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

进行了上述标题的研究，结果总结如下：从骨髓（BM）移植后27个月至死亡，对患有白细胞粘附缺陷的骨髓（BM）移植小母牛的临床和病理学结果进行了1.6年的评价。对一例白细胞粘附缺陷的骨髓移植母牛血清细胞因子水平及其在中性粒细胞上的mRNA表达进行了研究。受影响的小母牛的临床状况在BM移植后相对稳定。纵向监测持续性高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？在受影响的小母牛的血清中，范围为15.8至321.7 ng/ml，最大浓度出现在与IL-6峰值相一致的时间。血清IL-6水平在0.32 ~ 27.9ng/ml之间，并与血清IgG水平的升高有关 ...更多信息 IL-1 E？的NA，IL-6，IL-8和粒细胞和巨噬细胞集落刺激因子（GM-CSF）增加的中性粒细胞从受影响的小母牛压缩到对照组。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表达增强相关。这些结果表明，受影响的母牛的体液免疫被激活，中性粒细胞的产生似乎是在E？2整合素介导的吞噬细胞功能比较了正常牛和牛白细胞粘附缺陷症（BLAD）牛中性粒细胞的杀伤活性（KA）和抗体依赖性细胞毒性（ADCC），以评价CD 18缺陷牛中性粒细胞的细胞毒性。与正常中性粒细胞相比，CD 18缺陷型中性粒细胞的KA显著降低（P =< 0.01），而ADCC活性增加。本研究通过检测牛BLAD中性粒细胞对肾上腺素的反应性、L-选择素的表达、细胞内钙离子浓度的变化、硫代硫酸钠诱导的超氧阴离子产生和酪氨酸磷酸化，探讨L-选择素相关功能在牛BLAD中性粒细胞中的作用。4头正常犊牛体内注射肾上腺素后5-20分钟外周血中性粒细胞数显著增加（P < 0.05），而3头牛白细胞粘附缺陷症（BLAD）犊牛外周血中性粒细胞数无显著增加。3头BLAD犊牛中性粒细胞L-选择素表达量为正常犊牛的61- 77%。用佛波醇肉豆蔻酸酯醋酸酯（PMA）预处理中性粒细胞引起正常和BLAD小牛中性粒细胞上L-选择素表达的显着下降。结果表明，牛中性粒细胞表达的补体受体3（CR 3）和免疫球蛋白G受体（FCR）对超氧化物歧化酶（SOD）的产生和信号转导具有协同刺激作用。与CR 3 FCR共同刺激的正常中性粒细胞产生的超氧化物水平明显高于单独刺激，并与细胞内钙离子浓度和酪氨酸磷酸化100 kDa蛋白量的升高密切相关。CR 3和FCR对牛中性粒细胞产生超氧阴离子的共刺激作用不明显，CR 3和FCR对牛中性粒细胞产生超氧阴离子的共刺激作用与中性粒细胞活性的消耗有关，DEMPO可用于检测细胞外（包括吞噬体内）的超氧阴离子，而不能检测细胞内的超氧阴离子。p38 MARK抑制剂和PKC抑制剂的作用是抑制中性粒细胞产生超氧化物。这两种抑制剂也造成了显着衰减的调理酵母多糖诱导的磷酸化p47 phox，而吞噬作用被发现抑制MAPK抑制剂，但不是PKC抑制剂。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PI 3-K，而不依赖于PKC、ERK和p38 MARK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