Characterization of CD18-Deficient Neutrophil Functions and Some Modulations of Bovine Neutrophils from Cattle with Bovine Leukocyte Adhesion Deficiency

我们进行了上述研究，结果总结如下：从骨髓移植后27个月到死亡，对骨髓移植后白细胞粘附缺陷的小母牛进行了1.6年的临床和病理观察。研究了白细胞粘附缺陷小母牛骨髓移植后血清细胞因子水平及其对中性粒细胞mRNA的表达。小母牛脑移植后临床情况相对稳定。纵向监测持续高Ei -球蛋白血症和血清免疫球蛋白G （IgG）浓度升高。白细胞介素(IL) -1E？感染母牛血清中IL-6的浓度范围为15.8 ~ 321.7 ng/ml，最高浓度出现在IL-6峰值出现的时间。血清IL-6水平在0.32 ~ 27.9 ng/ml之间，似乎与母牛mR中血清IgG的增加有关。与对照组压实的母牛中性粒细胞中IL-6、IL-8、粒细胞和巨噬细胞集落刺激因子（GM-CSF）均升高。受影响的小母牛的持续高ei -球蛋白血症似乎与IL-6及其血清水平的mRNA表达增强有关。研究结果表明，受感染的小母牛的体液免疫被激活，中性粒细胞的产生似乎在E？2整合素介导的吞噬细胞功能。为了评价cd18缺陷牛中性粒细胞的杀伤活性（KA）和抗体依赖细胞毒性（ADCC），比较了正常牛中性粒细胞和牛白细胞粘附缺陷牛中性粒细胞的杀伤活性（KA）和抗体依赖细胞毒性（ADCC）。与正常中性粒细胞相比，缺乏cd18的中性粒细胞KA显著降低（P =< 0.01），而ADCC活性升高。免疫球蛋白G (FcyR-IgG) Fc受体介导的中性粒细胞ADCC在BLAD中性粒细胞中增强。在体内对肾上腺素的反应、中性粒细胞上l -选择素的表达、细胞内钙（[Ca2+]i）的变化、硫脂诱导的超氧化物产生和中性粒细胞中酪氨酸磷酸化被评估，以阐明正常和CD18缺陷牛中性粒细胞中l -选择素相关功能的作用。4头正常犊牛体内给药后5 ~ 20 min外周血中性粒细胞数量显著（P < 0.05）增加，而3头牛白细胞粘附缺陷（BLAD）犊牛外周血中性粒细胞数量无显著增加。3头BLAD犊牛中性粒细胞中l -选择素的表达量为正常犊牛的61 ~ 77%。肉豆蔻酸酯phorbol acetate （PMA）预处理中性粒细胞导致正常犊牛和bld犊牛中性粒细胞中l-选择素的表达显著降低。硫脂诱导的BLAD犊牛中性粒细胞持续期[Ca2+]i浓度显著（P < 0.05）降低；追随者。研究了补体受体3型（CR3）和Fc受体在牛中性粒细胞上表达的免疫球蛋白G （FCR）对超氧化物产生和信号转导的共刺激作用。与CR3 FCR一起刺激的正常中性粒细胞超氧化物的产生远远大于单独刺激CR3或FCR，并且与细胞内钙（[Ca2+]i）浓度的升高和酪氨酸磷酸化100kDa蛋白的量密切相关。没有发现这些受体对cr3缺陷中性粒细胞超氧化物产生的共刺激作用。CR3和FCR对牛中性粒细胞的共同刺激导致超氧化物的产生和信号转导增强，并且似乎与中性粒细胞活性的消耗有关。DEMPO可用于检测胞外空间（包括吞噬体内）的超氧化物，但不能检测胞内空间的超氧化物。p38 MARK抑制剂和PKC抑制剂的作用抑制了中性粒细胞超氧化物的产生。这两种抑制剂都能显著减弱调理酶蛋白诱导的p47 phox磷酸化，而吞噬作用被MAPK抑制剂抑制，而PKC抑制剂不被抑制。结果表明p38 MARK参与了超氧化物产生和吞噬的信号通路。F-actin的形成依赖于PI3-K，不依赖于PKC、ERK和p38MARK以及细胞内钙的增加，而NADPH氧化酶的激活部分依赖于PKC的激活和细胞内钙的增加。少

The above-entitled study was performed and the results were summarized as follows :Clinical and pathobiological findings of the bone marrow (BM) -transplanted heifer with leukocyte adhesion deficiency were evaluated for 1.6 years, from 27 months after BM transplantation until deceased. Serum cytokine levels and their expression of mRNA on neutrophils from a bone marrow (BM) transplanted heifer with leukocyte adhesion deficiency were evaluated. the clinical condition of the affected heifer was relatively stable after BM-transplantation. Persistent hyper Ei - globulinemia and increased serum immunoglobulin G (IgG) concentrations were monitored longitudinally. The concentration of interleukin (IL) -1E? In serum from the affected heifer ranged from 15.8 to 321.7 ng/ml, and maximum concentration occurred at the time which coincided with peak IL-6. Serum Levels of IL-6 ranged from 0.32 to 27.9 ng/ml, and they appeared to be associated with the increment of serum IgG in the affected heifer mR … More NAs for IL-1E?, IL-6, IL-8 and granulocyte and macrophage colony stimulating factor (GM-CSF) were increased in neutrophils from the affected heifer compacted to controls. Persistent hyper Ei-globulinemia of the affected heifer appeared to be associated with enhanced mRNA expression for IL-6 and its serum levels. There findings suggesting that humoral immunity of the affected heifer is activated and the production of neutrophils appears to be enhanced under the incapability of E?2 integrin-mediated functions of phagocytic cells.Killing activity (KA) and