The defect of biodefense mechanisms in bovine leukocyte adhesion deficiency syndrome (BLAD) as observed from signal transduction pathway

本项目旨在通过正常中性粒细胞和与补体iC3b受体对应的β -整合素CR3基因缺失的牛白细胞粘附缺陷（BLAD）中性粒细胞，阐明NADPH氧化酶激活和吞噬的信号转导机制。为此，使用了各种试剂来抑制NADPH氧化酶相关的信号转导。我们发现，蛋白激酶C （PKC）、磷脂酰肌醇3-激酶（PI 3-激酶）和p38丝裂原活化蛋白激酶（p38 MAPK）的抑制剂可以剂量依赖性地抑制血清活化酶（s-OZ）刺激BLAD和正常犊牛中性粒细胞产生的超氧化物，尽管s-OZ刺激BLAD中性粒细胞产生的超氧化物比s-OZ刺激正常中性粒细胞产生的超氧化物要少。这些结果表明，缺乏β -整合素CR3不会影响NADPH氧化酶的信号转导途径，但会减少NA和DPH氧化酶的超氧化物产生。通过向患病小牛输注cd18阳性粒细胞，可部分恢复BLAD中NADPH氧化酶活性的降低。此外，PI 3-激酶和p38 MAPK（而非PKC）被证明是正常中性粒细胞吞噬活性所必需的。关于NADPH氧化酶激活的细胞内机制，已知NADPH氧化酶的一个组成部分p47phox在其c端区域的丝氨酸上被广泛磷酸化，磷酸化是NADPH氧化酶激活的触发因素。对人慢性肉芽肿病（CGD）的p47phox和p47phox缺陷B细胞进行定点诱变，结果表明，为了激活氧化酶，必须发生丝氨酸303/304、359/370和可能的丝氨酸379的磷酸化。这些结果似乎不仅对理解NADPH氧化酶活性的信号转导机制，而且对通过粒细胞或基因输血治疗BLAD和p47phox缺乏症的CGD具有重要意义。少

This project was performed to clarify the signal transduction mechanisms for NADPH oxidase activation and phagocytotis by using normal neutrophils and those with bovine leukocyte adhesion deficiency (BLAD) which was genetic deficient in beta2-integrin CR3 corresponding to the receptor of complement iC3b. Various reagents to inhibit NADPH oxidase-related signal transduction were used for this purpose. We found that inhibitors of protein kinase C (PKC), phosphatidyl inositol 3-kinase (PI 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 stimulation of BLAD neutrophils with s-OZ brought about the lower generation of superoxide than that of normal neutrophils with s-OZ.These results indicated that the lack of beta2-integrin CR3 did not influence signal transduction pathways of NADPH oxidase but reduced superoxide production of NA … More DPH oxidase. This reduced NADPH oxidase activity in BLAD was partially recovered by transfusion of CD 18-positive granulocytes to diseased calf. Furthennore, PI 3-kinase and p38 MAPK but not PKC were shown to be required for phagocytotic activity in normal neutrophils. Concerning the intracellular mechanisms of NADPH oxidase activation, the p47phox, one component of NADPH oxidase, is known to be extensively phosphorylated at serines that are located among its C-terminal region and the phosphorylation is a trigger for the activation of NADPH oxidase. Using site-directed mutagenesis of p47phox and p47phox-deficient B cells from human chronic granulomatous disease (CGD), it was shown that the phosphorylation of serines 303/304, 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 for therapy of BLAD and p47phox-deficient CGD by the granulocyte or gene transfusion. Less