Neutrophils, a major type of blood leukocytes, are indispensable for host defense of bacterial infections. Directed migration in a gradient of chemotactic stimuli enables these cells to rapidly find the site of infection and destroy the invading pathogens. PMNs can exist in three different activation states, namely, resting, primed, and activated states. Activated neutrophils produce reactive oxygen species (ROS), in what is known as respiratory burst, increase phagocytosis, delay apoptosis, among other functions. These cells also can be activated in vitro by particles, bacterial signal peptides (fMLP) and substances as phorbol- myrystate-acetate (PMA). The PMA is a diacylglicerol (DAG) analog that bypasses membrane receptors and directly activates intracellular protein kinase C (PKC). A comparative proteomic analysis of normal and stimulated neutrophils can lead to the identification of relevant diagnostic markers or potential drug targets.
In this study, human neutrophils were prepared from peripheral venous blood of healthy volunteers collected in syringes with heparin. Neutrophils (greater than 96% purity, greater than 97% viability) were obtained from enriched blood by density centrifugation on percoll gradient. Remaining erythrocytes were lysed with water and the pellet of PMN was resuspended in PBS. 1,85 10⁶ cells were activated with 110ng PMA for 20 minutes in 1 mL of PBS. Finally, cells were pelleted and incubated in lyses buffer for 12hours. Three hundred and fifty micrograms of proteins of resting and PMA activated neutrophils were submitted to 2D eletrophoresis, pH4-7. The gels were analyzed through Image Master Platinum version 5.0 (Amersham Pharmacia Biotech).
Up to this moment, 78,86 % of the spots were matched among the reference neutrophil normal gel and PMA activated. The correlation coeficient was 0,829 between the two gels. 180 spots were regarded as belonging only to normal gel, and 191 spots were exclusive of the PMA activated neutrophil map.