Inhibition of allergic inflammation in the airways using aerosolized antisense to Syk kinase

Activation of the protein tyrosine kinase Syk is an early event that follows cross-linking of Fc gamma R and Fc epsilon R, leading to the release of biologically active molecules in inflammation. We reported previously that aerosolized Syk antisense oligodeoxynucleotides (ASO) depresses Syk expression in inflammatory cells, the release of mediators from alveolar macrophages, and pulmonary inflammation. To study the effect of Syk ASO in allergic inflammation and airway hyperresponsiveness, we used the Brown Norway rat model of OVA-induced allergic asthma. Syk ASO, delivered in a liposome, carrier/lipid complex by aerosol to rats, significantly inhibited the Ag-induced inflammatory cell infiltrate in the bronchoalveolar space, decreasing both neutrophilia and eosinophilia. The number of eosinophils in the lung parenchyma was also diminished. Syk ASO also depressed up-regulation of the expression of beta(2) integrins, alpha(4) integrin, and ICAM-1 in bronchoalveolar lavage leukocytes and reversed the Ag-induced decrease in CD62L expression on neutrophils. Furthermore, the increase in TNF levels in bronchoalveolar lavage following Ag challenge was significantly inhibited. Syk ASO also suppressed Ag-mediated contraction of the trachea in a complementary model. Thus, aerosolized Syk ASO suppresses many of the central components of allergic asthma and inflammation and may provide a new therapeutic approach.     

G-CSF suppresses allergic pulmonary inflammation, downmodulating cytokine, chemokine and eosinophil production

AimsGranulocyte Colony-Stimulating Factor (G-CSF), which mobilizes hemopoietic stem cells (HSC), is believed to protect HSC graft recipients from graft-versus-host disease by enhancing Th2 cytokine secretion. Accordingly, G-CSF should aggravate Th2-dependent allergic pulmonary inflammation and the associated eosinophilia. We evaluated the effects of G-CSF in a model of allergic pulmonary inflammation.Main methodsAllergic pulmonary inflammation was induced by repeated aerosol allergen challenge in ovalbumin-sensitized C57BL/6J mice. The effects of allergen challenge and of G-CSF pretreatment were evaluated by monitoring: a) eosinophilia and cytokine/chemokine content of bronchoalveolar lavage fluid, pulmonary interstitium, and blood; b) changes in airway resistance; and c) changes in bone-marrow eosinophil production.Key findingsContrary to expectations, G-CSF pretreatment neither induced nor enhanced allergic pulmonary inflammation. Instead, G-CSF: a) suppressed accumulation of infiltrating eosinophils in bronchoalveolar, peribronchial and perivascular spaces of challenged lungs; and b) prevented ovalbumin challenge-induced rises in airway resistance. G-CSF had multiple regulatory effects on cytokine and chemokine production: in bronchoalveolar lavage fluid, levels of IL-1 and IL-12 (p40), eotaxin and MIP-1a were decreased; in plasma, KC, a neutrophil chemoattractant, was increased, while IL-5 was decreased and eotaxin was unaffected. In bone-marrow, G-CSF: a) prevented the increase in bone-marrow eosinophil production induced by ovalbumin challenge of sensitized mice; and b) selectively stimulated neutrophil colony formation.SignificanceThese observations challenge the view that G-CSF deviates cytokine production towards a Th2 profile in vivo, and suggest that this neutrophil-selective hemopoietin affects eosinophilic inflammation by a combination of effects on lung cytokine production and bone-marrow hemopoiesis.     

