Synthetic prostacyclin analogs differentially regulate macrophage function via distinct analog-receptor binding specificities

PGI(2) (prostacyclin) is a lipid mediator with vasodilatory and antithrombotic effects used in the treatment of vasoconstrictive/ischemic diseases including pulmonary artery hypertension. However, emerging research supports a role for PGs, including PGI(2), in the regulation of both innate and acquired immunity. As PGI(2) is unstable, we sought to define the effects of various PGI(2) analogs on resident alveolar macrophage (AM) and peritoneal macrophage (PM) innate immune functions. The effects of iloprost, carbaprostacyclin, and treprostinil on the regulation of phagocytosis, bacterial killing, and inflammatory mediator production were determined in both macrophage populations from rats. Iloprost failed to suppress AM functions to the same degree that it did in PMs, a characteristic shared by carbaprostacyclin. This difference reflected greater expression of the G(alphas) protein-coupled I prostanoid receptor and greater cAMP generation in PMs than AMs. Treprostinil inhibited phagocytosis, bacterial killing, and cytokine generation in AMs to a much greater degree than the other PGI(2) analogs and more closely resembled the effects of PGE(2). Studies with the E prostanoid (EP) 2 receptor antagonist AH-6809 and EP2-null macrophages indicated that this was due in part to the previously unknown ability of treprostinil to stimulate the EP2 receptor. The present investigation for the first time identifies differences in immunoregulatory properties of clinically administered PGI(2) analogs. These studies are the first to explore the capacity of PGI(2) to regulate bacterial killing and phagocytosis in macrophages, and our findings may hold important consequences regarding the risk of infection for patients receiving such agents.     

Prostaglandin E2 inhibits alveolar macrophage phagocytosis through an E-prostanoid 2 receptor-mediated increase in intracellular cyclic AMP

Prostaglandin E(2) is a potent lipid mediator of inflammation that effects changes in cell functions through ligation of four distinct G protein-coupled receptors (E-prostanoid (EP)1, EP2, EP3, and EP4). During pneumonia, PGE(2) production is enhanced. In the present study, we sought to assess the effect of endogenously produced and exogenously added PGE(2) on FcRgamma-mediated phagocytosis of bacterial pathogens by alveolar macrophages (AMs), which are critical participants in lung innate immunity. We also sought to characterize the EP receptor signaling pathways responsible for these effects. PGE(2) (1-1000 nM) dose-dependently suppressed the phagocytosis by rat AMs of IgG-opsonized erythrocytes, immune serum-opsonized Klebsiella pneumoniae, and IgG-opsonized Escherichia coli. Conversely, phagocytosis was stimulated by pretreatment with the cyclooxygenase inhibitor indomethacin. PGE(2) suppression of phagocytosis was associated with enhanced intracellular cAMP production. Experiments using both forskolin (adenylate cyclase activator) and rolipram (phosphodiesterase IV inhibitor) confirmed the inhibitory effect of cAMP stimulation. Immunoblot analysis of rat AMs identified expression of only EP2 and EP3 receptors. The selective EP2 agonist butaprost, but neither the EP1/EP3 agonist sulprostone nor the EP4-selective agonist ONO-AE1-329, mimicked the effects of PGE(2) on phagocytosis and cAMP stimulation. Additionally, the EP2 antagonist AH-6809 abrogated the inhibitory effects of both PGE(2) and butaprost. We confirmed the specificity of our results by showing that AMs from EP2-deficient mice were resistant to the inhibitory effects of PGE(2). Our data support a negative regulatory role for PGE(2) on the antimicrobial activity of AMs, which has important implications for future efforts to prevent and treat bacterial pneumonia.     

