Alveolar Macrophage Cell Line NR8383 Research Articles

LncRNA HCG18 loaded by polymorphonuclear neutrophil-secreted exosomes aggravates sepsis acute lung injury by regulating macrophage polarization.

Polymorphonuclear neutrophils (PMNs) exert significant roles in septic acute lung injury (ALI). Accumulating evidence suggests that PMN-derived exosomes (PMN-exo) are a novel subcellular entity that is the fundamental link between PMN-driven inflammation and tissue damage. However, the role of PMN-exo in septic ALI and the underlying mechanisms remain unclear. Tumor necrosis factor-α (TNF-α), a key regulator of innate immunity in septic ALI, was used to induce PMN activation in vitro. Using an in vitro co-culture system, the rat alveolar macrophage cell line NR8383 was co-cultured with TNF-α-stimulated PMN-released exosomes (TNF-α-exo) to further confirm the results of the in vitro studies and explore the underlying mechanisms involved. A septic lung injury model was established by cecal ligation and puncture surgery, and PMN-exo were injected into septic mice through the tail vein, and then lung injury, inflammatory release, macrophage polarization, and apoptosis were examined. The results reported that TNF-α-exo promoted the activation of M1 macrophages after i.p. injection in vivo or co-culture in vitro. Furthermore, TNF-α-exo affected alveolar macrophage polarization by delivering HCG18. Mechanistic studies indicated that HCG18 mediated the function of TNF-α-exo by targeting IL-32 in macrophages. In addition, tail vein injection of si-HCG18 in septic mice significantly reduced TNF-α-exo-induced M1 macrophage activation and lung macrophage death, as well as histological lesions. In conclusion, TNF-α-exo-loaded HCG18 contributes to septic ALI by regulating macrophage polarization. These findings may provide new insights into novel mechanisms of PMN-macrophage polarization interactions in septic ALI and may provide new therapeutic strategies for patients with sepsis.

MiR-155 contribute to airway inflammation in COPD by regulating autophagy via targeting TLR4/NF-ΚB

To evaluate the expression of miR-155 and autophagy in COPD and the related mechanism. Alveolar macrophages were extracted from COPD patients. COPD dynamic mouse model was established. Mouse alveolar macrophages were also isolated and cultured. Rat alveolar macrophage cell line NR8383 was introduced. The expression of TLR4 and NF-κB in NR8383 cells with CSE treatment was also evaluated. miR-155 was upregulated in alveolar macrophages from bronchoalveolar lavage fluid of COPD patients, COPD dynamic mouse model and CSE treated NR8383 cell line (p < 0.05). Overexpression of miR‐155 led to dysregulation of cell autophagy and was closely. miR‐155 promoted cell autophagy by directly targeting TLR4/NF-κB pathway in NR8383 cell line. Our study revealed a novel inflammatory role of miR-155 in COPD and the related mechanisms. We provide a new perspective to understand the pathogenesis of COPD, and miR-155 might be useful as potential target for the treatment of COPD.

Overexpressing long non-coding RNA OIP5-AS1 ameliorates sepsis-induced lung injury in a rat model via regulating the miR-128-3p/Sirtuin-1 pathway

ABSTRACT Sepsis, resulting from infections, is a systemic inflammatory response syndrome with a high fatality rate. The present study revolves around probing into the function and molecular mechanism of long non-coding RNA OIP5 antisense RNA 1 (lncRNA OIP5-AS1) in modulating acute lung injury (ALI) mediated by sepsis. Here, a sepsis model was constructed using cecal ligation and puncture (CLP) surgery in vivo. The alveolar macrophage cell line NR8383 and the alveolar type II cell line RLE-6TN were dealt with lipopolysaccharide (LPS) for in-vitro experiments. We discovered that OIP5-AS1 and Sirtuin1 (SIRT1) were markedly down-regulated in sepsis models elicited by CLP or LPS, while miR-128-3p experienced a dramatic up-regulation. OIP5-AS1 overexpression attenuated NR8383 and RLE-6TN cell apoptosis triggered by LPS and suppressed the expressions of nuclear factor kappa B (NF-κB), inducible nitric oxide synthase (iNOS), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in NR8383 and RLE-6TN cells, whereas miR-128-3p overexpression resulted in the opposite phenomenon. Moreover, OIP5-AS1 overexpression relieved lung edema, lung epithelial cell apoptosis, infiltration of myeloperoxidase (MPO)-labeled polymorphonuclear neutrophils (PMN), inflammatory responses triggered by CLP in vivo. Mechanistically, miR-128-3p, which targeted SIRT1, was hobbled by OIP5-AS1. All in all, OIP5-AS1 overexpression enhanced sepsis-induced ALI by modulating the miR-128-3p/SIRT1 pathway, which helps create new insights into sepsis treatment.