Effects of cannabinoids on LPS-stimulated inflammatory mediator release from macrophages: involvement of eicosanoids

Delta(9)-Tetrahydrocannabinol (Delta(9)-THC) is the major psychoactive component of marijuana and elicits pharmacological actions via cannabinoid receptors. Anandamide (AEA) and 2-arachidonoyl-glycerol (2-AG) are endogenous ligands for cannabinoid receptors, which because of their structural similarities to arachidonic acid (AA), AEA, and 2-AG could serve as substrates for lipoxygenases and cyclooxygenases (COXs) that metabolize polyunsaturated fatty acids to potent bioactive molecules. In this study, we have compared the effects of Delta(9)-THC, AEA, 2-AG, and another cannabinoid agonist, indomethacin morpholinylamide (IMMA), on lipopolysaccharide (LPS)-induced NO, IL-6, and PGE(2) release from J774 macrophages. Delta(9)-THC, IMMA, and AEA diminish LPS-induced NO and IL-6 production in a concentration-dependent manner. 2-AG inhibits the production of IL-6 but slightly increases iNOS-dependent NO production. Delta(9)-THC and IMMA also inhibit LPS-induced PGE(2) production and COX-2 induction, while AEA and 2-AG have no effects. These discrepant results of 2-AG on iNOS and COX-2 induction might be due to its bioactive metabolites, AA and PGE(2), whose incubation cause the potentiation of both iNOS and COX-2 induction. On the contrary, the AEA metabolite, PGE(2)-ethanolamide, influences neither the LPS-induced NO nor IL-6 production. Taken together, direct cannabinoid receptor activation leads to anti-inflammatory action via inhibition of macrophage function. The endogenous cannabinoid, 2-AG, also serves as a substrate for COX-catalyzing PGE(2) production, which in turn modulates the action of CB2.     

Differential effects of ebselen on neutrophil recruitment,chemokine, and inflammatory mediator expression in a rat model of lipopolysaccharide-induced pulmonary inflammation

We postulated that the seleno-organic compound ebselen would attenuate neutrophil recruitment and activation after aerosolized challenge with endotoxin (LPS) through its effect as an antioxidant and inhibitor of gene activation. Rats were given ebselen (1-100 mg/kg i.p.) followed by aerosolized LPS exposure (0.3 mg/ml for 30 min). Airway inflammatory indices were measured 4 h postchallenge. Bronchoalveolar lavage (BAL) fluid cellularity and myeloperoxidase activity were used as a measure of neutrophil recruitment and activation. RT-PCR analysis was performed in lung tissue to assess gene expression of TNF-alpha, cytokine-induced neutrophil chemoattractant-1 (CINC-1), macrophage-inflammatory protein-2 (MIP-2), ICAM-1, IL-10, and inducible NO synthase. Protein levels in lung and BAL were also determined by ELISA. Ebselen pretreatment inhibited neutrophil influx and activation as assessed by BAL fluid cellularity and myeloperoxidase activity in cell-free BAL and BAL cell homogenates. This protective effect was accompanied by a significant reduction in lung and BAL fluid TNF-alpha and IL-1 beta protein and/or mRNA levels. Ebselen pretreatment also prevented lung ICAM-1 mRNA up-regulation in response to airway challenge with LPS. This was not a global effect of ebselen on LPS-induced gene expression, because the rise in lung and BAL CINC-1 and MIP-2 protein levels were unaffected as were lung mRNA expressions for CINC-1, MIP-2, IL-10, and inducible NO synthase. These data suggest that the anti-inflammatory properties of ebselen are achieved through an inhibition of lung ICAM-1 expression possibly through an inhibition of TNF-alpha and IL-1 beta, which are potent neutrophil recruiting mediators and effective inducers of ICAM-1 expression.     