Expression of N-methyl-d-aspartate receptor and its effect on nitric oxide production of rat alveolar macrophages

We early show that glutamate (Glu) mediate hyperoxia-induced newborn rat lung injury through N-methyl-d-aspartate receptor (NMDAR). In this study, we search for evidence of NMDAR expression on newborn rat alveolar macrophages (AMs) and the difference between newborn and adult rat AMs, and the possible effect on nitric oxide (NO) production of AMs by exogenous NMDA. The protein of NMDAR was showed by immunocytochemistry, and the mRNA was examined by RT-PCR and real-time PCR. The results show that: (i) both newborn and adult rat AMs express NMDAR1 and the four NMDAR2 subtypes and newborn rat AMs are higher expression. (ii) NMDA administration increase NO production, inducible nitric oxide synthase (iNOS) activity and iNOS mRNA expression of AMs. (iii) NMDAR activation elevates NO secretion of AMs, which suggests that AM may be one of the key cellular origin of the elevated NO secretion in hyperoxia-induced lung injury.     

Assessment of training effects on activity levels of alveolar macrophage in matured rats using chemiluminescent technique

Chemiluminescence responses have been used for the evaluation of phagocyte function. In this study, to evaluate effects of training started after maturation on pulmonary immunity, the activity levels of rat alveolar macrophages (AMs) were assessed as reactive oxygen species (ROS) generating capacity, measured by lucigenin‐ and luminol‐dependent chemiluminescence, using a parallel luminometer. One group of male Wistar rats started training at 11 weeks old and another group at 17 weeks old. The experimental period was 12 weeks, and about half of the rats were sacrificed after 6 weeks. The forced and voluntary exercises affect the mean levels of body weights and cell populations in the bronchoalveolar lavage fluid in younger animals; however, the voluntary exercise group in younger animals seemed to adapt after 12 weeks. By contrast, chemiluminescence responses in older rats observed after 6 weeks suggest that AMs are primed, and the maximum releasing activities of ROS are reduced. These changes in AM activity may be caused by the exercise and separation stresses and the rats may adapt to those stressors after 12 weeks. The chemiluminescent technique is thought to be useful to evaluate the changes of AM activity. Copyright © 2002 John Wiley & Sons, Ltd.     

Development and Functions of Alveolar Macrophages

Macrophages residing in various tissue types are unique in terms of their anatomical locations, ontogenies, developmental pathways, gene expression patterns, and immunological functions. Alveolar macrophages (AMs) reside in the alveolar lumen of the lungs and serve as the first line of defense for the respiratory tract. The immunological functions of AMs are implicated in the pathogenesis of various pulmonary diseases such as allergic asthma, chronic obstructive pulmonary disorder (COPD), pulmonary alveolar proteinosis (PAP), viral infection, and bacterial infection. Thus, the molecular mechanisms driving the development and function of AMs have been extensively investigated. In this review article, we discuss the roles of granulocyte-macrophage colony-stimulating factor (GM-CSF) and transforming growth factor (TGF)-β in AM development, and provide an overview of the anti-inflammatory and pro-inflammatory functions of AMs in various contexts. Notably, we examine the relationships between the metabolic status of AMs and their development processes and functions. We hope that this review will provide new information and insight into AM development and function.     

House dust mite Dermatophagoides farinae augments proinflammatory mediator productions and accessory function of alveolar macrophages: implications for allergic sensitization and inflammation

In this study, we examine the effects of Dermatophagoides farinae (Der f), a major source of airborne allergens, on alveolar macrophages (AMs), and we also test its contribution to allergic responses in mice. Der f activated NF-kappaB of AMs and, unlike OVA or LPS stimulation, up-regulated IL-6, TNF-alpha, and NO. In addition, it down-regulated antioxidants, but affected neither the expression nor production of IL-12. Der f-stimulated AMs expressed enhanced levels of costimulatory B7 molecules, supported T cell proliferation, and promoted Th2 cell development. The enhanced accessory function was suppressed by blockade mAbs to B7.2, IL-6, and TNF-alpha and by N-monomethyl-L-arginine, an NO synthase inhibitor, and N-acetylcysteine, a thiol antioxidant, whereas it was augmented by (+/-)-S-nitroso-N-acetylpenicillamine, an NO donor. Arg-Gly-Asp-Ser peptide and neo-glycoproteins galactose-BSA and mannose-BSA inhibited the Der f-induced IL-6 and TNF-alpha productions and enhanced accessory function of AMs. Der f was more potent than OVA for inducing pulmonary eosinophilic inflammation, NO, and serum allergen-specific IgG1 Ab production in mice. AMs from Der f-challenged mice expressed enhanced levels of B7 and augmented T cell proliferation ex vivo. In Der f-challenged mice, respiratory syncytial virus infection (5 x 10(5) pfu; 3 days before Der f instillation) augmented Der f-specific Ab production, whereas dexamethasone (50 mg/kg; 1 h before Der f instillation) diminished the allergic airway inflammation and Ab response. We conclude that AMs are sensitive targets for Der f and that the Der f-induced proinflammatory responses may represent an important mechanism in mediating the development of allergic sensitization and inflammation.     

