prostaglandin E2

Prostaglandin (PG) E₂, which exerts its actions via the PG receptors EP1–4, is produced from arachidonic acid by cyclooxygenase (COX)-1 and COX-2. The aim of this study was to investigate the mechanisms by which interleukin (IL)-1β induces the expression of PG receptors in cultured human chondrocytes and to explore the role of PGE₂ in this process. The cells were cultured with 0, 10, or 100 U/mL IL-1β with or without 1 μM celecoxib, a specific inhibitor of COX-2, for up to 28 days. Expression of the genes encoding COX-1, COX-2, and EP1–4 was quantified using real-time PCR, and expression of the corresponding proteins was examined using immunohistochemical staining. PGE₂ production was determined using ELISA. IL-1β treatment caused a marked dose- and time-dependent increase in the levels of PGE₂, COX-2, and EP4 as compared with the untreated control. It did not affect the expression of COX-1, and it decreased the expression of EP1 and EP2. EP3 expression was not detected in either the absence or the presence of IL-1β. When celecoxib was also present, IL-1β failed to stimulate PGE₂ production and EP4 expression, but its stimulatory effect on COX-2 expression and its inhibitory effect on EP1 and EP2 expression were unchanged. IL-1β increases the production of PGE₂, COX-2, and the PG receptor EP4 in cultured human chondrocytes. The increase in EP4 expression appears to be a result of the increased PGE₂ production.     

Cholera Toxin Induces a Shift from Inactive to Active Cyclooxygenase 2 in Alveolar Macrophages Activated by Mycobacterium bovis BCG

Intranasal vaccination stimulates formation of cyclooxygenases (COX) and release of prostaglandin E₂ (PGE₂) by lung cells, including alveolar macrophages. PGE₂ plays complex pro- or anti-inflammatory roles in facilitating mucosal immune responses, but the relative contributions of COX-1 and COX-2 remain unclear. Previously, we found that Mycobacterium bovis BCG, a human tuberculosis vaccine, stimulated increased release of PGE₂ by macrophages activated in vitro; in contrast, intranasal BCG activated no PGE₂ release in the lungs, because COX-1 and COX-2 in alveolar macrophages were subcellularly dissociated from the nuclear envelope (NE) and catalytically inactive. This study tested the hypothesis that intranasal administration of BCG with cholera toxin (CT), a mucosal vaccine component, would shift the inactive, NE-dissociated COX-1/COX-2 to active, NE-associated forms. The results showed increased PGE₂ release in the lungs and NE-associated COX-2 in the majority of COX-2⁺ macrophages. These COX-2⁺ macrophages were the primary source of PGE₂ release in the lungs, since there was only slight enhancement of NE-associated COX-1 and there was no change in COX-1/COX-2 levels in alveolar epithelial cells following treatment with CT and/or BCG. To further understand the effect of CT, we investigated the timing of BCG versus CT administration for in vivo and in vitro macrophage activations. When CT followed BCG treatment, macrophages in vitro had elevated COX-2-mediated PGE₂ release, but macrophages in vivo exhibited less activation of NE-associated COX-2. Our results indicate that inclusion of CT in the intranasal BCG vaccination enhances COX-2-mediated PGE₂ release by alveolar macrophages and further suggest that the effect of CT in vivo is mediated by other lung cells.     

Lansoprazole inhibits nitric oxide and prostaglandin E(2) production in murine macrophage RAW 264.7 cells

Aberrantly activated macrophages, which overproduce inflammatory mediators, are involved in the pathogenesis of many inflammatory diseases. We analyzed the anti-inflammatory activity of lansoprazole (LPZ), a typical proton pump (P-ATPase) inhibitor, on RAW264.7 murine macrophages. Treatment of lipopolysaccharide (LPS)-stimulated RAW264.7 cells with LPZ inhibited the production of nitric oxide (NO) and prostaglandin E₂ (PGE₂). Since P-ATPase expression was not observed in RAW264.7 cells, the anti-inflammatory effect of LPZ was independent of ATPase. In contrast, diphenylene iodonium (DPI), an inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, decreased NO but not PGE₂ levels. LPZ suppressed the LPS-stimulated production by RAW264.7 cells of reactive oxygen species (ROS), which plays an important role in inflammatory responses. ROS elevation in these cells was associated with NO but not PGE₂ production, suggesting that LPZ inhibits NO production by suppressing NADPH oxidase activity. These findings suggest that LPZ may be useful in the treatment of many inflammatory diseases associated with activated macrophages.     