Establishment of an in vitro model using the rat alveolar macrophage cell line NR8383.

To establish an in vitro model of radiation-induced lung injury using rat lung alveolar macrophages (NR8383). Using a medical electronic linear accelerator, cells were irradiated with either 0 Gy or 6 Gy X-rays. At 6, 12, 24, 30 and 48 h, the DNA damage index (8-OHdG) and lipid damage index (MDA) were measured in the two groups. We also determined the levels of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and transforming growth factor-β (TGF-β). The levels of 8-OHdG and MDA in the 6 Gy irradiation group were higher than those in the 0 Gy group at 6, 12, 24, 30 and 48 h after irradiation. The levels reached the highest value -6 h after irradiation, and then gradually decreased. The levels of the inflammatory factors TNF-α, TGF-β and IL-6 were higher in the 6 Gy irradiation group than those in the 0 Gy group at 6, 12, 24, 30 and 48 h after irradiation. Six Gy X-ray irradiated NR8383 cells can be used to establish an in-vitro model of radiation-induced lung injury. The levels of 8-OHdG, MDA, TNF-α, TGF-β and IL-6 can be used as effective evaluation indicators.

Evaluation of the NLRP3 Inflammasome Activating Effects of a Large Panel of TiO2 Nanomaterials in Macrophages.

TiO2 nanomaterials are among the most commonly produced and used engineered nanomaterials (NMs) in the world. There is controversy regarding their ability to induce inflammation-mediated lung injuries following inhalation exposure. Activation of the NACHT, LRR and PYD domains-containing protein 3 (NALP3) inflammasome and subsequent release of the cytokine interleukin (IL)-1β in pulmonary macrophages has been postulated as an essential pathway for the inflammatory and associated tissue-remodeling effects of toxic particles. Our study aim was to determine and rank the IL-1β activating properties of TiO2 NMs by comparing a large panel of different samples against each other as well as against fine TiO2, synthetic amorphous silica and crystalline silica (DQ12 quartz). Effects were evaluated in primary bone marrow derived macrophages (BMDMs) from NALP3-deficient and proficient mice as well as in the rat alveolar macrophage cell line NR8383. Our results show that specific TiO2 NMs can activate the inflammasome in macrophages albeit with a markedly lower potency than amorphous SiO2 and quartz. The heterogeneity in IL-1β release observed in our study among 19 different TiO2 NMs underscores the relevance of case-by-case evaluation of nanomaterials of similar chemical composition. Our findings also further promote the NR8383 cell line as a promising in vitro tool for the assessment of the inflammatory and inflammasome activating properties of NMs.

A multi-omics approach reveals mechanisms of nanomaterial toxicity and structure–activity relationships in alveolar macrophages

In respect to the high number of released nanomaterials and their highly variable properties, novel grouping approaches are required based on the effects of nanomaterials. Proper grouping calls for a combination of an experimental setup with a higher number of structurally similar nanomaterials and for employing integrated omics approaches to identify the mode of action. Here, we analyzed the effects of seven well-characterized NMs comprising different chemical compositions, sizes and chemical surface modifications on the rat alveolar macrophage cell line NR8383. The NMs were investigated at three doses ranging from 2.5 to 10 µg/cm2 after 24 h incubation using an integrated multi-omics approach involving untargeted proteomics, targeted metabolomics, and src homology 2 (SH2) profiling. By using Weighted Gene Correlation Network Analysis (WGCNA) for the integrative data, we identified correlations of molecular pathways with physico-chemical properties and toxicological endpoints. The three investigated SiO2 variants induced strong alterations in all three omics approaches and were, therefore, be classified as “active.” Two organic phthalocyanines showed minor responses and Mn2O3 induced a different molecular response pattern than the other NMs. WGCNA revealed that agglomerate size and surface area as well as LDH release are among the most important parameters correlating with nanotoxicology. Moreover, we identified key drivers that can serve as representative biomarker candidates, supporting the value of multi-omics approaches to establish integrated approaches to testing and assessment (IATAs).

