Nitrated proteins in bronchoalveolar lavage fluid of patients at risk of ventilator-associated bronchopneumonia

Abstract

The study was designed to identify markers of oxidative injury, related to the nitric oxide derived cascade, in bronchoalveolar lavage (BAL) fluid from intensive care patients suspected of ventilator-associated pneumonia (VAP) and/or acute respiratory distress syndrome (ARDS).Thirty-eight patients developing VAP and/or ARDS (VAP/ARDS group) were compared to 20 ventilated patients without VAP/ARDS (control group). Myeloperoxidase (MPO) and elastase, taken as markers of neutrophil activation were measured by enzymatic techniques, and nitrated proteins (NTPs) by an immunological method. The cytotoxicity of the BAL fluid was tested using cultured human epithelial alveolar cells by the release of pre-incorporated 51 Cr.Mean NTP concentration and, MPO and elastase activities were different between the VAP/ARDS and control groups (p<0.05 for NTPs; p<0.005 for MPO; p<0.005 for elastase). NTP concentration correlated with MPO and elastase activity and neutrophil number (r=0.93, 0.91 and 0.87, respectively), but not to protein concentration and arterial oxygen tension/inspiratory oxygen fraction. The cytotoxicity of BAL correlated with NTP concentration (r=0.92) and MPO activity (r=0.89).It was concluded that the concentrations of nitrated proteins in bronchoalveolar lavage fluid correlated with the oxidant activity of neutrophils and that, bronchoalveolar lavage fluid cytotoxicity was correlated with the nitrated protein concentration and may be mediated by oxidants. Eur Respir J 2000; 16: 296±301. In patients with or at risk of acute lung injury or acute respiratory distress syndrome (ARDS), bronchoalveolar lavage (BAL) permits exploration of distal lung regions, which are mainly implicated in the inflammatory reaction and in acute respiratory failure. This bronchoscopy technique allows access to damaged tissues and gives the possibility of early detection of inflammatory mediators that have either accumulated or were produced in the lung. From studies already performed on BAL fluids, it appears that neutrophils constitute the majority of the cell population present in the lungs of ARDS patients, and that products of phagocyte degranulation such as myeloperoxidase (MPO) and elastase (free or complexed to a 1 -proteinase inhibitor) can also be found [1,2].These degranulation products argued in favour of activation of phagocytes in the lungs, involving release of reactive oxygen species, especially products derived from the activities of reduced nicotinamide adenine dinucleotide phoshate-oxidase and MPO: superoxide anion, hydrogen peroxide, and hypochlorous acid. Phagocytes also produce nitric oxide. The simultaneous production of NO . and O 2 . -allows in situ generation of peroxynitrite, a potent oxidative species which reacts quickly by incompletely determined mechanisms [3±5]. In the lung, ONOO -is reported to be responsible for alterations to surfactant of type II alveolar cells [6]. ONOO -is involved in the oxidation of lipoproteins, nucleic acids, proteins and peptides, and in the nitration of tyrosine an...

Bronchoalveolar lavage fluids of patients with lung injury activate the transcription factor nuclear factor-κB in an alveolar cell line

“…We previously demonstrated that an oxidant activity is present in BAL fluid from ventilated patients and that this BAL fluid is cytotoxic to normal alveolar cells in culture [11]. In the present study, we demonstrate that these BAL fluids can induce activation of the transcription factor NF-κB in alveolar cells.…”

“…As described previously [11], the occurrence of nitrated proteins in BAL fluid was quantified using a competitive nitrotyrosine ELISA kit (TCS Cell Works). The lower limit of detection was 0.1 µg\ml nitrated BSA equivalents.…”

“…The local production of peroxynitrite, resulting from the reaction of NO with superoxide anion, has been suggested, and was demonstrated by measuring levels of nitrated proteins in alveoli [9,10]. We recently demonstrated that increased concentrations of nitrated proteins are present in the BAL fluid of ventilated patients, related to the presence of stimulated neutrophils [11].…”

Total Antioxidant Status and Severity of Community-Acquired Pneumonia: Are They Correlated?

“…These results were similar to those of Nowak et al [10] on lipid peroxidation products and blood neutrophils. On the other hand, TAS change was correlated with total WBC, suggesting that other sources of ROS may be involved in peripheral blood oxidant/antioxidant imbalance, except neutrophils, such as monocytes or even lung alveolar macrophages [18, 20]. …”

“…For example, increased ROS production was found in bronchoalveolar lavage fluid from these patients, especially in those that were severely immunocompromised [18]. This oxidative response was mainly due to neutrophil recruitment and activation, as a positive correlation was found with the percentage of neutrophils and release of myeloperoxidase, the main enzyme for their respiratory burst and antibacterial activity [18, 19]. Increased markers of oxidant injury, such as nitrated proteins, have been found in bronchoalveolar lavage fluid in patients with ventilator-associated pneumonia.…”