Abstract 1838 Neonatal Pulmonology II: Oxidant and Inflammatory Lung Injury Poster Symposium, Tuesday, 5/4

Background: Intrapulmonary neutrophils and alveolar macrophages play a role in tissue injury in neonatal lung diseases by releasing proteases. In this context, the role of MMPs and TIMPs and their modulation by pharmacological agents has not been defined. Aims: 1) To study the extracellular matrix degradative activity and inhibition in bronchoalveolar lavage fluid (BAL) from mechanically ventilated newborns. 2) To evaluate the modulation of these activities in cultured BAL cells by Dexamethasone (Dex), and by non selective (Ibuprofen (Ibu)) and selective (Nimesulide (Nime)) inhibitors of cyclo-oxygenase-2, which may be involved in MMPs synthesis via a GTP-dependent signal transduction pathway. 3) To study in a model of human monocytic cell line (HL-60), constitutively expressing MMPs, the effects of GTP depletion on differentiation (associated with MMPs production) and other cellular functions (proliferation and apoptosis). Materials and methods: BAL samples collected from 20 ventilated newborns (GA 24-41 wks, BW 550-1040 g, PNA 0-31 d, with RDS, CDH, BPD). Separation of BALs cell pellets (CP) from fluid phase (FP), CP resuspension in RPMI medium and incubation c and s LPS (10 mM), Dex (1mM), Ibu (50 mM), and Nime (10 mM). Gelatinolytic activity of MMPs isoforms and degree of inhibition by TIMPs evaluated by zymograms and reverse-zymograms on FP and conditioned media of cultured BAL cells. GTP modulation in HL-60 cells tested with mycophenolic acid (MPA) (a specific IMP-dehydrogenase inhibitor) and with guanosine (which reverses MPA effects by increasing intracellular GTP level through the salvage pathway). Exponentially growing HL-60 cells (5-10 × 104 cells/ml) were cultured in RPMI medium containing MPA (0-10 mM) c and s guanosine (50 mM). Cell differentiation was evaluated by FACS analysis of CD11b and superoxide generation (slide test), cellular proliferation by BrdU incorporation (ELISA), apoptosis by flow cytometry using the tunel reaction, and intracellular GTP levels by HPLC. Results: Under basal conditions, FPs and CM of cultured BAL cells showed high MMPs (mainly MMP-9) and low TIMPs activities. LPS stimulation of cultured BAL cells for 24 h. lead to a marked induction of MMP-9 and to a consistent reduction of TIMPs activities. A marked decrease in MMPs and an increase in TIMPs activities were observed with Ibu but not with Dex. Nime gave variable results, mostly increasing both activities. In HL-60 cells MPA induced a time- and dose-dependent inhibition of proliferation and a decrease in GTP pools. Cell death by apoptosis was observed at MPA concentrations higher than 2 (M and with incubation times longer than 48 h, while lower MPA levels were associated with an increase of both differentiation indices. All these MPA-induced effects were completely reversed by guanosine. Conclusions: The imbalance between MMPs and their inhibitors in BALs of ventilated newborns could be an important factor in the production of lung damage. At least in cultured BAL cells Ibu appears to modulate this process suggesting alternative mechanisms of action for some anti-inflammatory drugs, while the variable response to Nime could be related to the cells differentiation phase. Low GTP induces differentiation in HL-60 cells. The induction of MMP-9 gene expression during macrophage differentiation and the involvement of a GTP-dependent pathway in this and in other cellular activities, indicate that HL-60 cell line is a good model for studying the effects of drugs on MMPs and TIMPs production, and suggests a possible target for pharmacological interventions in inflammatory lung diseases.