Activation of NLRP3 inflammasome in peripheral nerve contributes to paclitaxel-induced neuropathic pain

BackgroundPaclitaxel is commonly used as a cancer chemotherapy drug that frequently causes peripheral neuropathic pain. Inflammasome is a multiprotein complex consisting of Nod-like receptor proteins (NLRPs), apoptosis-associated speck-like protein, and caspase-1, which functions to switch on the inflammatory process and the release of interleukin-1β. Growing evidences have supported that peripheral interleukin-1β is critical in enhancing paclitaxel-induced neuropathic pain. However, whether activation of NLRP3 inflammasome in peripheral nerve contributes to paclitaxel-induced neuropathic pain is still unclear.ResultsPaclitaxel induced mechanical allodynia of rats from day 3 and worsened gradually till 3 weeks after injection. Paclitaxel resulted in expression of NLRP3 and activated fragments of caspase-1 and interleukin-1β in L4-6 dorsal root ganglia and sciatic nerve three weeks after injection, indicating activation of NLRP3 inflammasome. The expression of NLRP3 was located in CD68-labeled macrophages infiltrating in L4-6 dorsal root ganglia and sciatic nerve, and paclitaxel increased the expression of NLRP3 in macrophage. Moreover, the paclitaxel elicited mitochondria damage, which became swollen and enlarged in macrophages and axons of sciatic nerve three weeks after injection. In vitro, paclitaxel increased the number of damaged mitochondria and mitochondrial reactive oxygen species production in the rat alveolar macrophage cell line NR8383. The administration of a non-specific reactive oxygen species scavenger, phenyl-N-tert-butylnitrone, markedly alleviated mechanical allodynia and inhibited the activation of NLRP3 inflammasome in L4-6 dorsal root ganglia and sciatic nerve of the paclitaxel-induced neuropathic pain model.ConclusionsPaclitaxel induced mechanical allodynia and activation of NLRP3 inflammasome in infiltrated macrophages of L4-6 dorsal root ganglia and sciatic nerve. Paclitaxel elicited mitochondria damage and reactive oxygen species production may result in activation of NLRP3 inflammasome in peripheral nerve, which contributes to paclitaxel-induced neuropathic pain.

Calcitonin Peptide Family Members Are Differentially Regulated by LPS and Inhibit Functions of Rat Alveolar NR8383 Macrophages.

Members of the calcitonin peptide family—calcitonin gene-related peptide (CGRP), adrenomedullin (AM), and adrenomedullin2/intermedin (IMD)–exert modulatory effects upon monocytes and macrophages of various extrapulmonary origins. Utilizing the rat alveolar macrophage (AMφ) cell line NR8383, we here set out to determine to which extent these three peptides and their receptors are differentially regulated in AMφ and what specific effects they have on AMφ key functions. LPS treatment differentially up-regulated expression of the peptides and receptors. Among the three peptides, IMD mRNA content was lowest both in primary rat AMφ and NR8383 cells, whereas IMD peptide dominated in basal and LPS-stimulated secretion from NR8383 cells. Fcγ receptor-mediated phagocytosis and TNF-α production were inhibited by AM, IMD, and CGRP, whereas pro-IL-1β mRNA was slightly down-regulated exclusively by CGRP. Neither of these peptides affected IL-6 or IL-10 production. None increased intracellular calcium concentration, but AM significantly inhibited store-operated calcium entry. In conclusion, the rat AMφ cell line NR8383 is both a source and a target of the calcitonin peptide family members AM, IMD, and CGRP. Despite sharing proteins of the receptor complexes, AM, IMD, and CGRP each showed a characteristic pattern of effects and regulation, suggesting that these closely related peptides are not just redundant members of one common signaling pathway but act in concert by addressing parallel signaling cascades. Since peptide and receptor expression are up-regulated by LPS, these signaling pathways might act as inhibitory feedback mechanisms in pulmonary bacterial infection.

In Vitro Assessment of Uptake and Lysosomal Sequestration of Respiratory Drugs in Alveolar Macrophage Cell Line NR8383.