Critical role of prostaglandin E2 overproduction in impaired pulmonary host response following bone marrow transplantation

The success of bone marrow transplantation (BMT) as a therapy for malignant and inherited disorders is limited by infectious complications. We previously demonstrated syngeneic BMT mice are more susceptible to Pseudomonas aeruginosa pneumonia due to defects in the ability of donor-derived alveolar macrophages (AMs), but not polymorphonuclear leukocytes (PMNs), to phagocytose bacteria. We now demonstrate that both donor-derived AMs and PMNs display bacterial killing defects post-BMT. PGE2 is a lipid mediator with potent immunosuppressive effects against antimicrobial functions. We hypothesize that enhanced PGE2 production post-BMT impairs host defense. We demonstrate that lung homogenates from BMT mice contain 2.8-fold more PGE2 than control mice, and alveolar epithelial cells (2.7-fold), AMs (125-fold), and PMNs (10-fold) from BMT animals all overproduce PGE2. AMs also produce increased prostacyclin (PGI2) post-BMT. Interestingly, the E prostanoid (EP) receptors EP2 and EP4 are elevated on donor-derived phagocytes post-BMT. Blocking PGE2 synthesis with indomethacin overcame the phagocytic and killing defects of BMT AMs and the killing defects of BMT PMNs in vitro. The effect of indomethacin on AM phagocytosis could be mimicked by an EP2 antagonist, AH-6809, and exogenous addition of PGE2 reversed the beneficial effects of indomethacin in vitro. Importantly, in vivo treatment with indomethacin reduced PGE2 levels in lung homogenates and restored in vivo bacterial clearance from the lung and blood in BMT mice. Genetic reduction of cyclooxygenase-2 in BMT mice also had similar effects. These data clearly demonstrate that overproduction of PGE2 post-BMT is a critical factor determining impaired host defense against pathogens.     

Lung-restricted macrophage activation in the pearl mouse model of Hermansky-Pudlak syndrome

Pulmonary inflammation, abnormalities in alveolar type II cell and macrophage morphology, and pulmonary fibrosis are features of Hermansky-Pudlak Syndrome (HPS). We used the naturally occurring "pearl" HPS2 mouse model to investigate the mechanisms of lung inflammation observed in HPS. Although baseline bronchoalveolar lavage (BAL) cell counts and differentials were similar in pearl and strain-matched wild-type (WT) mice, elevated levels of proinflammatory (MIP1gamma) and counterregulatory (IL-12p40, soluble TNFr1/2) factors, but not TNF-alpha, were detected in BAL from pearl mice. After intranasal LPS challenge, BAL levels of TNF-alpha, MIP1alpha, KC, and MCP-1 were 2- to 3-fold greater in pearl than WT mice. At baseline, cultured pearl alveolar macrophages (AMs) had markedly increased production of inflammatory cytokines. Furthermore, pearl AMs had exaggerated TNF-alpha responses to TLR4, TLR2, and TLR3 ligands, as well as increased IFN-gamma/LPS-induced NO production. After 24 h in culture, pearl AM LPS responses reverted to WT levels, and pearl AMs were appropriately refractory to continuous LPS exposure. In contrast, cultured pearl peritoneal macrophages and peripheral blood monocytes did not produce TNF-alpha at baseline and had LPS responses which were no different from WT controls. Exposure of WT AMs to heat- and protease-labile components of pearl BAL, but not WT BAL, resulted in robust TNF-alpha secretion. Similar abnormalities were identified in AMs and BAL from another HPS model, pale ear HPS1 mice. We conclude that the lungs of HPS mice exhibit hyperresponsiveness to LPS and constitutive and organ-specific macrophage activation.     