Prostaglandin production and cellular aging

Prostaglandin (PG) production by human embryo lung fibroblasts (HELF) is stimulated by a number of effectors including angiotensin, thrombin, bradykinin and ascorbic acid. The types of prostaglandins produced are to a degree effector-dependent. For example, angiotensin stimulates mainly PGE₂ synthesis, thrombin stimulates production of both PGE₂ and prostacyclin while bradykinin and ascorbic acid stimulate production of PGE₂, PGF_2α, prostacyclin and thromboxane A₂. Upon senescence in culture, prostacyclin (PGI₂) production by HELF drops dramatically in response to ascorbic or arachidonic acids. An overall drop in prostaglandin synthesis is observed with bradykinin stimulation. Prostaglandin production is also related to senescence in human skin fibroblasts (HSF). These cells produce predominantly PGI₂. Prostacyclin production in response to bradykinin drops in HSF as they are obtained from individuals of increasing chronologic age. Thus our results indicate changes in prostaglandin production upon senescence, a dependency of these changes on the given stimulus and a correlation between in vivo and in culture aging with respect to prostaglandin production.     

Interleukin-1β induces cytosolic PLA2 in parallel with prostaglandin E2 in rheumatoid synovial fibroblasts

We investigated the temporal relationship between the increase in enzymatic activity and protein of a high molecular weight (100 kDa), cytosolic PLA₂ (cPLA₂) in interleukin-1β (IL-1β)-treated rheumatoid synovial fibroblasts (RSF). Both of these responses increased according to a similar time-course which correlates with PGE₂ production by these cells. In contrast, 14 kDa, secreted PLA₂ (sPLA₂), which was also produced by RSF, was not affected by IL-1β treatment. These findings support that an augmentation of cPLA₂ activity, caused by an induction of cPLA₂ protein, rather than sPLA₂, is temporally associated with increased PGE₂ production in IL-1β-treated RSF.     

Francisella tularensis LVS Induction of Prostaglandin Biosynthesis by Infected Macrophages Requires Specific Host Phospholipases and Lipid Phosphatases

Francisella tularensis induces the synthesis of prostaglandin E₂ (PGE₂) by infected macrophages to alter host immune responses, thus providing a survival advantage to the bacterium. We previously demonstrated that PGE₂ synthesis by F. tularensis-infected macrophages requires cytosolic phospholipase A2 (cPLA₂), cyclooxygenase 2 (COX-2), and microsomal prostaglandin E synthase 1 (mPGES1). During inducible PGE₂ synthesis, cPLA₂ hydrolyzes arachidonic acid (AA) from cellular phospholipids to be converted to PGE₂. However, in F. tularensis-infected macrophages we observed a temporal disconnect between Ser505-cPLA₂ phosphorylation (a marker of activation) and PGE₂ synthesis. These results suggested to us that cPLA₂ is not responsible for the liberation of AA to be converted into PGE₂ by F. tularensis-infected macrophages. Utilizing small-molecule inhibitors, we demonstrated that phospholipase D and diacylglycerol lipase were required for providing AA for PGE₂ biosynthesis. cPLA₂, on the other hand, was required for macrophage cytokine responses to F. tularensis. We also demonstrated for the first time that lipin-1 and PAP2a contribute to macrophage inflammation in response to F. tularensis. Our results identify both an alternative pathway for inducible PGE₂ synthesis and a role for lipid-modifying enzymes in the regulation of macrophage inflammatory function.     

汉黄芩素对流感病毒感染肺泡巨噬细胞炎症相关因子的影响

目的: 研究汉黄芩素对甲型流感病毒鼠肺适应株A/FM/1/47(H1N1)感染的大鼠肺泡巨噬细胞(NR8383)产生促炎症细胞因子、炎症介质及氧自由基的影响。 方法: 流感病毒感染 NR8383细胞1 h后,加入含汉黄芩素的培养基(终浓度16 mg/L),药物作用后6 h、12 h和24 h,ELISA法检测细胞上清中肿瘤坏死因子α(TNF-α)和单核细胞趋化蛋白 1(MCP-1)的含量,放射免疫测定法检测细胞上清中前列腺素E₂(PGE₂)、磷脂酸A₂(PLA₂)和白三烯B₄(LTB₄)的含量;药物作用后8 h、24 h、36 h和48 h,生化法检测细胞内一氧化氮(NO)含量和诱导型一氧化氮合酶(iNOS)活性,4 h、8 h、18 h和24 h,生化法检测超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量;药物作用后24 h,real-time PCR检测细胞内TNF-α和MCP-1的mRNA水平。 结果: 汉黄芩素抑制了流感病毒感染NR8383细胞后TNF-α、MCP-1的转录和表达(P<0.01),降低了PGE₂、PLA₂、LTB₄和MDA的含量(P<0.05);减少了NO和iNOS的产生(P<0.05),增强了SOD的活性(P<0.05)。 结论: 汉黄芩素明显抑制了流感病毒感染后肺泡巨噬细胞内各种炎症相关因子的产生,具有抗炎作用。