PurposeTo assess accumulation and lysosomal sequestration of 9 drugs used in respiratory indications (plus imipramine as positive control) in the alveolar macrophage (AM) cell line NR8383.MethodsFor all drugs, uptake at 5 μM was investigated at 37 and 4°C to delineate active uptake and passive diffusion processes. Accumulation of basic clarithromycin, formoterol and imipramine was also assessed over 0.1–100 μM concentration range. Lysosomal sequestration was investigated using ammonium chloride (NH4Cl), monensin and nigericin. Impact of lysosomal sequestration on clarithromycin accumulation kinetics was investigated.ResultsBoth cell-to-medium concentration ratio (Kp) and uptake clearance (CLuptake) ranged > 400-fold for the drugs investigated. The greatest Kp was observed for imipramine (391) and clarithromycin (82), in contrast to no accumulation seen for terbutaline. A concentration-dependent accumulation was evident for the basic drugs investigated. Imipramine and clarithromycin Kp and CLuptake were reduced by 59–85% in the presence of NH4Cl and monensin/nigericin, indicating lysosomal accumulation, whereas lysosomal sequestration was not pronounced for the other 8 respiratory drugs. Clarithromycin uptake rate was altered by NH4Cl, highlighting the impact of subcellular distribution on accumulation kinetics.ConclusionsThis study provides novel evidence of the utility of NR8383 for investigating accumulation and lysosomal sequestration of respiratory drugs in AMs.Electronic supplementary materialThe online version of this article (doi:10.1007/s11095-015-1753-8) contains supplementary material, which is available to authorized users.

MiR-132 inhibits lipopolysaccharide-induced inflammation in alveolar macrophages by the cholinergic anti-inflammatory pathway

ABSTRACTObjective: Although microRNA-132 (miR-132) has been shown to be involved in the inflammatory regulation, its role in sepsis-induced lung injury is unknown. We hypothesized that miR-132 attenuated lipopolysaccharide (LPS)-induced inflammation of alveolar macrophages by targeting acetylcholinesterase (AChE) and enhancing the acetylcholine (ACh)-mediated cholinergic anti-inflammatory response. Methods: The LPS-treated rat alveolar macrophage cell line NR8383 was used as the inflammatory model. To assess the effect of miR-132, alveolar macrophages were transfected with miR-132 mimic or inhibitor. Results: We found that miR-132 was upregulated in LPS-stimulated alveolar macrophages. Induction of AChE mRNA showed an inverse pattern with respect to AChE protein and activity, suggesting posttranscriptional regulation of AChE. Utilizing miR-132 mimic transfection, we found that overexpression of miR-132 enhanced the ACh-mediated cholinergic anti-inflammatory reaction by targeting AChE mRNA in LPS-treated alveolar macrophages. Blockage of miR-132 using miR-132 inhibitor reversed the Ach action upon LPS-induced release of inflammatory mediators and reduction in AchE protein/activity. Moreover, in the presence of ACh, upregulation of miR-132 suppressed LPS-induced nuclear translocation of NF-κB and production of STAT3 and phosphorylated STAT3, while downregulation of miR-132 enhanced the nuclear translocation of NF-κB. Conclusion: We propose that miR-132 functions as a negative regulator of the inflammatory response in alveolar macrophages by potentiating the cholinergic anti-inflammatory pathway, and represents a potential therapeutic leverage point in modulating inflammatory responses.

Proprotein Convertase 1/3 (PC1/3) in the Rat Alveolar Macrophage Cell Line NR8383: Localization, Trafficking and Effects on Cytokine Secretion

The proprotein convertase 1/3 (PC1/3) is an important post-translational processing enzyme for the activation of precursor proteins within the regulated secretory pathway. Well characterized for its role in the neural and endocrine systems, we recently reported an unconventional role of PC1/3 as a modulator of the Toll-like receptor innate immune response. There are only a few reports that have studied PC1/3 expression in macrophages, and more investigation is needed to better characterize its function. These studies would greatly benefit from model cell lines. Our study aims to identify and characterize PC1/3 in a relevant model macrophage cell line and to determine the links between PC1/3 and innate immune cellular responses. We describe the rat alveolar cell line, NR8383, as expressing PC1/3 and the most common Toll-like receptors. In NR8383 cells, PC1/3 is localized at the Trans-Golgi network and traffics to lysosome related vesicles upon lipopolysaccharide stimulation. Moreover, we report the co-localization of PC1/3 and Toll-like receptor 4 upon lipopolysaccharide stimulation. Down regulation of PC1/3 by shRNA produce a similar phenotype in NR8383 to what we previously reported in isolated peritoneal macrophages. PC1/3 shRNA induced changes in the cellular organization and expression of the specific trafficking regulator RAB GTPase. As a consequence, NR8383 down-regulated for PC1/3, present an abnormal cytokine secretion profile. We conclude that the NR8383 cell line represents a good model to study PC1/3 in macrophages and we present PC1/3 as an important regulator of vesicle trafficking and secretion in macrophages.

