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Effects of bronchoalveolar lavage (BAL) fluids of patients with ventilator-associated bronchopneumonia (VAP) on alveolar cells in culture

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Lungs of patients with VAP or ARDS are invaded by neutrophils which are activated and degranulate in situ, releasing granulocytic enzymes and reactive oxygen species (ROS) that could be responsible for alterations of neighbouring alveolar cells. When clinical and radiological signs suggesting VAP were encountered, we performed 71 bronchoalveolar lavages (BAL) in 58 ventilated patients (42 men, 16 women with a mean age of 55.59± 16.87 years; an APACHE II score at entry of 15.85± 5.49; a mean intubation duration of 19.93± 15.49 days; a mean ICU stay of 29.45± 23.99 days; and 17 deaths). VAP was confirmed by quantitative bacteriological culture >104 CFU/ml of BAL and non-infectious ARDS was confirmed by classical criteria including PaO2/FiO2 ratio <200. In these BAL fluids, we measuredthe concentrations of nitrated proteins (NTP) as an indicator of oxidative activity (ELISA technique), and the concentrations of active myeloperoxidase and elastase, two markers of phagocyte degranulation (enzymatic measure). The effects of BAL fluid on human alveolar cells (A549) in culture were analyzed by measuring the release of 51Cr pre-incorporated by the cells (cytotoxicity test) and correlated with NTP, myeloperoxidase and elastase values (Pearson's correlation and Mann-Whitney test with P<0.05 being significant). Preliminary studies were also performed to determine the mechanism of cytotoxicity of BAL by analysing the capacity of these fluids to activate the nuclear transcription factor NFκB.

VAP and/or ARDS was diagnosed in 38 patients (VAP/ARDS group: 29 VAP and 9 ARDS), and the 20 others formed the control group. No significant difference was observed between the two groups for clinical parameters. There was no significant difference in the mean protein value of BAL between the two groups (1.49± 0.17 mg/ml in VAP/ARDS versus 1.38± 0.34 in controls). On the contrary, a significant difference was found for the neutrophil count, NTP, myeloperoxidase and elastase values, and for cytotoxicity (IC: cytotoxicity index expressed in %).

The cytotoxicity of BAL was correlated with the values of NTP (r=0.92; P<0.001) and MPO (r=0.88; P<0.001). In 19 BAL, NFκB was found to be activated, and this activation was correlated with the BAL value of IL8 (P<0.005).

From these data, we concluded that MPO and elastase were released in the alveoli by activated neutrophils and that NTP were formed in situ by the oxidant activity of stimulated neutrophils (in situ production of peroxynitrite and/or activity of MPO on nitrite or peroxynitrite). This intra-alveolar oxidant activity led to the production of BAL fluids which were cytotoxic on alveolar cells and which enable the activation of the signal transduction pathway. However, the exact consequences of this NFκB activation and the particular compounds of BAL responsible for cytotoxicity remain to determine.

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Nys, M., Damas, P., Deby, G. et al. Effects of bronchoalveolar lavage (BAL) fluids of patients with ventilator-associated bronchopneumonia (VAP) on alveolar cells in culture. Crit Care 4, P96 (2000). https://doi.org/10.1186/cc816

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Keywords

  • Bronchoalveolar Lavage
  • Peroxynitrite
  • Oxidant Activity
  • Alveolar Cell
  • Nuclear Transcription Factor