Phenotypic comparison of allergic airway responses to house dust mite in three rat strains

Brown Norway (BN) rats develop a robust response to antigens in the lung, characterized by a large increase in allergen-specific immune function and pulmonary eosinophilia. The objective of this study was to investigate alternative models by determining whether other rat strains could be sensitized to house dust mite (HDM) antigen and whether the allergic disease process could be worsened with repeated allergen exposure. In general, BN rats sensitized by either subcutaneous or intratracheal routes exhibited increased pulmonary allergy compared with Sprague-Dawley (SD) and Lewis (L) rats. Multiple intratracheal allergen exposures incrementally increased HDM-specific immune function in BN rats but progressively decreased eosinophil recruitment and markers of lung injury. SD rats had more moderate responses, whereas L rats were relatively unresponsive. Because BN rats developed stronger clinical hallmarks of allergic asthma under various immunization regimes compared with SD and L rats, we conclude that the BN is the most appropriate strain for studying allergic asthma-like responses in rats. Phenotypic differences in response to HDM were associated with differences in the Th1/Th2 cytokine balance and antioxidant capacity.     

Hypoxia-induced pulmonary endothelin-1 expression is unaltered by nitric oxide.

Nitric oxide (NO) attenuates hypoxia-induced endothelin (ET)-1 expression in cultured umbilical vein endothelial cells. We hypothesized that NO similarly attenuates hypoxia-induced increases in ET-1 expression in the lungs of intact animals and reasoned that potentially reduced ET-1 levels may contribute to the protective effects of NO against the development of pulmonary hypertension during chronic hypoxia. As expected, hypoxic exposure (24 h, 10% O(2)) increased rat lung ET-1 peptide and prepro-ET-1 mRNA levels. Contrary to our hypothesis, inhaled NO (iNO) did not attenuate hypoxia-induced increases in pulmonary ET-1 peptide or prepro-ET-1 mRNA levels. Because of this surprising finding, we also examined the effects of NO on hypoxia-induced increases in ET peptide levels in cultured cell experiments. Consistent with the results of iNO experiments, administration of the NO donor S-nitroso-N-acetyl-penicillamine to cultured bovine pulmonary endothelial cells did not attenuate increases in ET peptide levels resulting from hypoxic (24 h, 3% O(2)) exposure. In additional experiments, we examined the effects of NO on the activity of a cloned ET-1 promoter fragment containing a functional hypoxia inducible factor-1 binding site in reporter gene experiments. Whereas moderate hypoxia (24 h, 3% O(2)) had no effect on ET-1 promoter activity, activity was increased by severe hypoxic (24 h, 0.5% O(2)) exposure. ET-1 promoter activity after S-nitroso-N-acetyl-penicillamine administration during severe hypoxia was greater than that in normoxic controls, although activity was reduced compared with that in hypoxic controls. These findings suggest that hypoxia-induced pulmonary ET-1 expression is unaffected by NO.     

Surfactant protein A prevents silica-mediated toxicity to rat alveolar macrophages

Silicosis is a serious occupational lung disease associated with irreversible pulmonary fibrosis. The interaction between inhaled crystalline silica and the alveolar macrophage (AM) is thought to be a key event in the development of silicosis and fibrosis. Silica can cause direct injury to AMs and can induce AMs to release various inflammatory mediators. Acute silicosis is also characterized by a marked elevation in surfactant apoprotein A (SP-A); however, the role of SP-A in silicosis is unknown. We investigated whether SP-A directly affects the response of AMs to silica. In this study, the degree of silica toxicity to cultured rat AMs as assessed by a (51)Cr cytotoxicity assay was shown to be dependent on the time of exposure and the concentration and size of the silica particles. Silica directly injured rat AMs as evidenced by a cytotoxic index of 32.9 +/- 2.5, whereas the addition of rat SP-A (5 microg/ml) significantly reduced the cytotoxic index to 16.6 +/- 1.2 (P < 0. 001). This effect was reversed when SP-A was incubated with either polyclonal rabbit anti-rat SP-A antibody or D-mannose. These data indicate that SP-A mitigates the effect of silica on AM viability, and this effect may involve the carbohydrate recognition domain of SP-A. The elevation of SP-A in acute silicosis may serve as a normal host response to prevent lung cell injury after exposure to silica.     