Role of M3 mAChR in in vivo and in vitro models of LPS-induced inflammatory response

ObjectiveWe tested the potential role of the mAChR in lipopolysaccharide (LPS)‐induced inflammatory response in in vivo and in vitro models and a possible signaling pathway involved in the inflammatory process. MethodsAnesthetized mice were challenged with intratracheal LPS to induce acute lung injury. The cytology and histopathology changes, expression of cytokines and pulmonary vascular permeability were used to evaluate the effects of the cholinergic agent. Alveolar macrophage cell line NR8383 was also used to confirm the role of mAChRs and the molecular mechanisms underlying the LPS-induced events. ResultsLPS-induced acute lung injury (ALI) was significantly improved by atropine (a non-selective mAChR antagonist) and 4-DAMP (a M3 mAChR antagonist), as indicated by the diminution of neutrophil infiltration, pulmonary vascular permeability and IL-6 and TNF-α production. LPS-induced TNF-α production from the alveolar macrophage was significantly inhibited by atropine and 4-DAMP, but not pirenzepine (a M1 mAChR antagonist) and methoctramine (a M2 mAChR antagonist). Interestingly, LPS-induced TNF-α production was enhanced by the muscarinic receptor agonist pilocarpine, and treatment with pilocarpine alone was able to trigger TNF-α production from the alveolar macrophage, which was effectively attenuated by 4-DAMP. Western blot analysis showed that LPS-induced degradation of IκBα was strongly blocked by atropine/4-DAMP both in vivo and in vitro, indicating that M3 mAChR was involved in LPS-induced lung inflammation by mediating the NF-κB signaling pathway. ConclusionOur findings bring the evidence that the blockage of mAChR exerts anti-inflammatory properties, in which the M3 mAChR plays an important role in the LPS-induced lung inflammation.

Acylated triterpenoid saponins from roots of Gypsophila trichotoma

Eleven new triterpenoid saponins were isolated from the roots of Gypsophila trichotoma Wend. (Caryophyllaceae) together with one known compounds. The structures were established on the basis of extensive 1D and 2D NMR analysis, completed by analysis of HR-ESI-MS and ESI-MSn. The cytotoxicity of the saponin extract from G. trichotoma was evaluated on a rat alveolar macrophage cell line NR8383 and a human macrophage cell line U937. The synergistic effect of the aminoacyl saponins, previously isolated from G. trichotoma, was tested for its ability to enhance the cytotoxicity of the targeted toxin in HER14 cells.

Myeloid differentiation protein 2 silencing decreases LPS-induced cytokine production and TLR4/MyD88 pathway activity in alveolar macrophages

Lipopolysaccharides (LPSs) activate the innate immune response during Gram-negative bacterial infections through the Toll-like receptor 4 (TLR4)/myeloid differentiation protein 2 (MD-2) complex. MD-2 binds LPS with high affinity and is critical for TLR4-dependent signal transduction. However, the exact role of MD-2 on LPS signal transduction and cytokine production in alveolar macrophages (AMs) remains unclear. This study showed that the transcription levels of MD-2, TLR4 and MyD88 in the NR8383 cell line were up-regulated after LPS stimulation and that the increased transcript levels were attenuated after RNA interference of MD-2. Similarly, LPS induced increases in TNF-α, IL-1β and IL-6 protein levels in NR8383 cell supernatants was significantly inhibited by MD-2 silencing. These results suggest that in association with the TLR4/MyD88 signaling pathway LPS-induced cytokine production can be partially reduced by MD-2 silencing in the rat pulmonary alveolar macrophage cell line NR8383. MD-2 silencing was proved to be a useful tool for testing the role of MD-2 in the LPS signaling pathway and may be a potential therapeutic tool against LPS-induced lung inflammation.

