| Introduction: Polymorphonuclear leukocytes (PMN) are the major effectors of immunity, which play a key role in microbicidal killing and inflammatory activities. PMN exist in one of three states: quiescent, primed, or active. Of the three, the primed state is a critical component of adult respiratory distress syndrome (ARDS) and multiple organ failure (MOF). Airway inflammation caused by PMN infiltration plays an important role in the initiation and development of asthma. Greater leukocyte reaction is induced by agonist in patient with asthma. These observations suggest that priming of leukocytes may be of great importance in severe asthma. But whether antigen can prime PMN and the priming mechanism have yet to be uncovered. Recently the mechanism of signal transduction in priming has been focused on and the relationship between PMN priming and phospholipase D activation has been concerned. PLD is widely distributed inmammalian cells, where it is regulated by a variety of extracelluar signals. The major substrate of PLD is phosphatidylcholine (PC), which is hydrolyzed to phosphatidic acid and choline. The PLD-catalysed PC hydrolysis is an important mechanism of signal transduction in cells. The effects of priming by GM-CSF and TNF- a on PLD activity were studied and demonstrated that treatment of neutrophils with the priming agent alone did not result in accumulation of phosphatidic acid, however the subsequent fMLP-induced accumulation was augmented and prolonged in primed cells. Recently it was also found that PLD activity significantly increased in leukocytes primed by cardiopulmonary bypass (CPB). All these studies suggest that the activation of PLD may be of vital importance in priming. But the role of PLD in priming remains disputed. AIM: To investigate, (1) whether or not LPS and OA prime rat peripheral leukocytes. (2) The effect of sensitization on the priming. (3) The role of phospholipase D in the priming.Methods: The peripheral leukocytes were separated and purified from sensitized or unsensitized rats. LPS or OA was used as a priming agent. fMLP was used as an activating agent. Degraduation of leukocyte was determined by measurement of elasetase release and MPO activity. PLD activity was assayed by the generation of choline, which was measured by choline-oxidase-catalyzed formation of HaCh and HjC^ was detected by oxidation of 4-aminoantipyrine in the present of horseradish peroxidase. The PLD activity can be quantified by calculation of producted choline according to a standard curve. All data were analyzed by Sigma State using one-way ANOVA, Dunnett's and Spearman Correlation test. Data were expressedas x+s. Result:1) Compared with cells treated by LPS, fMLP or vehicle alone, leukocytes from unsensitized rat challenged with fMLP(l u mol-L"1) for 10 min after incubated with LPS(100ng/ml LPS) for 45 min release more elastase. (P<0.05) .MPO activity of leukocytes challenged with fMLP after incubated with LPS was significantly higher than cells treated by LPS, fMLP or vehicle alone too (PO.05).There was no significant difference between leukocytes treated by LPS and vehicle for elastase release and MPO activity. Compared with vehicle controls, fMLP treated alone did not cause significant release of elastase (P>0.05). However, MPO activity of cells treated by fMLP is significantly higher than vehicle control (PO.05). Both elastase release and MPO activity of leukocytes challenged by fMLP were lower than cells challenged by fMLP after LPS incubation, which suggest that LPS alone can not induce elastase or MPO release, but LPS can amplify the elastase and MPO release induced by fMLP.Leukocytes from unsensitized rat challenged with fMLP(l v mol-L"1) for 10 min after incubation with OA(4 u g/ml) for 45 min released significantly more elastase than cells treated by OA alone(P<0.05). But compared with cells treated by fMLP, which did not cause more elastase release (P>0.05). The similar results were also obtained for MPO activity.OA incubation alone did not cause elastase and MPO...
|
PDF Full Text Request