Journal List > Tuberc Respir Dis > v.63(6) > 1001175

Kwon, Hyun, and Lee: Cytosolic Phospholipase A2 Activity in Neutrophilic Oxidative Stress of Platelet-activating Factor-induced Acute Lung Injury

Abstract

Background

The present investigation was performed in rats and isolated human neutrophils in order to confirm the presumptive role of the positive feedback loop of cytosolic phospholipase A2 (cPLA2) activation by platelet-activating factor (PAF).

Methods

The possible formation of the positive feedback loop of the cPLA2 activation and neutrophilic respiratory burst was investigated in vivo and in vitro by measurement of the parameters denoting acute lung injury. In addition, morphological examinations and electron microscopic cytochemistry were performed for the detection of free radicals in the lung.

Results

Five hours after intratracheal instillation of PAF (5µg/rat), the lung leak index, lung myeloperoxidase (MPO) activity, the number of neutrophils and the concentration of cytokine-induced neutrophil chemoattractant (CINC) in bronchoalveolar lavage fluid were increased by PAF as compared with those of control rats. The NBT assay and cytochrome-c reduction assay revealed an increased neutrophilic respiratory burst in isolated human neutrophils following exposure to PAF. Lung and neutrophilic cPLA2 activity were increased following PAF exposure and exposure to hydrogen peroxide increased cPLA2 activity in the lung. Histologically, inflammatory findings of the lung were observed after PAF treatment. Remarkably, as determined by CeCl3 cytochemical electron microscopy, increased production of hydrogen peroxide was identified in the lung after PAF treatment.

Conclusion

PAF mediates acute oxidative lung injury by the activation of cPLA2, which may provoke the generation of free radicals in neutrophils.

Figures and Tables

Figure 1
Histological changes in the lung after instillation of PAF (5µg) into the trachea. Perivascular cuffing (asterik, A), interstitial edema and infiltration of inflammatory cells were found (A, B). Accumulation of inflammatory cells in vascular lumen, interstitium and alveoli were found (arrow head, C, D). ×200.
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Figure 2
Electron microscopic finding of normal lung (A). Lamellar bodies, endothelial cells and type I alveolar cells were well preserved. In CeCl3 electron microscopic cytochemistry (B), deposits of cerrous perhydroxide were not found indicating oxidants were not generated in the normal lung. Bar indicates 2.5µm.
LB: lamellar body; En: endothelial cell; AL: alveolar lumen.
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Figure 3
Ultrastructural changes in the lung of the rat given PAF (5µg) intratracheally. In routine electron microscopic finding (A), the infiltration of neutrophils in interstitium (NP), degeneration of lamellar bodies (arrow head), vacuolization of lamellar bodies (asterik) were noted. NP: neutrophil. Bar indicates 2.0µm. In CeCl3 cytochemical electron microscopy, deposits of cerrous perhydroxide granules were indentified in the interstitium and to the proximity of neutrophils (B). NP: neutrophil; Bar indicates 1.0µm.
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Table 1
Comparisons of lung leak index, lung MPO, BAL PMNLs, CINC concentration between sham and PAF-treated rats
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Values are given as mean±S.E.

n indicates number of experiments.

MPO: myeloperoxidase; BAL PMNL: bronchoalveolar lavage polymorphonuclear leukocyte; CINC: cytokine induced neutrophil chemoatractant; PAF: platelet-activating factor; LLI: lung leak index.

*p<0.001, sham vs. PAF.

Table 2
Comparison of indeces of neutrophilic oxidative stress between sham and PAF-treated neutrophils
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Values are given as mean±S.E.

n indicates number of experiments.

NBT assay was carried out with collected neutrophil from BALF of rats and cytochrome-c reduction assay was performed with isolated human neutrophils.

PAF: platelet-activating factor.

*p<0.001, sham vs. PAF.

Table 3
Stimulating effects of PAF on the cPLA2 activity in the lung and isolated human neutrophils
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Values are given as mean±S.E.

n indicates number of experiments.

PAF: platelet-activating factor; cPLA2: cytosolic phospholipase A2.

*p<0.001, sham vs. PAF.

Table 4
Effect of hydrogen peroxide on the lung cPLA2 activity
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Values are given as mean±S.E.

n indicates number of experiments.

PAF: platelet-activating factor; cPLA2: cytosolic phospholipase A2.

*p<0.001, sham vs. PAF.

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