calcimycin and Pneumonia

The pulmonary effects of inhaled A23187 are reviewed. Guinea pigs challenged with this divalent cationic ionophore rapidly develop airway obstruction, which is maintained for at least 4 h. Pulmonary inflammation and increased airway responsiveness are also observed. Pharmacologic manipulations suggest that these actions are due to the release of multiple mediators. We have found A23187 challenge to be valuable as an approach for testing potential asthma drugs.

Topics: Administration, Inhalation; Animals; Calcimycin; Guinea Pigs; Lung; Male; Pneumonia

The accumulation of neutrophils and proinflammatory mediators, such as leukotriene B4 (LTB4), is a classic marker of inflammatory disease. The clearance of apoptotic neutrophils, inhibition of proinflammatory signaling, and production of proresolving lipids (including lipoxins, such as lipoxin A4 [LXA4]) are imperative for resolving inflammation. Tulathromycin (TUL), a macrolide used to treat bovine respiratory disease, confers immunomodulatory benefits via mechanisms that remain unclear. We recently reported the anti-inflammatory properties of TUL in bovine phagocytes in vitro and in Mannheimia haemolytica-challenged calves. The findings demonstrated that this system offers a powerful model for investigating novel mechanisms of pharmacological immunomodulation. In the present study, we examined the effects of TUL in a nonbacterial model of pulmonary inflammation in vivo and characterized its effects on lipid signaling. In bronchoalveolar lavage (BAL) fluid samples from calves challenged with zymosan particles (50 mg), treatment with TUL (2.5 mg/kg of body weight) significantly reduced pulmonary levels of LTB4 and prostaglandin E2 (PGE2). In calcium ionophore (A23187)-stimulated bovine neutrophils, TUL inhibited phospholipase D (PLD), cytosolic phospholipase A2 (PLA2) activity, and the release of LTB4. In contrast, TUL promoted the secretion of LXA4 in resting and A23187-stimulated neutrophils, while levels of its precursor, 15(S)-hydroxyeicosatetraenoic acid [15(S)-HETE], were significantly lower. These findings indicate that TUL directly modulates lipid signaling by inhibiting the production of proinflammatory eicosanoids and promoting the production of proresolving lipoxins.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Bronchoalveolar Lavage Fluid; Calcimycin; Cattle; Dinoprostone; Disaccharides; Heterocyclic Compounds; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Lipoxins; Lung; Male; Neutrophils; Particulate Matter; Phospholipases A2; Pneumonia; Primary Cell Culture; Zymosan

The serine proteases released by activated polymorphonuclear neutrophils [NSPs (neutrophil serine proteases)] contribute to a variety of inflammatory lung diseases, including CF (cystic fibrosis). They are therefore key targets for the development of efficient inhibitors. Although rodent models have contributed to our understanding of several diseases, we have previously shown that they are not appropriate for testing anti-NSP therapeutic strategies [Kalupov, Brillard-Bourdet, Dade, Serrano, Wartelle, Guyot, Juliano, Moreau, Belaaouaj and Gauthier (2009) J. Biol. Chem. 284, 34084-34091). Thus NSPs must be characterized in an animal model that is much more likely to predict how therapies will act in humans in order to develop protease inhibitors as drugs. The recently developed CFTR-/- (CFTR is CF transmembrane conductance regulator) pig model is a promising alternative to the mouse model of CF [Rogers, Stoltz, Meyerholz, Ostedgaard, Rokhlina, Taft, Rogan, Pezzulo, Karp, Itani et al. (2008) Science 321, 1837-1841]. We have isolated blood neutrophils from healthy pigs and determined their responses to the bacterial pathogens Pseudomonas aeruginosa and Staphylococcus aureus, and the biochemical properties of their NSPs. We used confocal microscopy and antibodies directed against their human homologues to show that the three NSPs (elastase, protease 3 and cathepsin G) are enzymatically active and present on the surface of triggered neutrophils and NETs (neutrophil extracellular traps). All of the porcine NSPs are effectively inhibited by human NSP inhibitors. We conclude that there is a close functional resemblance between porcine and human NSPs. The pig is therefore a suitable animal model for testing new NSP inhibitors as anti-inflammatory agents in neutrophil-associated diseases such as CF.