Potential role of apoptotic macrophages in pulmonary inflammation and fibrosis

Induction of apoptosis has been associated with a variety of exposures which result in inflammatory and fibrotic lung disorders. Macrophages are key regulatory cells in the lung; however, the role of apoptotic macrophages in those pulmonary disorders is not well characterized. In the present investigation, apoptotic macrophages were instilled into the lungs of rats to study directly the pulmonary responses to apoptotic cells. The effects of apoptotic macrophages on lung inflammation and fibrosis, as well as associated protein expression of TNF-alpha, TGF-beta, and matrix metalloproteinases (MMPs) were examined. Induction of macrophage apoptosis was carried out in vitro using a variety of known apoptosis inducers. Intratracheal administration of apoptotic macrophages (5 x 10(6) cells/rat) into the lung of rats caused an increase in pulmonary infiltration of macrophages and lung cell apoptosis 4 weeks after the treatment as indicated by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. In contrast, pulmonary instillation of saline or normal control macrophages had no effect. Histological analysis of lung sections showed collagen deposition and fibrotic lesions after apoptotic cell treatment but not in control groups. Immunohistochemical studies revealed increased expression of TNF-alpha, TGF-beta, MMP2, and MMP9 in the treatment group 4 weeks after the treatment. These results suggest a role for macrophage apoptosis in the initiation of these lung disorders. This study provides direct evidence that apoptotic macrophages can induce lung inflammation and fibrosis and that this induction may be associated with increased expression of TNF-alpha, TGF-beta, MMP2, and MMP9. Published 2002 Wiley-Liss, Inc.     

Maleylated-BSA suppresses IFN-gamma-mediated Ia expression in murine peritoneal macrophages

Maleylated bovine serum albumin (maleyl-BSA) and other polyanionic polymers that are recognized by cell surface receptors on macrophages have been shown to induce chemotaxis, protease secretion, and tumoricidal function in this cell type. In this paper the effect of maleyl-BSA on Ia antigen expression has been evaluated. In a fashion similar to LPS, maleyl-BSA suppressed IFN-gamma-induced expression of Ia in a time- and dose-dependent manner. Also like LPS, maleyl-BSA stimulated the production and secretion of substantial amounts of PGE2 over a 24-hr period. This did not, however, appear to be the primary mechanism by which expression of Ia was suppressed, because co-treatment of the cells with indomethacin, which totally inhibited the production of PGE2, only minimally affected the suppressive activity. Surprisingly, the suppressive activity of both maleyl-BSA and LPS could be largely abrogated by co-treatment of the cells with cyclohexamide during the time period when Ia expression was sensitive to suppression. This effect was selective in that PGE2- or dibutyryl cyclic AMP-induced suppression of Ia expression was not affected by cyclohexamide treatment. The data support the concept that there are multiple molecular mechanisms involved in the negative regulation of IFN-gamma-induced Ia expression in macrophages. Such mechanisms may include, in addition to the synthesis of PGE2 and consequent elevation in intracellular levels of cyclic AMP, one or more proteins made early after treatment with either maleyl-BSA or LPS. Thus the function of some of these early gene products may be to regulate expression of functional genes such as that encoding Ia antigen.     

Relationships between pulmonary inflammation, plasma transudation, and oxygen metabolite secretion by alveolar macrophages

We have previously shown that alveolar macrophages from normal rabbit lungs do not synthesize reactive oxygen intermediates unless first conditioned by culture in vitro in the presence of serum for 24 to 48 hr. This conditioning process is mediated by a serum constituent that partitions on gel exclusion columns with an apparent m.w. of 30,000 to 50,000 daltons. Alveolar macrophage conditioning in vitro requires protein synthesis, is associated with the generation of membrane NADPH oxidase activity, and is reversible. We have predicted therefore that during the course of pulmonary inflammation, as observed 3 wk after i.v. injection of M. butyricum in oil, alveolar macrophages might similarly become conditioned in vivo through exposure to plasma protein transudates reaching the alveolus. In support of this hypothesis we show that after experimental production of granulomatous pulmonary inflammation in rabbits, alveolar macrophages showed an augmented capacity to secrete superoxide anion when stimulated with phorbol ester, and this enhancement increases exponentially with increased plasma transudation. This augmented enhancement was reversible, and decreased after culture in vitro in the absence of serum. Mature alveolar macrophages were responsible for this enhanced superoxide anion production rather than freshly emigrated monocytes. Moreover, superoxide anion production in this model of pulmonary inflammation appears to be an "all-or-none" phenomenon, with superoxide anion production associated with a subpopulation of optimally conditioned alveolar macrophages, whereas the remaining unconditioned alveolar macrophages produce little or none. We feel that these two classes of alveolar macrophages may be derived from inflamed and noninflamed regions of the lung, respectively, thereby reflecting the discontinuous nature of the inflammatory lesions themselves. Thus we propose that measurements of reactive oxygen intermediate production by lavaged alveolar macrophages may provide a semi-quantitative measure of chronic pulmonary inflammation.     

