Amplified nitric oxide production by pulmonary alveolar macrophages of newborn rats.

Oxygen (O2)-dependent and O2-independent antimicrobial mechanisms are used by alveolar macrophages (AM) to maintain lung sterility, but these mechanisms are underdeveloped in neonatal AM. Nitric oxide (NO(.)), a more recently described antimicrobial and immunomodulating molecule, has not been studied in neonatal AM. Lavaged AM from 3-day-old, 10-day-old, maternal and adult rats were treated with or without lipopolysaccharide (LPS) and/or interferon-γ (IFN-γ) and NO(.) synthase activity was measured as its L-arginine metabolites: NO2(-), NO3(-), and citrulline. Superoxide anion (O2(.-)) production by suspended macrophages, initiated by either opsonized zymosan or phorbol, was used as a marker of O2-dependent antimicrobial activity. Lysozyme content of AM was measured as a component of O2-independent antimicrobial activity. Unstimulated 3-day-old macrophages generated >10-fold more NO2(-) + NO3(-) than did 10-day-old, maternal or adult AM. Twenty hours after LPS and IFN-γ stimulation, 3-day-old AM produced > 2 times more NO2(-) and NO3(-) than did the more mature macrophages. Basal and stimulated O2(.-) release was similar among 3-day-old, 10-day-old and adult AM, while lysozyme concentrations were > 4-fold higher in adult macrophages compared to AM from 3-day-old pups. Rather than having a role in NO(.)-dependent antimicrobial activity, we propose that newborn AM have amplified NO(.) production to modulate their own differentiation and replication after birth. The age-dependent differences in NO(.) synthase expression by AM may lend insight into the regulation of this important enzyme.

Effects of smoking marijuana, tobacco or cocaine alone or in combination on DNA damage in human alveolar macrophages.

This study examined the role of marijuana smoking in the pathogenesis of human lung cancer by measuring DNA damage in alveolar macrophages (AM). The alkaline unwinding method was used to determine DNA single-strand breaks in AM lavaged from non-smokers [NS] and smokers of marijuana [MS], tobacco [TS] or cocaine [CS], either alone or in combination. DNA damage was related to superoxide anion (O2-) production by AM stimulated with phorbol myristate acetate (PMA) and to nitric oxide content of smoke using cellular nitrite (NO2-) concentrations. The percentage of double-stranded DNA present after alkaline unwinding was higher in AM of NS (41 +/- 5% [11]) and CS (41 +/- 4% [9]) versus that of MS (31 +/- 4% [8]), TS (35 +/- 3% [11]), MTS (26 +/- 4% [3]), and CTS (27 +/- 5%* [10]), mean +/- SEM [n], * = p < 0.1 vs. NS). PMA stimulated O2- production by AM from NS and CS was lower than that of other smokers, but the differences were not significant. O2- release, however, had an inverse correlation with DNA single-strand breaks (r = -0.38, p = 0.009). Nitrite content of AM from NS and CS was less than that of other smokers' cells (p < 0.1 for TS & CTS vs. NS), but DNA damage had no relationship to NO2- concentration. We conclude that AM recovered from MS, either alone or in combination with tobacco smoking, show a trend towards DNA damage. Studies utilizing a larger population should verify our findings and further define its relationship to enhanced oxidant production by macrophages.

Pyrrolidine dithiocarbamate inhibits induction of nitric oxide synthase activity in rat alveolar macrophages.

Since nuclear factor kappa B (NF-kappa B) is activated during many inflammatory conditions, we assessed its role in expression of the L-arginine-nitric oxide pathway by rat alveolar macrophages. Pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-kappa B activation, was added to cultured macrophages stimulated with lipopolysaccharide (LPS) and interferon-gamma (IFN gamma). Inducible nitric oxide synthase (iNOS) activity was determined by measuring the stable nitrogen oxide end products of L-arginine oxidation: nitrite (NO2-) and nitrate (NO3-). Ten, 25 and 50 microM PDTC progressively inhibited iNOS activity by macrophages. When 50 microM PDTC was added 2 h before LPS + IFN gamma, L-arginine oxidation by macrophages was inhibited by > 99%; L-arginine oxidation was reduced by 70% if 50 microM PDTC and the stimuli were introduced together; NO2- and NO3- were not decreased significantly if 50 microM PDTC was added 6 h after LPS + IFN gamma. Cycloheximide added along with LPS + IFN gamma totally inhibits iNOS activity, while cycloheximide added 6 h after LPS + IFN gamma did not reduce NO2- and NO3- in tissue culture supernatants. These findings suggest iNOS activity in macrophages treated with LPS + IFN gamma requires NF-kappa B activation.

Cytokine- and Pneumocystis carinii- induced L-arginine oxidation by murine and human pulmonary alveolar macrophages.

Lipopolysaccharide plus interferon gamma stimulated the L-arginine-.NO pathway of murine, but not human pulmonary alveolar macrophages. Pneumocystis carinii induced .NO production by both murine and human pulmonary alveolar macrophages suggesting that the parasite stimulates L-arginine oxidation in these cells. The potential anti-Pneumocystis activity of .NO warrants further study.