| Research Abstract |
This project was performed to clarify the signal transduction mechanisms for NADPH oxidase activation and phagocytotis by using normal neutrophil, human clonic granulomaous disease (CGD) and bovine leukocyte adhesion deficiency (BLAD) which were genetic deficient in p47phox and beta2-integrin CR3 corresponding to the receptor of complement iC3b, respectively The various reagents to inhibit NADPH oxidase-related signal transduction were used for this purpose. We found that inhibitors for protein kinase C (PKC), phosphatidyl inositol 3 kinase (P1 3-kinase) and p38 mitogen-activated protein kinase (p38 MAPK) were dose-dependently inhibited superoxide generation from serum-opsonized zymosan (s-OZ)-stimulated neutrophils from BLAD and normal calves, although the stimulation of BLAD neutrophils with s-OZ brought about lower generation of superoxide than that of normal neutrophil. These results indicated that the lack of beta2-integrin CR3 did not influence signal transduction pathways of NAD
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PH oxidase but reduced superoxide production from NADPH oxidase. This reduced NADPH oxidase activity in BLAD was partially recovered by transfusion of CD 18-positive granulocytes to disease animal. Furthermore, P1 3-kinase and p38 MAPK but not PKC are shown to be required for phagocytotic activity in normal neutrophil. Concerning the intracellular mechanisms of NADPH oxidase activation, the p47phox, one component of NADPH oxidase. is known to be phosphorylated extensively on serines that are located among its C-terminal amino acids and this phosphorylation is a trigger for the activation of NADPH oxidase. By using site-directed mutagenesis of p47phox and p47phox-deficien B cells from human clonic granulomaous disease (COD), it was showed that the phosphorylation of serines 3031304, 359(370 and possibly serine 379 must take place in order to activate the oxidase. These results seem to be important in not only understanding the signal transduction mechanism of NADPH oxidase activity but also development of therapy for BLAD and p47phox-deficien COD such as granulocyte transfusion and gene therapy. Less
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