Depletion of pulmonary intravascular macrophages inhibits acute lung inflammation

Pulmonary intravascular macrophages (PIMs) are present in ruminants and horses. These species are highly sensitive to acute lung inflammation compared with non-PIM-containing species such as rats and humans. There is evidence that rats and humans may also recruit PIMs under certain conditions. We investigated precise contributions of PIMs to acute lung inflammation in a calf model. First, PIMs were recognized with a combination of in vivo phagocytic tracer Monastral blue and postembedding immunohistology with anti-CD68 monoclonal antibody. Second, gadolinium chloride depleted PIMs within 48 h of treatment (P < 0.05). Finally, PIMs contain TNF-alpha, and their depletion reduces cells positive for IL-8 (P < 0.05) and TNF-alpha (P < 0.05) and histopathological signs of acute lung inflammation in calves infected with Mannheimia hemolytica. The majority of IL-8-positive inflammatory cells in lung septa of infected calves were platelets. Platelets from normal cattle contained preformed IL-8 that was released upon in vitro exposure to thrombin (P < 0.05). These novel data show that PIMs, as the source of TNF-alpha, promote recruitment of inflammatory cells including IL-8-containing platelets to stimulate acute inflammation and pathology in lungs. These data may also be relevant to humans due to our ability to recruit PIMs.     

Hypoxic stress suppresses lung tumor-secreted exosomal miR101 to activate macrophages and induce inflammation

Hypoxia promotes inflammation in the tumor microenvironment. Although hypoxia-inducible factor 1α (HIF1α) is a master modulator of the response to hypoxia, the exact mechanisms through which HIF1α regulates the induction of inflammation remain largely unclear. Using The Cancer Genome Atlas Lung Squamous Cell Carcinoma (TCGA-LUSC) database, we divided patients with LUSC into two groups based on low or high HIF1α expression. After analyzing the differentially expressed genes in these two groups, we found that HIF1α was positively correlated with interleukin 1A (IL1A) and IL6 expression. Our in vitro study showed that hypoxic stress did not induce IL1A or IL6 expression in tumor cells or macrophages but dramatically enhanced their expression when co-cultured with tumor cells. We then investigated the effect of tumor-derived exosomes on macrophages. Our data suggested that the changes in miR101 in the tumor-derived exosomes played an important role in IL1A and IL6 expression in macrophages, although the hypoxic stress did not change the total amount of exosome secretion. The expression of miR101 in exosomes was suppressed by hypoxic stress, since depletion of HIF1α in tumor cells recovered the miR101 expression in both tumor cells and exosomes. In vitro, miRNA101 overexpression or uptake enriched exosomes by macrophages suppressed their reprogramming into a pro-inflammatory state by targeting CDK8. Injection of miR101 into xenografted tumors resulted in the suppression of tumor growth and macrophage tumor infiltration in vivo. Collectively, this study suggests that the HIF1α-dependent suppression of exosome miR101 from hypoxic tumor cells activates macrophages to induce inflammation in the tumor microenvironment.Subject terms: Small-cell lung cancer, Small-cell lung cancer     

Inflammasome activators induce fibronectin expression and release in macrophages

Extracellular fibronectin (Fn) can activate pro‐inflammatory pathways and serves as an endogenous danger signalling molecule; thus, it has been suggested as a biomarker for several diseases. In the present study, we found that pathogen‐derived activators of the inflammasomes induce the expression and secretion of Fn in macrophages through a mechanism involving adenosine triphosphate and caspase‐1 activation. We also found that plasma Fn induces caspase‐1 activation and cell death in macrophages, epithelial cells, and fibroblasts. Together, these results indicate that Fn plays a critical role in inflammasome‐activated cells by amplifying caspase‐1 activation and inducing inflammatory cell death.     