Paradoxical role of alveolar macrophage-derived granulocyte-macrophage colony-stimulating factor in pulmonary host defense post-bone marrow transplantation

Impaired host defense post-bone marrow transplant (BMT) is related to overproduction of prostaglandin E(2) (PGE(2)) by alveolar macrophages (AMs). We show AMs post-BMT overproduce granulocyte-macrophage colony-stimulating factor (GM-CSF), whereas GM-CSF in lung homogenates is impaired both at baseline and in response to infection post-BMT. Homeostatic regulation of GM-CSF may occur by hematopoietic/structural cell cross talk. To determine whether AM overproduction of GM-CSF influenced immunosuppression post-BMT, we compared mice that received BMT from wild-type donors (control BMT) or mice that received BMT from GM-CSF-/- donors (GM-CSF-/- BMT) with untransplanted mice. GM-CSF-/- BMT mice were less susceptible to pneumonia with Pseudomonas aeruginosa compared with control BMT mice and showed antibacterial responses equal to or better than untransplanted mice. GM-CSF-/- BMT AMs displayed normal phagocytosis and a trend toward enhanced bacterial killing. Surprisingly, AMs from GM-CSF-/- BMT mice overproduced PGE(2), but expression of the inhibitory EP(2) receptor was diminished. As a consequence of decreased EP(2) receptor expression, we found diminished accumulation of cAMP in response to PGE(2) stimulation in GM-CSF-/- BMT AMs compared with control BMT AMs. In addition, GM-CSF-/- BMT AMs retained cysteinyl leukotriene production and normal TNF-alpha response compared with AMs from control BMT mice. GM-CSF-/- BMT neutrophils also showed improved bacterial killing. Although genetic ablation of GM-CSF in hematopoietic cells post-BMT improved host defense, transplantation of wild-type bone marrow into GM-CSF-/- recipients demonstrated that parenchymal cell-derived GM-CSF is necessary for effective innate immune responses post-BMT. These results highlight the complex regulation of GM-CSF and innate immunity post-BMT.     

Serum amyloid P-component in murine tuberculosis: induction kinetics and intramacrophage Mycobacterium tuberculosis growth inhibition in vitro

Serum amyloid P-component (SAP), a pentraxin, is known to play an important role in innate immunity to microbial infections; however, nothing is known about it during tuberculosis (TB). Mice intratracheally infected with Mycobacterium tuberculosis Erdman, showed peak SAP levels (442+/-58.2 microg/ml) on day 21, which declined to background levels by day 60. Their serum interleukin-6 levels paralleled SAP levels, whereas, their serum transforming growth factor-beta levels were paradoxical. During the acute phase of infection, the SAP levels positively correlated with the lung mycobacterial load. Purified mouse SAP (1-50 microg/ml) treatment of M. tuberculosis-infected alveolar macrophages (AMs), in vitro, inhibited their intracellular mycobacterial growth; maximum inhibition (1.1 log10 CFU reduction) occurred at 10 microg/ml, and a 4-day treatment appeared optimal. Treatment of AMs with both rabbit anti-mouse SAP polyclonal antibody and mannose-derived simple sugars, separately, blocked the SAP-induced inhibition of mycobacterial growth. The mycobacterial growth inhibition appeared to be nitric oxide (NO)-dependent as NO synthase inhibitors, both aminoguanidine and N(G)-monomethyl-L-arginine, annulled it. Further, SAP treatment of infected AMs induced significant (P<0.05) elaboration of nitrite (72.1+/-8.3 nM/ml), compared to the controls, and these AMs showed augmented expression of inducible NO synthase. This first study demonstrates that during murine TB the SAP levels were increased, and purified mouse SAP inhibited the intra-AM M. tuberculosis growth, in vitro, apparently via NO-dependent mechanism(s). SAP may thus contribute both to the pathogenesis and pulmonary innate immunity in TB.     

