Lipopolysaccharide-induced lung injury in mice. I. Concomitant evaluation of inflammatory cells and haemorrhagic lung damage.

Intratracheal instillation of lipopolysaccharide (LPS) induces an inflammatory response characterized by infiltration of polymorphonuclear neutrophils (PMNs) into the extracellular matrix and by the release of mediators that play a fundamental role in lung damage. In the present study, we developed a mouse model which allows correlation of the inflammatory response and haemorrhagic tissue injury in the same animal. In particular, the different steps of the inflammatory response and tissue damage were evaluated by the analysis of three parameters: myeloperoxidase (MPO) activity in the parenchyma, reflecting PMNs accumulation into the lung, inflammatory cells count in the bronchoalveolar lavage fluid (BALF), reflecting their extravasation, and total haemoglobin estimation in BALF, a marker of haemorrhagic tissue damage consequent to PMNs degranulation. In our experimental conditions, intra-tracheal administration of 10 microg/mouse of LPS evoked an increase of MPO activity in the lung at 4 h (131%) and 6 h (147%) from endotoxin challenge. A significant increase of PMNs in the BALF was noticed at these times with a plateau between the 12nd and 24th h. PMN accumulation produced a time-dependent haemorrhagic lung damage until 24 h after LPS injection (4 h: +38%; 6 h: +23%; 12 h: +44%; 24 h: +129% increase of haemoglobin concentration in the BALF vs. control). Lung injury was also assessed histopathologically. Twenty-four hours after the challenge, diffuse alveolar haemorrhage, as well as PMN recruitment in the interstitium and alveolus were observed in the LPS group. This model was pharmacologically characterized by pretreatment of LPS-treated mice with antiinflammatory drugs acting on different steps of the <>. We demonstrated that: a) betamethasone (1, 3, 10, 30 mg/kg p.o.) reduced in a dose-dependent manner the MPO activity, the number of inflammatory cells and, at the same time, lung injury; b) pentoxifylline, a TNFalpha production inhibitor (200 mg/kg i.p.), inhibited PMN extravasation and lung haemorrhage but it was not able to reduce MPO activity in the lung; c) L-680,833, an anti-elastase compound (30 mg/kg po), decreased significantly only the haemorrhagic lung damage; d) indomethacin, a non steroidal antiinflammatory drug (5 mg/kg p.o.), did not show any effect on any of the parameters considered. In conclusion, our in vivo mouse model is a practical alternative to animal models of ARDS (Adult Respiratory Distress Syndrome) recently described and it permits to dissect and to characterize the different steps of PMNs infiltration and, at the same time, the damage caused by their activation.

Lipopolysaccharide-induced lung injury in mice. II. Evaluation of functional damage in isolated parenchyma strips.

Pulmonary inflammatory diseases are characterized by changes in airway responsiveness. This phenomenon is commonly related to the action of inflammatory mediators produced by infiltrated leukocytes. The aim of this study was to investigate in an ex vivo experimental model the effect of acute instillation of lipopolysaccharide (bacterial endotoxin; LPS) on lung parenchyma contractility. We firstly characterized the responsiveness of isolated murine lung to airway stimuli. Murine parenchymal strips were found to be mainly sensitive to 5-hydroxytryptamine (5-HT) while the cholinergic agonist, methacholine (MCh), evoked a smaller contractile response. 5-HT responsiveness was inhibited by methysergide. No significant parenchymal contraction was evoked by histamine, substance P and bradykinin. Lung responsiveness to 5-HT was significantly reduced by in vivo LPS treatment and this effect was only partially paralleled by leukocyte infiltration. In addition, LPS-induced hyporesponsiveness was significantly inhibited by betamethasone (BMS) or pentoxifylline (PTX) pretreatment suggesting that 5-HT lung hyporesponsiveness could be mediated by LPS-induced inflammatory mediators such as inflammatory cytokines.

A dominant negative RAS-specific guanine nucleotide exchange factor reverses neoplastic phenotype in K-ras transformed mouse fibroblasts.

Ras proteins are small GTPases playing a pivotal role in cell proliferation and differentiation. Their activation state depends on the competing action of GTPase Activating Proteins (GAP) and Guanine nucleotide Exchange Factors (GEF). A tryptophan residue (Trp1056 in CDC25Mm-GEF), conserved in all ras-specific GEFs identified so far has been previously shown to be essential for GEF activity. Its substitution with glutamic acid results in a catalytically inactive mutant, which is able to efficiently displace wild-type GEF from p21ras and to originate a stable ras/GEF binary complex due to the reduced affinity of the nucleotide-free ras/GEF complex for the incoming nucleotide. We show here that this 'ras-sequestering property' can be utilized to attenuate ras signal transduction pathways in mouse fibroblasts transformed by oncogenic ras. In fact overexpression of the dominant negative GEFW1056E in stable transfected cells strongly reduces intracellular ras-GTP levels in k-ras transformed fibroblasts. Accordingly, the transfected fibroblasts revert to wild-type phenotype on the basis of morphology, cell cycle and anchorage independent growth. The reversion of the transformed phenotype is accompanied by DNA endoreduplication. The possible use of dominant negative ras-specific GEFs as a tool to down-regulate tumor growth is discussed.