Topics: Animals; Calcimycin; Calcium Ionophores; Cell Degranulation; Disease Models, Animal; Humans; In Vitro Techniques; Neutrophil Activation; Neutrophils; Pneumonia; Pseudomonas aeruginosa; Serine Proteases; Serine Proteinase Inhibitors; Species Specificity; Staphylococcus aureus; Swine

Microparticles (MP) are phospholipid vesicles shed by cells upon activation or apoptosis. Monocyte-derived MP upregulate the synthesis of proinflammatory mediators by lung epithelial cells; the molecular bases of such activity are unknown. Peroxisome proliferator-activated receptors (PPAR) have been demonstrated to be involved in the modulation of nuclear factor (NF)-κB transcriptional activity and inflammation. We investigated whether the upregulation of the synthesis of proinflammatory cytokines by human lung epithelial cells induced by monocyte/macrophage-derived MP involves NF-κB activation and is modulated by PPAR-γ. MP were generated by stimulation of human monocytes/macrophages with the calcium ionophore, A23187. MP were incubated with human lung epithelial cells. NF-κB translocation was assessed by electrophoretic mobility shift assay. Interleukin (IL)-8 and monocyte chemotactic protein (MCP)-1 synthesis was assessed by ELISA and RT-PCR. Stimulation of A549 alveolar cells with monocyte/macrophage-derived MP caused an increase in NF-κB activation and IL-8 and MCP-1 synthesis that was inhibited by pre-incubation with the PPAR-γ agonists, rosiglitazone and 15-deoxy-Δ12,14-prostaglandin-J2. Parallel experiments with normal human bronchial epithelial cells largely confirmed the results. The effects of PPAR-γ agonists were reversed by the specific antagonist, GW9662. Upregulation of the synthesis of proinflammatory mediators by human lung epithelial cells induced by monocyte/macrophage-derived MP is mediated by NF-κB activation through a PPAR-γ dependent pathway.

Topics: Anilides; Bronchi; Calcimycin; Cell Line; Cell-Derived Microparticles; Cells, Cultured; Chemokine CCL2; Humans; Interleukin-8; Ionophores; Monocytes; NF-kappa B; Pneumonia; PPAR gamma; Prostaglandin D2; Rosiglitazone; Thiazolidinediones; Up-Regulation

We report here that 4-dibenzo[a,d]cyclohepten-5-ylidene-1-[4-(2H-tetrazol-5-yl)-butyl]-piperidine (AT-56) is an orally active and selective inhibitor of lipocalin-type prostaglandin (PG) D synthase (L-PGDS). AT-56 inhibited human and mouse L-PGDSs in a concentration (3-250 microm)-dependent manner but did not affect the activities of hematopoietic PGD synthase (H-PGDS), cyclooxygenase-1 and -2, and microsomal PGE synthase-1. AT-56 inhibited the L-PGDS activity in a competitive manner against the substrate PGH(2) (K(m) = 14 microm) with a K(i) value of 75 microm but did not inhibit the binding of 13-cis-retinoic acid, a nonsubstrate lipophilic ligand, to L-PGDS. NMR titration analysis revealed that AT-56 occupied the catalytic pocket, but not the retinoid-binding pocket, of L-PGDS. AT-56 inhibited the production of PGD(2) by L-PGDS-expressing human TE-671 cells after stimulation with Ca(2+) ionophore (5 microm A23187) with an IC(50) value of about 3 microm without affecting their production of PGE(2) and PGF(2alpha) but had no effect on the PGD(2) production by H-PGDS-expressing human megakaryocytes. Orally administered AT-56 (<30 mg/kg body weight) decreased the PGD(2) production to 40% in the brain of H-PGDS-deficient mice after a stab wound injury in a dose-dependent manner without affecting the production of PGE(2) and PGF(2alpha) and also suppressed the accumulation of eosinophils and monocytes in the bronco-alveolar lavage fluid from the antigen-induced lung inflammation model of human L-PGDS-transgenic mice.