Deletion of PIKfyve alters alveolar macrophage populations and exacerbates allergic inflammation in mice

Alveolar macrophages (AMs) are specialized tissue‐resident macrophages that orchestrate the immune responses to inhaled pathogens and maintain organ homeostasis of the lung. Dysregulation of AMs is associated with allergic inflammation and asthma. Here, we examined the role of a phosphoinositide kinase PIKfyve in AM development and function. Mice with conditionally deleted PIKfyve in macrophages have altered AM populations. PIKfyve deficiency results in a loss of AKT activation in response to GM‐CSF, a cytokine critical for AM development. Upon exposure to house dust mite extract, mutant mice display severe lung inflammation and allergic asthma accompanied by infiltration of eosinophils and lymphoid cells. Moreover, they have defects in production of retinoic acid and fail to support incorporation of Foxp3⁺ T_reg cells in the lung, resulting in exacerbation of lung inflammation. Thus, PIKfyve plays a role in preventing excessive lung inflammation through regulating AM function.     

Quantification of isoniazid and acetylisoniazid in rat plasma and alveolar macrophages by liquid chromatography-tandem mass spectrometry with on-line extraction

To evaluate if pulmonary delivery of microparticles loaded with a prodrug of isoniazid (INH), isoniazid methanesulfonate (INHMS), can target alveolar macrophages (AM) and reduce metabolism of INH, an HPLC-MS/MS assay with automated online extraction for quantification of INH and its metabolite acetylisoniazid (AcINH) in plasma and AMs was developed and validated. Reproducibility in rat plasma and homogenate of a rat AM cell line, NR8383, for INH and AcINH showed excellent precision and accuracy with calibration curves exhibiting linearity within a range of 1-250ng/ml of INH and 0.05-50ng/ml of AcINH (r(2)>0.99). The validated methods were successfully applied to pharmacokinetic study of INHMS-loaded microparticles in rats, demonstrating efficient targeting of AMs and reduction of INH metabolism.     

Ethanol induces oxidative stress in alveolar macrophages via upregulation of NADPH oxidases

Chronic alcohol abuse is a comorbid variable of acute respiratory distress syndrome. Previous studies showed that, in the lung, chronic alcohol consumption increased oxidative stress and impaired alveolar macrophage (AM) function. NADPH oxidases (Noxes) are the main source of reactive oxygen species in AMs. Therefore, we hypothesized that chronic alcohol consumption increases AM oxidant stress through modulation of Nox1, Nox2, and Nox4 expression. AMs were isolated from male C57BL/6J mice, aged 8-10 wk, which were treated with or without ethanol in drinking water (20% w/v, 12 wk). MH-S cells, a mouse AM cell line, were treated with or without ethanol (0.08%, 3 d) for in vitro studies. Selected cells were treated with apocynin (300 μM), a Nox1 and Nox2 complex formation inhibitor, or were transfected with Nox small interfering RNAs (20-35 nM), before ethanol exposure. Human AMs were isolated from alcoholic and control patients' bronchoalveolar lavage fluid. Nox mRNA levels (quantitative RT-PCR), protein levels (Western blot and immunostaining), oxidative stress (2',7'-dichlorofluorescein-diacetate and Amplex Red analysis), and phagocytosis (Staphylococcus aureus internalization) were measured. Chronic alcohol increased Nox expression and oxidative stress in mouse AMs in vivo and in vitro. Experiments using apocynin and Nox small interfering RNAs demonstrated that ethanol-induced Nox4 expression, oxidative stress, and AM dysfunction were modulated through Nox1 and Nox2 upregulation. Further, Nox1, Nox2, and Nox4 protein levels were augmented in human AMs from alcoholic patients compared with control subjects. Ethanol induces AM oxidative stress initially through upregulation of Nox1 and Nox2 with downstream Nox4 upregulation and subsequent impairment of AM function.     