Synthesis, inhibitory activity towards human leukocyte elastase and molecular modelling studies of 1-carbamoyl-4-methyleneaminoxyazetidinones.

Some monocyclic beta-lactam derivatives of type 3 (MAOAs) in which the leaving group (LG) on the C(4) is a methyleneaminoxy moiety, were synthesised and tested in vitro and in vivo for their inhibitory activity towards human leukocyte elastase (HLE). Some compounds showed an appreciable in vitro inhibitory activity against this enzyme. Effects on the anti-HLE activity due to the nature of the substituents R and R(1) present on their LG were observed and rationalised by means of molecular modelling techniques. The results of in vivo pharmacological tests indicated that MAOAs, while showing an inhibitory activity on the haemorrhage induced by HLE, did not exhibit any effects due to the R and R(1) substituents.

Synthesis and in vitro and in vivo evaluation of the 2-(6'methoxy-3',4'-dihydro-1'-naphtyl)-4H-3,1-benzoxazin-4- one as a new potent substrate inhibitor of human leukocyte elastase.

The title 2-vinyl-4H-3,1-benzoxazin-4-one has been synthesised and tested for inhibitory activity against human leukocyte elastase. The compound has shown activity both in vitro towards human sputum elastase and in vivo in an hemorrhagic assay.

Differential contribution of R and S isomers in ketoprofen anti-inflammatory activity: role of cytokine modulation.

Among nonsteroidal anti-inflammatory drugs (NSAIDs), 2-arylpropionic acids exist as a racemic mixture of its enantiomeric forms, with S-enantiomers primarily responsible for inhibition of prostaglandin synthesis and of inflammatory events. The aim of this study was to compare the anti-inflammatory effects of R- and S-ketoprofen in vitro and in vivo. S-Ketoprofen efficiently inhibited carrageenan-induced edema formation, but it could also amplify the LPS-induced production of the inflammatory cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1), in close correlation with its ability to inhibit prostaglandin synthesis. Because these inflammatory cytokines are among the factors involved in carrageenan-induced inflammation and also are possibly involved in gastric damage, enhanced cytokine production could partially mask the analgesic effect of S-ketoprofen, and it can be associated with the clinical evidence of its gastric toxicity. On the other hand, R-ketoprofen contributes to the overall activity of the racemate, by playing the main role in ketoprofen-induced analgesia. Unlike the S-isomer, R-ketoprofen did not induce a significant increase of cytokine production even at cyclooxygenase-blocking concentrations. It is concluded that the R-isomer directly contributes to the anti-inflammatory effects of ketoprofen, being more analgesic, and because it does not amplify inflammatory cytokine production.

Effectiveness of carbocysteine lysine salt monohydrate on models of airway inflammation and hyperresponsiveness.

We investigated the possible effects of the mucoactive drug Carbocysteine lysine salt monohydrate (CLS.H2O) on experimentally-induced airway inflammation and hyperresponsiveness. CLS.H2O given by the oral route (300 mg kg(-1)) significantly reduced neutrophil infiltration into the airway lumen induced by intratracheal injection of IL-1 beta in rats. In addition, CLS.H2O inhibited dose-dependently (100-300 mg kg(-1) p.o.) the formation of pleural exudate and leukocyte recruitment induced by intrapleural injection of carrageenan in rats. Because of the close interaction between the inflammatory process and the development of airway hyperresponsiveness we also tested CLS.H2O on cigarette-smoke-induced inflammation and hyperreactivity in anaesthetized guinea-pigs. The drug, given either by oral (300 mg kg(-1)) or aerosol route (30-100 mg ml(-1)), was able to reduce the increase in airway responsiveness induced by smoke and the associated cell recruitment detected in the bronchoalveolar lavage (BAL) fluids. These results suggest that CLS.H2O can exert an anti-inflammatory action in addition to its mucoregulatory activity. The anti-inflammatory and anti-hyperreactivity effect of the drug within the airways may be of advantage in the treatment of inflammatory lung diseases where mucus secretion together with airway inflammation and hyperreactivity contribute to airway obstruction.