Phagostimulatory effect of uptake of PLGA microspheres loaded with rifampicin on alveolar macrophages

Our previous results on the phagocytic activity of alveolar macrophages (Mϕs) toward poly(lactic-co-glycolic) acid microspheres (PLGA MS) loaded with the anti-tuberculosis agent rifampicin (R-PLGA MS) suggest that the phagocytosis of R-PLGA MS enhances the phagocytic activity of Mϕ cells. To confirm this possibility, we examined the effect of phagocytosis of R-PLGA MS and polystyrene latex (PSL) MS on the phagocytic uptake of fluorescent PSL (F-PSL) MS by cells of the rat alveolar macrophage cell line NR8383 at 37°C. Phagocytic activity was examined in terms of the population of Mϕ cells that had phagocytosed MS (N(total)) and the total number of MS phagocytosed (n(total)) by counting the phagocytic Mϕ cells and the MS ingested in optical microscopic fields. Phagocytosis of R-PLGA MS enhanced about 1.5 times the values of N(total) and n(total) of the phagocytosis of F-PSL MS under the conditions where the phagocytosis of F-PSL MS did not attain the saturated level. In contrast, the phagocytosis of PSL MS did not enhance the phagocytic activity of Mϕ cells toward F-PSL MS. In conclusion, R-PLGA MS are favorable for drug delivery of anti-tuberculosis agents into alveolar Mϕs due to their ability to up-regulate the phagocytosis of MS.

The role of macrophage mediators in respirable quartz-elicited inflammation

The instigation and persistence of an inflammatory response is widely considered to be critically important in quartz-induced lung cancer and fibrosis. Macrophages have been long recognised as a crucial player in pulmonary inflammation, but evidence for the role of type II epithelial cells is accumulating. Investigations were performed in the rat lung type II cell line RLE and the rat alveolar macrophage cell line NR8383 using Western blotting, NF-κB immunohistochemistry and qRT-PCR of the pro-inflammatory genes iNOS and COX-2, as well as the cellular stress gene HO-1. The direct effect of quartz on pro-inflammatory signalling cascades and gene expression in RLE cells was compared to the effect of conditioned media derived from quartz-treated NR8383 cells. Conditioned media activated the NF-κB signalling pathway and induced a far stronger upregulation of iNOS mRNA than quartz itself. Quartz elicited a stronger, progressive induction of COX-2 and HO-1 mRNA. Our results suggest a differentially mediated inflammatory response, in which reactive particles themselves induce oxidative stress and activation of COX-2, while mediators released from particle-activated macrophages trigger NF-κB activation and iNOS expression in type II cells.

Inhibition of Ca2+ influx by surfactant in NR8383 alveolar macrophages

Pulmonary surfactants reduce alveolar surface tension and alter inflammatory cell function. We studied the effects of surfactant preparations on Ca2+ influx regulated by protein kinase C (PKC) and mitogen-activated protein kinases (MAPK) and cytokine secretion in the alveolar macrophage (AM) cell line NR8383. Fura-2-loaded AMs were stimulated with zymosan (200 microg/ml), 1,2-dioctanoyl-sn-glycerol (DOG, 20 microM) or C6-ceramide (C6C, 10 microM) in the presence of exogenous surfactants (beractant, calfactant or colfosceril) or surfactant phospholipid (dipalmitoyl phosphatidylcholine, DPPC), at 250 microg/ml phospholipid and changes in cytosolic free Ca2+ (Delta[Ca2+]i) and cytokines were measured. Zymosan-induced Delta[Ca2+]i (117 +/- 5 nM) at 3 min was reduced (p <0.001) by beractant (50 +/- 6 nM), colfosceril (61 +/- 2 nM), calfactant (46 +/- 5 nM), and DPPC (52 +/- 5 nM). Beractant inhibited the Delta[Ca2+]i by PKC stimulation with DOG and all preparations reduced the MAPK-induced Ca2+ influx by C6C. Beractant and Ca2+ channel blocker SKF 96365 (10 microM) together abolished the zymosan-stimulated Delta[Ca2+]i. Zymosan-stimulated TNF-alpha and IL-1beta secretion was also inhibited by surfactant pretreatment. These results indicate that exogenous surfactant inhibits Ca2+ influx and cytokine secretion in zymosan-stimulated AMs. This anti-inflammatory activity may be through an interaction with downstream signaling elements or Ca2+ channels.