E-prostanoid 2 receptor signaling suppresses lung innate immunity against Streptococcus pneumoniae

Pneumonia is a major global health problem. Prostaglandin (PG) E(2) is an immunomodulatory lipid with anti-inflammatory, immunosuppressive, and pro-resolving actions. Data suggest that the E-prostanoid (EP) 2 receptor mediates immunomodulatory effects of PGE(2), but the extent to which this occurs in Streptococcus pneumoniae infection is unknown. Intratracheal lung infection of C57BL/6 mice possessing (EP2(+/+)) or lacking (EP2(-/-)) the EP2 receptor was performed, as were in vitro studies of alveolar macrophage (AM) host defense functions. Bacterial clearance and survival were significantly improved in vivo in EP2(-/-) mice and it correlated with greater neutrophilic inflammation and higher lung IL-12 levels. Upon ex vivo challenge with pneumococcus, EP2(-/-)cells expressed greater amounts of TNF-α and MIP-2 than did EP2(+/+) AMs, and had improved phagocytosis, intracellular killing, and reactive oxygen intermediate generation. These data suggest that PGE(2)-EP2 signaling may provide a novel pharmacological target for treating pneumococcal pneumonia in combination with antimicrobials.     

Cutting edge: macrophage inhibition by cyclic AMP (cAMP): differential roles of protein kinase A and exchange protein directly activated by cAMP-1

cAMP has largely inhibitory effects on components of macrophage activation, yet downstream mechanisms involved in these effects remain incompletely defined. Elevation of cAMP in alveolar macrophages (AMs) suppresses FcgammaR-mediated phagocytosis. We now report that protein kinase A (PKA) inhibitors (H-89, KT-5720, and myristoylated PKA inhibitory peptide 14-22) failed to prevent this suppression in rat AMs. We identified the expression of the alternative cAMP target, exchange protein directly activated by cAMP-1 (Epac-1), in human and rat AMs. Using cAMP analogs that are highly specific for PKA (N6-benzoyladenosine-3',5'-cAMP) or Epac-1 (8-(4-chlorophenylthio)-2'-O-methyladenosine-3',5'-cAMP), we found that activation of Epac-1, but not PKA, dose-dependently suppressed phagocytosis. By contrast, activation of PKA, but not Epac-1, suppressed AM production of leukotriene B(4) and TNF-alpha, whereas stimulation of either PKA or Epac-1 inhibited AM bactericidal activity and H(2)O(2) production. These experiments now identify Epac-1 in primary macrophages, and define differential roles of Epac-1 vs PKA in the inhibitory effects of cAMP.     

Nimesulide inhibits lipopolysaccharide-induced production of superoxide anions and nitric oxide and iNOS expression in alveolar macrophages

The study was designed to investigate the effect of nimesulide on lipopolysaccharide (LPS)-induced proinflammatory oxidants production by rat alveolar macrophages (AMs). Effects of LPS and nimesulide on antioxidant defense and the expression of inducible nitric oxide synthase (iNOS) were also studied. It was found that nimesulide could scavenge superoxide anions (O2*-), nitric oxide (NO*) and total oxidant burden induced by LPS in AMs in vitro. Approximately 850 nmoles of nimesulide had activity equivalent to one IU of superoxide dismutase (SOD). Further, to confirm the in vitro observation, Male Wistar rats were orally administered with nimesulide (9 mg/kg b. wt. twice daily) for one week followed by intratracheal instillation of 2 microg LPS to stimulate lung inflammation. AMs from bronchoalveolar lavage fluid were collected 18 h after instillation of LPS. Nimesulide pretreatment could inhibit O2*-, NO() and lipid peroxidation in AMs. Nimesulide also suppressed LPS-induced iNOS expression in AMs in vivo and in vitro. Nimesulide could also normalize LPS-induced changes in the levels of superoxide dismutase (SOD), glutathione reductase (GR) and reduced glutathione (GSH) in AMs. Inhibition in production of oxidants in LPS-challenged AMs by nimesulide could be one of the pathways for its anti-inflammatory action.