Capsaicin, a spicy component of hot peppers, modulates adipokine gene expression and protein release from obese-mouse adipose tissues and isolated adipocytes, and suppresses the inflammatory responses of adipose tissue macrophages

Adipokines are involved in the obesity-induced chronic inflammatory response that plays a crucial role in the development of obesity-related pathologies such as type II diabetes and atherosclerosis. We here demonstrate that capsaicin, a naturally occurring phytochemical, can suppress obesity-induced inflammation by modulating adipokine release from and macrophage behavior in obese mice adipose tissues. Capsaicin inhibited the expressions of IL-6 and MCP-1 mRNAs and protein release from the adipose tissues and adipocytes of obese mice, whereas it enhanced the expression of the adiponectin gene and protein. The action of capsaicin is associated with NF-kappaB inactivation and/or PPARgamma activation. Moreover, capsaicin suppressed not only macrophage migration induced by the adipose tissue-conditioned medium, but also macrophage activation to release proinflammatory mediators. Capsaicin may be a useful phytochemical for attenuating obesity-induced inflammation and obesity-related complications.     

Extra-cellular matrix suppresses expression of the apoptosis mediator Fas by epigenetic DNA methylation

The extracellular matrix (ECM) of bone consists mainly of collagen type I, which induces osteoblastic differentiation and prevents apoptosis. Fas induces apoptosis in cells improperly adhering to ECM. Recently, it was described that Fas expression is modulated by epigenetic DNA methylation. Mouse MC3T3-E1 pre-osteoblastic cells were cultured either on collagen coated or on uncoated culture dishes for control. mRNA was isolated and gene expression was analyzed by quantitative RT–PCR. Furthermore, we measured global and specific DNA methylation. Compared to controls, cells cultured on collagen-coated dishes increased the expression of Runx2 and OCN indicating differentiation of pre-osteoblastic cells. Additionally, collagen up-regulated cyclin-A2 and down-regulated Fas expression suggesting increased cell multiplication. Furthermore, the expression of Dnmt1 and Hells, key mediators of the DNA-methylation process, was increased. As a consequence, we demonstrate that global DNA methylation and specific methylation of the Fas promoter was higher in MC3T3-E1 cells cultured on collagen when compared to controls. Investigation of signal transduction pathways by mean of inhibitors suggests that focal adhesion kinase, MAP- and Jun-kinases and AP-1 are involved in this process. In summary, we demonstrate that ECM prevents activation of Fas by epigenetic DNA-methylation.     

A sesquiterpenol extract potently suppresses inflammation in macrophages and mice skin and prevents chronic liver damage in mice through JNK-dependent HO-1 expression

This study aimed to elucidate the anti-inflammatory and hepatoprotective bioactivities of a sesquiterpenol, (1S,6R)-2,7(14),10-bisabolatrien-1-ol-4-one (BSL), isolated from Cryptomeria japonica (Taxodiaceae) wood extract. BSL markedly suppressed TNF-α and IL-6 secretion, PGE(2) production, and mRNA expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), in lipopolysaccharide (LPS)-stimulated mouse macrophages. BSL also potently inhibited the 12-O-tetradecanoylphorbol-13-acetate (TPA) induced protein levels of nitrotyrosine and COX-2 in mouse skin with dermatitis. Conversely, the stress protein heme oxygenase-1 (HO-1) was found upregulated in the same BSL-treated macrophages, probably through activation of the JNK-dependent pathway. LPS-induced activation of NF-κB and mitogen-activated protein kinase signaling pathways, however, was not responsive to BSL treatment. A BSL-enriched extract (BSL-E; 10mg/kg) significantly prevented CCl(4)-induced chronic liver injury, lipid accumulation, and cell necrosis and inhibited aminotransferase activities and iNOS and COX-2 overexpression in mice liver tissues, an effect comparable with that of silymarin, a hepatoprotective drug.     