Topics: Administration, Oral; Animals; Calcimycin; Cyclooxygenase 1; Cyclooxygenase 2; Dinoprost; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Enzyme Inhibitors; Eosinophils; Humans; Intramolecular Oxidoreductases; Ionophores; Lipocalins; Male; Megakaryocytes; Membrane Proteins; Mice; Mice, Knockout; Monocytes; Pneumonia; Prostaglandin D2; Wound Healing; Wounds, Stab

To investigate whether FK506 (tacrolimus) can inhibit Fas- or A23187-induced interleukin (IL)-8 expression and cell death in A549 human alveolar epithelial cells, plus Fas-mediated acute lung injury in vivo.. Assays for IL-8, cell death, and caspase-3 activity were performed. A549 cells were treated with 25 micromol A23187 or 0.2 microg/ml agonistic anti-Fas antibody plus 5 ng/ml interferon-gamma (IFN-gamma). Tacrolimus was treated at 0.1-10 ng/ml. For in vivo experiment, agonistic anti-Fas antibody (Jo2) at 2.5 microg/g was intratracheally instilled into C57BL/6 mice. Neutrophils and protein contents in bronchoalveolar lavage (BAL) fluid were measured within 24 h of instillation. Mice were orally treated with 32 mg/kg of tacrolimus 24 h and 1 h prior to instillation.. Both Fas and A23187 caused significant IL-8 expression and cell death in A549 cells. Tacrolimus inhibited A23187-induced IL-8 expression alone while it protected all Fas-mediated responses. Mice instilled intratracheally with Jo2 at 2.5 microg/g had significant increases in neutrophils, protein contents in BAL fluid and in expression of chemoattractants for neutrophils. These increases were reversed by tacrolimus.. Tacrolimus serves as a therapeutic option for improving lung injury through inhibition of Fas-mediated inflammation.

Topics: Animals; Antibodies, Monoclonal; Calcimycin; Caspase 3; Cell Death; Cell Line; Dose-Response Relationship, Drug; fas Receptor; Gene Expression Regulation; Gene Expression Regulation, Enzymologic; Humans; Immunosuppressive Agents; Inflammation; Interleukin-8; Male; Mice; Mice, Inbred C57BL; Pneumonia; Pulmonary Alveoli; Respiratory Mucosa; RNA, Messenger; Tacrolimus

The intracellular regulation and kinetics of prostaglandin (PG)E(2) synthesis in human airway epithelial (NCI-H292) cells was investigated. Interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha and lipopolysaccharide (LPS) all induced PGE(2) synthesis (p<0.001) and transient (5-15 min) phosphorylation of extracellular signal-regulated kinase (ERK). Phorbol myristate acetate (PMA) and calcium ionophore, A23187 further enhanced PGE(2) synthesis (p<0.001) and caused phosphorylation of ERK that was sustained for up to 16 h. COX-2 protein expression and PGE(2) synthesis were increased following exposure to combinations of stimuli that increased intracellular Ca(2+), and activated protein kinase C as well as ERK. Inhibition of ERK almost completely abrogated PGE(2) synthesis in response to all stimuli. Sustained, maximum PGE(2) synthesis was observed when cells were stimulated such that ERK phosphorylation was concomitant with increased COX-2 protein expression. These results argue against redundancy in pathways for PGE(2) synthesis, and suggest that at various stages of inflammation different stimuli may influence ERK activation and COX-2 expression, so as to tightly regulate the kinetics and amount of PGE(2) produced by airway epithelial cells in response to lung inflammation.