Immunosuppressive properties of surfactant in alveolar macrophage NR8383

To evaluate the anti-inflammatory effects of exogenous surfactants and surfactant phospholipid without surfactant proteins (SP-A and SP-D) on the lipopolysaccharide- (LPS) stimulated rat alveolar macrophage (AM) cell line NR8383. Exogenous surfactants (beractant, calfactant or colfosceril) and surfactant phospholipid (dipalmitoyl phosphatidylcholine, DPPC), standardized to phospholipid content of 25-1,000 microg/ml were incubated with LPS- (1 microg/ml) stimulated NR8383 AMs. TNF-alpha and IL-1beta secretion and nitric oxide (NO) formation following LPS stimulation were inhibited by treatment with surfactants or DPPC. Furthermore, LPS-dependent NO production and iNOS protein levels were significantly suppressed in cells pretreated for one hour with beractant compared to beractant added simultaneously with or following LPS. Additionally, LPS-stimulated oxidative burst, measured by flow cytometry, was significantly decreased by beractant. Finally, beractant inhibited the translocation of NF-kappaB from cytoplasmic into nuclear extract in LPS-stimulated NR8383 AMs. Exogenous surfactants and surfactant phospholipid inhibit secretion of proinflammatory cytokines and NO in NR8383 AMs. The inhibitory effects of beractant on oxygen radical and LPS-induced NO formation may result from unique mechanisms of decreasing cell signaling. The anti-inflammatory activity of surfactant products used in the treatment of neonatal respiratory distress syndrome (RDS) may depend upon the specific preparation or dose used.

Ozone Exposure of Macrophages Induces an Alveolar Epithelial Chemokine Response through IL-1α

Ozone is known to produce an acute influx of neutrophils, and alveolar epithelial cells can secrete chemokines and modulate inflammatory processes. However, direct exposure of alveolar epithelial cells and macrophages to ozone (O(3)) produces little chemokine response. To determine if cell-cell interactions might be responsible, we investigated the effect of alveolar macrophage-conditioned media after ozone exposure (MO(3)CM) on alveolar epithelial cell chemokine production. Serum-free media were conditioned by exposing a rat alveolar macrophage cell line NR8383 to ozone for 1 hour. Ozone stimulated secretion of IL-1alpha, IL-1beta, and IL-18 from NR8383 cells, but there was no secretion of chemokines or TNF-alpha. Freshly isolated type II cells were cultured, so as to express the biological markers of type I cells, and these cells are referred to as type I-like cells. Type I-like cells were exposed to diluted MO(3)CM for 24 hours, and this conditioned medium stimulated secretion of cytokine-induced neutrophil chemattractant-1 (CXCL1) and monocyte chemoattractant protein-1 (CCL2). Secretion of these chemokines was inhibited by the IL-1 receptor antagonist. Although both recombinant IL-1alpha and IL-1beta stimulated alveolar epithelial cells to secrete chemokines, recombinant IL-1alpha was 100-fold more potent than IL-1beta. Furthermore, neutralizing anti-rat IL-1alpha antibodies inhibited the secretion of chemokines by alveolar epithelial cells, whereas neutralizing anti-rat IL-1beta antibodies had no effect. These observations indicate that secretion of IL-1alpha from macrophages stimulates alveolar epithelial cells to secrete chemokines that can elicit an inflammatory response.

Ionotropic glutamate receptors in lungs and airways: molecular basis for glutamate toxicity.

We earlier showed that the ionotropic glutamate receptor agonist N-methyl D-aspartate (NMDA) induces excitotoxic pulmonary edema, and that endogenous activation of NMDA receptors (NMDAR) could mediate lung injury caused by oxidative stress. In this study, we searched for evidence of NMDAR expression in the rat lung and in the alveolar macrophage (AM) cell line NR8383, and for possible regulation of receptor expression by NMDA. The presence of mRNA for NMDAR 1 and the four known NMDAR 2 subtypes (A, B, C, and D) was examined by reverse transcriptase-polymerase chain reaction using isoform-specific primers. NMDAR 1 was expressed in all lung regions examined (peripheral, midlung, and mainstem), as well as in trachea and the AMs. Expression of NMDAR 2A and 2B subtypes was not detected, whereas NMDAR 2C was present only in peripheral and mid-lung samples. NMDAR 2D was the dominant subtype expressed in the peripheral, gas-exchange zone of lung and in alveolar macrophages, and this expression was upregulated in lungs treated with NMDA. Western blot confirmed the presence of NMDAR 1 protein in all lung regions and in AMs. These findings provide a molecular-biological basis for the excitotoxic actions of glutamate in rat lungs and airways, and raise the question of a possible physiologic role for NMDAR in lung and airway function.