antibody-dependent cell cytotoxicity (ADCC) of neutrophils from normal cattle and with bovine leukocyte adhesion deficiency (BLAD) were compared in order to evaluate the cytotoxicity of CD18-deficient bovine neutrophils. The KA of CD18-deficient neutrophils was significantly lowered (P =< 0.01) when compared to normal neutrophils, whereas ADCC activity was increased. Fc receptor for immunoglobulin G (FcyR-IgG) mediated ADCC of neutrophils appeared to be enhanced in BLAD neutrophils.In vivo responsiveness to epinephrine, expression of L-selectin on neutrophils, changes in intracellular calcium ([Ca2+]i), sulfatide-induced superoxide production and tyrosine phosphorylation in neutrophils were evaluated to elucidate the role of L-selectin-associated functions of normal and CD18 deficient bovine neutrophils. The number of neutrophils in peripheral blood was significantly (P < 0.05) increased in 4 normal calves at 5-20 minutes after in vivo administration of epinephrine, however, no significant increase of neutrophils was found in 3 calves with bovine leukocyte adhesion deficiency (BLAD). Expression of L-selectin on neutrophils from 3 calves with BLAD was 61-77 % of that of normal calves. Pretreatment of neutrophils with phorbol myristate acetate (PMA) caused a marked decrease in the expression of L-selectin on neutrophils from both normal- and BLAD-calves. Sulfatide-induced sustained phase of [Ca2+]i concentration in neutrophils from calves with BLAD was significantly (P < 0.05) decreased ; following.Costimulatory effects of complement receptor type 3 (CR3) and Fc receptor for immunoglobulin G (FCR) expressed on bovineneutropfils on superoxide production and signal transduction were evaluated. Superoxide production of normal neutrophils stimulated together with CR3 FCR was much greater than that stimulated with CR3 or FCR alone, and was closely associated with the rise of intracellular calcium ([Ca2+]i) concentration and the amount of tyrosine phosphorylated 100kDa protein. The costimulatory effect of there receptors on superoxide production in CR3-deficient neutrophils was not found. The costimulation of CR3 and FCR on bovine neutrophils leads to enhanced superoxide production and signal transduction, and appears to be ralated to the comsummation of neutrophil activity.DEMPO is useful to detect superoxide of extracellular space including the intraphagosome but not that of intracellular space in opsonized zymosan-stimulated phagocytes. The roles of p38 MARK inhibitor and PKC inhibitor suppressed the superoxide production of neutrophils. Both inhibitors also caused significant attenuation of opsonized zymosan-induced phosphorylation of p47 phox, whereas phagocytosis was found to be inhibited by MAPK inhibitor but not by PKC inhibitor. The results suggest that p38 MARK participates in both signaling pathways of superoxide production and phagocytosis. The formation of F-actin is dependent on PI3-K and independent of PKC, ERK and p38MARK as well as the increase in intracellular calcium, whereas the activation of NADPH oxidase is partly dependent on the activation of PKC and the increase in intracelluar calcium. Less