Albino Bonazzi

Cyclo-oxygenase-2 mediates P2Y receptor-induced reactive astrogliosis

Excessive cyclo‐oxygenase‐2 (COX‐2) induction may play a role in chronic neurological diseases characterized by inflammation and astrogliosis. We have previously identified an astroglial receptor for extracellular nucleotides, a P2Y receptor, whose stimulation leads to arachidonic acid (AA) release, followed, 3 days later, by morphological changes resembling reactive astrogliosis. Since COX‐2 may be upregulated by AA metabolites, we assessed a possible role for COX‐2 in P2Y receptor‐mediated astrogliosis. A brief challenge of rat astrocytes with the ATP analogue α,β‐methylene ATP (α,βmeATP) resulted, 24 h later, in significantly increased COX‐2 expression. The selective COX‐2 inhibitor NS‐398 completely abolished α,βmeATP‐induced astrocytic activation. Constitutive astroglial COX‐1 or COX‐2 did not play any role in purine‐induced reactive astrogliosis. PGE2, a main metabolite of COX‐2, also induced astrocytic activation. These data suggest that a P2Y receptor mediates reactive astrogliosis via induction of COX‐2. Antagonists selective for this receptor may counteract excessive COX‐2 activation in both acute and chronic neurological diseases.

Monoclonal Anti-CD18 Antibody Prevents Transcellular Biosynthesis of Cysteinyl Leukotrienes In Vitro and In Vivo and Protects Against Leukotriene-Dependent Increase in Coronary Vascular Resistance and Myocardial Stiffness

BACKGROUND Cysteinyl leukotrienes (cys-LT) can constrict small and large vessels and increase vascular permeability. Formation of cys-LT arising from polymorphonuclear leukocytes (PMNL) and endothelial cell cooperation (transcellular synthesis) led to the hypothesis that PMNL-endothelial cell adhesion may represent a key step toward the formation of vasoactive cys-LT. METHODS AND RESULTS We studied the effect of pretreatment with a monoclonal antibody directed against the CD18 subunit of PMNL beta(2)-integrin on the synthesis of cys-LT in a PMNL-perfused isolated rabbit heart in vitro and in a model of permanent ligature of the left descending coronary artery in the rabbit in vivo. Challenge of PMNL-perfused rabbit hearts with formyl-met-leu-phe (0.3 micromol/L) caused synthesis of cys-LT and increase in coronary perfusion pressure that were prevented by the anti-CD18 antibody. Similar results were obtained with the use of A-23187 (0.5 micromol/L) as a challenge. Persistence of PMNL-associated myeloperoxidase activity in the perfusion buffer was observed in the presence of the anti-CD18 antibody, indicating decreased PMNL infiltration. Coronary artery ligature in vivo increased urinary excretion of leukotriene E(4), supporting the activation of the 5-lipoxygenase pathway during experimental acute myocardial infarction. Pretreatment with the anti-CD18 antibody (1 mg/kg) prevented the increase in leukotriene E(4) excretion. CONCLUSIONS These data support the importance of adhesion in promoting cys-LT formation, originating from PMNL-endothelial cell cooperation, and contributing to myocardial stiffness and increased coronary resistance.

Nitric oxide modulation of transcellular biosynthesis of cys-leukotrienes in rabbit leukocyte-perfused heart

We have studied the role of nitric oxide (NO) in the regulation of the transcellular biosynthesis of sulphidopeptide leukotrienes (cys‐LT) generated upon neutrophil‐vascular wall interactions and their functional consequences, in the spontaneously beating, cell‐perfused, heart of the rabbit. Hearts were perfused under recirculating conditions (50 ml) with 5×106 purified human neutrophils (PMNL), and challenged with 0.5 μm A‐23187 for 30 min. Coronary perfusion pressure (CPP) and left‐ventricular end‐diastolic pressure (LVEDP) were monitored. Cys‐LT formation was measured by reversed phase high performance liquid chromatography (h.p.l.c.) and u.v. spectral analysis. Myeloperoxidase (MPO) enzyme activity, assayed in aliquots of the recirculating buffer, was used as a marker of PMNL adhesion to the coronary endothelium. Basal CPP and LVEDP values averaged 45±1.4 mmHg and 5±0.1 mmHg, respectively; A‐23187 triggered an increase in CPP (134±9 mmHg, at 30 min) which was significantly attenuated by pretreatment with l‐arginine, 100 μm (90±3 mmHg, at 30 min). Pretreatment with NG‐monomethyl‐l‐arginine, 10 μm (l‐NMMA), induced a marked increase in CPP (290±40 mmHg, at 20 min) and in LVEDP (47±16 mmHg), so pronounced that it caused cardiac arrest in systole in 5 out of 6 hearts and these were prevented by l‐arginine, 100 μm (CPP 115±10 mmHg, LVEDP 6±1.1 mmHg, at 30 min). The increase in CPP was accompanied by the release of cys‐LT in the circulating buffer, which was reduced significantly by l‐arginine. Pretreatment with l‐NMMA, caused a marked rise in cys‐LT concentrations which was prevented by l‐arginine. Neither l‐arginine nor l‐NMMA affected directly the A‐23187‐induced arachidonic acid (AA) metabolism in isolated PMNL alone. Pretreatment with l‐NMMA caused a prompt drop in myeloperoxidase (MPO) activity, suggesting rapid adhesion of PMNL to the coronary wall; this effect was significantly blunted by l‐arginine. This study suggests that NO provides cardioprotection in an organ model of transcellular metabolism of cys‐LT by preventing PMNL adhesion to the coronary intima.

Neutrophil-Endothelial Cells Cooperation in the Handling of Leukotrienes: Role in Coronary Inflammation

Sulphido-peptide leukotrienes (cys-LT, LTC4, D4 and E4), are products of the 5-lipoxygenase (5-LO) pathway of arachidonic acid (AA) metabolism and are intimately involved in the genesis of inflammatory responses, due to their vasopermeant properties that cause plasma extravasation and diapedesis of white cells into perivascular tissues [1]. The generation of cys-LT exhibits remarkable cellular specificity; polymorphonuclear leukocytes (PMNL) generate predominantly the unstable epoxide LTA4 which is then transformed to leukotriene B4 (LTB4) [2], whereas mast cells and eosinophils show preferential generation of cys-LT.

Cysteinyl-leukotriene receptors and transduction mechanisms in airway cells

A variety of inflammatory cells synthesize cys-leukotrienes (cys-LTs) C4, D4 and E4 in response to biological and non-biological stimuli1: eosinophils, basophils and mast cells2,3 are able to synthesize cys-LTs from arachidonic acid but cys-LTs can also be produced through transcellular metabolism from neutrophil-derived LTA4, by vascular endothelial cells4-6 and platelets7.