Alveolar hypoxia,alveolar macrophages,and systemic inflammation

Diseases featuring abnormally low alveolar PO₂ are frequently accompanied by systemic effects. The common presence of an underlying inflammatory component suggests that inflammation may contribute to the pathogenesis of the systemic effects of alveolar hypoxia. While the role of alveolar macrophages in the immune and defense functions of the lung has been long known, recent evidence indicates that activation of alveolar macrophages causes inflammatory disturbances in the systemic microcirculation. The purpose of this review is to describe observations in experimental animals showing that alveolar macrophages initiate a systemic inflammatory response to alveolar hypoxia. Evidence obtained in intact animals and in primary cell cultures indicate that alveolar macrophages activated by hypoxia release a mediator(s) into the circulation. This mediator activates perivascular mast cells and initiates a widespread systemic inflammation. The inflammatory cascade includes activation of the local renin-angiotensin system and results in increased leukocyte-endothelial interactions in post-capillary venules, increased microvascular levels of reactive O₂ species; and extravasation of albumin. Given the known extrapulmonary responses elicited by activation of alveolar macrophages, this novel phenomenon could contribute to some of the systemic effects of conditions featuring low alveolar PO₂.     

The human recombinant c-kit receptor ligand, rhSCF, induces mediator release from human cutaneous mast cells and enhances IgE-dependent mediator release from both skin mast cells and peripheral blood basophils.

The gene product of the steel locus of the mouse represents a growth factor for murine mast cells and a ligand for the c-kit proto-oncogene receptor, a member of the tyrosine kinase receptor class of oncogenes (for review, see O. N. Witte. 1990. Cell 63:5). We have studied the effect of the human recombinant c-kit receptor ligand stem cell factor (rhSCF) on the release of inflammatory mediators from human skin mast cells and peripheral blood basophils and compared its activity to that of rhIL-3, rhSCF (1 ng/ml to 1 microgram/ml) activated the release of histamine and PGD2 from mast cells isolated from human skin. Analysis by digital video microscopy indicated that purified human skin mast cells (84 +/- 5% pure) responded to rhSCF (0.1 to 1 microgram/ml) challenge with a rapid, sustained rise in intracellular Ca2+ levels that was accompanied by secretion of histamine. A brief preincubation (10 min) of mast cells with rhSCF (0.1 pg/ml to 1 ng/ml) significantly enhanced (100 +/- 35%) the release of histamine induced by anti-IgE (3 micrograms/ml), but was much less effective on IgE-mediated release of PGD2. In contrast, a short term incubation with rhSCF did not potentiate the secretion of histamine activated by substance P (5 microM). A 24-h incubation of mast cells with rhSCF did not affect the release of mediators induced by anti-IgE (3 micrograms/ml), probably due to receptor desensitization, rhSCF (1 ng/ml to 3 micrograms/ml) neither caused release of histamine or leukotriene C4 (LTC4) release from leukocytes of 14 donors, nor induced a rise in intracellular Ca2+ levels in purified (greater than 70%) basophils. Brief preincubation (10 min) of leukocytes with rhSCF (1 ng/ml to 3 micrograms/ml) caused an enhancement (69 +/- 11%) of anti-IgE-induced release of histamine that was significant at concentrations as low as 3 ng/ml (p less than 0.05), whereas it appeared less effective in potentiating IgE-mediated LTC4 release. In contrast, a prolonged incubation (24 h) with rhSCF (0.1 pg/ml to 100 ng/ml) did not enhance the release of histamine or LTC4 induced by anti-IgE (0.1 microgram/ml), whereas rhIL-3 (3 ng/ml) significantly potentiated the release of both mediators.(ABSTRACT TRUNCATED AT 400 WORDS)