Topics: Calcimycin; Calcium; Cyclooxygenase 2; Dinoprostone; Enzyme Induction; Epithelial Cells; Extracellular Signal-Regulated MAP Kinases; Humans; Interleukin-1beta; Ionophores; Kinetics; Lipopolysaccharides; Phospholipases A; Phosphorylation; Pneumonia; Protein Kinase C; Respiratory System; Signal Transduction; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha

Interleukin (IL)-11, like other members of the gp130 receptor class, possesses anti-inflammatory properties. We hypothesized that IL-11 pretreatment would attenuate endotoxin [lipopolysaccharide (LPS)]-induced lung inflammation and diminish injury to endothelium-dependent and -independent mechanisms of pulmonary vasorelaxation that require cGMP in Sprague-Dawley rats. LPS (20 mg/kg ip) increased lung tumor necrosis factor (TNF)-alpha compared with the saline control (0.7 +/- 0.15 ng/g lung wet wt for control vs. 3.5 +/- 0.09 ng/g lung wet wt for LPS; P < 0.05). IL-11 (200 mg/kg ip) injected 10 min before LPS administration attenuated the LPS-induced lung TNF-alpha levels (1.6 +/- 0.91 ng/g lung wet wt; P < 0.05 vs. LPS). IL-11 also diminished LPS-induced lung neutrophil sequestration as assessed by myeloperoxidase units (2.1 +/- 0.25 U/g lung wet wt for saline and 15.6 +/- 2.02 U/g lung wet wt for LPS vs. 7.07 +/- 1.65 U/g lung wet wt for LPS plus IL-11; P < 0.05). Similarly, TNF-alpha binding protein (175 mg/kg) attenuated LPS-induced myeloperoxidase activity (6.04 +/- 0.14 U/g lung wet wt; P < 0.05). Both IL-11 and TNF-alpha binding protein similarly attenuated LPS-induced endothelium-dependent vasomotor dysfunction with improved relaxation responses to 10(-7) and 10(-6) M acetylcholine and A-23187 in phenylephrine-preconstricted isolated pulmonary artery rings (P < 0.05 vs. LPS). Endothelium-independent relaxation responses to sodium nitroprusside were also improved after LPS at 10(-6) M (P < 0.05 vs. LPS). Moreover, IL-11 decreased endotoxin-induced mortality in CF1 mice from 90 to 50% (P

Topics: Acetylcholine; Animals; Antigens, CD; Calcimycin; Cyclic GMP; Cytokine Receptor gp130; Interleukin-11; Ionophores; Lipopolysaccharides; Lung; Male; Membrane Glycoproteins; Neutrophils; Nitroprusside; Peroxidase; Pneumonia; Pulmonary Circulation; Rats; Rats, Sprague-Dawley; Receptors, Tumor Necrosis Factor; Receptors, Tumor Necrosis Factor, Type I; Survival Analysis; Tumor Necrosis Factor Decoy Receptors; Tumor Necrosis Factor-alpha; Vasodilator Agents

A brief A23187 aerosol exposure produced prolonged airway obstruction with granulocyte accumulation in conscious guinea pigs. Aminophylline, atropine, pyrilamine, salbutamol, SC-41930 (a leukotriene B4 antagonist) and WEB 2086 (a platelet activating factor antagonist) were administered intravenously (i.v.) to evaluate their ability to prevent these changes. Inhaled salbutamol was also assessed. Aminophylline, atropine, and salbutamol (i.v. and aerosol) inhibited A23187-induced gas trapping (p less than 0.01). However, pyrilamine, SC-41930 and WEB 2086 did not influence this airway obstructive effect. Only atropine, inhaled salbutamol and SC-41930 inhibited the cell influx (p less than 0.01), while pyrilamine potentiated the inflammation (p less than 0.05). We conclude that A23187 produces a sustained bronchospasm and an intense granulocyte accumulation. The treatment agents tested differ considerably in their ability to alter A23187-induced airway obstruction and inflammation.

Topics: Albuterol; Aminophylline; Animals; Atropine; Bronchial Spasm; Calcimycin; Granulocytes; Guinea Pigs; Kinetics; Lung Diseases, Obstructive; Male; Pneumonia