中性粒细胞与哮喘非特异性炎症

已知多种细胞参与哮喘非特异性炎症的发生，如嗜酸性粒细胞、肥大细胞、巨噬细胞、辅助性Ｔ淋巴细胞中性粒细胞等，其中中性粒细胞的作用是近年来研究较多的一个课题。目前已证明中性粒细胞在哮喘非特异性炎症局部有明显浸润，并通过释放氧代射产物、蛋白酶以及阳离子物质造成局部组织损伤，还可产生多种炎症性介质来影响气道反应性，同时可加速哮喘非特异性炎症的发生和发展。     

中性粒细胞性支气管哮喘小鼠模型建立的探索

目的利用不同剂量脂多糖（lipopolysaccharides,LPS）干预卵清蛋白（ovalbumin,OVA）致敏和激发的小鼠,探索中性粒细胞性哮喘（neutrophilic asthma,NA）小鼠模型的建立。方法 80只BALB/c小鼠随机分为正常对照组（A组）、肺损伤对照组（B组：B1～B4）、嗜酸细胞性哮喘模型对照组（C组）、NA模型探索组（D组：D1～D4）。分致敏和激发两阶段建模,致敏阶段：A组PBS腹腔注射及PBS滴鼻,B组PBS腹腔注射及LPS滴鼻,C组OVA腹腔注射,D组OVA腹腔注射及LPS滴鼻;激发阶段：A、B组生理盐水雾化,C、D组5%OVA雾化。通过观察小鼠雾化时的症状、肺组织病理改变,检测支气管肺泡灌洗液（bronchoalveolar lavage fluid,BALF）细胞总数及分类计数和血清OVA-sIgE等评价NA小鼠模型建立。结果①D3组小鼠出现类似C组小鼠呼吸急促、大小便失禁等表现。②D3组BALF炎症细胞总数较A组显著升高（P〈0.01）;D3组中性粒细胞（neutrophil,NEU）百分比（NEU%）较A、C、D1、D2组显著升高（P值均〈0.01）,嗜酸粒细胞（eosinophil,EOS）百分比（EOS%）较A组显著升高而较C组显著低下（P值均〈0.01）。③D3组支气管管壁及肺泡间隔增厚变形,部分断裂,气道黏膜下除了EOS浸润外还存在明显NEU浸润。④D3组血清OVA-sIgE水平显著高于A组而低于C组（P值均〈0.05）。⑤D3组IL-4水平显著高于A组（P〈0.05）,与C组无显著差异（P〉0.05）;D3组IFN-γ水平显著低于A组（P〈0.05）,与C组无显著差异（P〉0.05）。结论 10μg LPS滴鼻吸入＋50μg OVA腹腔注射三次与5%OVA连续激发两周即D3组可成功建立NA小鼠模型。     

糖皮质激素对支气管哮喘患者痰嗜酸粒细胞和中性粒细胞凋亡的影响

为观察吸入糖皮质激素对支气管哮喘(简称哮喘)患者诱导痰中嗜酸粒细胞(EOS)和中性粒细胞凋亡的影响,我们对哮喘患者经糖皮质激素治疗前、后诱导痰中EOS和中性粒细胞凋亡进行对比研究。对象与方法　2 0例来自本院2 0 0 2年11月～2 0 0 3年3月门诊哮喘患者,男11例,女9例,平均年龄( 4 1±11)岁,符合1997年中华医学会呼吸病学分会哮喘学组的诊断标准[1] ,均为急性发作期(轻、中度) ,2 0例患者吸入糖皮质激素前为A组,给予吸入丙酸氟替卡松(商品名:辅舒酮,葛兰素史克公司) 2 5 0 μg ,每天2次,吸入4周后,视为B组,期间给以硫酸沙丁胺醇(商品名:…     

中性粒细胞性哮喘的危险因素研究进展

近些年来,中性粒细胞在支气管哮喘（简称哮喘）气道炎症中的作用已经引起了广泛关注,研究引发中性粒细胞哮喘的危险因素也取得了很大的成就。除了感染,长期使用糖皮质激素以外,肥胖、香烟烟雾、职业、空气污染等多种因素均可诱发中性粒细胞性哮喘。     

豚鼠中性粒细胞性哮喘模型的建立

目的建立豚鼠中性粒细胞性哮喘模型。方法 40只成年雄性豚鼠按随机数字表法分为对照组（A组）、嗜酸粒细胞性哮喘组（B组）、中性粒细胞性哮喘组（C组）、嗜酸粒细胞性哮喘治疗组（D组）和中性粒细胞性哮喘治疗组（E组）。用卵白蛋白（ovalbumin,OVA）和弗氏完全佐剂（Freund’s complete adjuvant,FCA）联合致敏或OVA单独致敏豚鼠后用OVA雾化吸入激发建立中性粒细胞性哮喘或嗜酸粒细胞性哮喘模型,治疗组在激发前腹腔注射地塞米松。观察各组豚鼠雾化激发后体征变化及支气管肺组织病理改变,并比较各组豚鼠气道阻力、血中白细胞分类计数、支气管肺泡灌洗液（brochial alveolar lavage fiuid,BALF）细胞总数及分类计数。结果 B、C组豚鼠激发后均出现典型哮喘症状,不同浓度乙酰甲胆碱激发后的气道阻力与A组相比均显著增高（P〈0.05）;B组豚鼠BALF总数、BALF及血中嗜酸粒细胞所占比例与A组比较显著增加（P〈0.05）;C组豚鼠BALF总数、BALF及血中中性粒细胞所占比例与A组比较均显著增加（P〈0.05）;C组豚鼠BALF中性粒细胞所占比例与B组相比显著增加（P〈0.05）;除B、D组血中性粒细胞外,D和E两组的上述其他各项指标分别与B组和C组相比明显降低（P〈0.05）,以上差异均有统计学意义。B、C两组豚鼠支气管肺组织病理均提示支气管管腔狭窄、黏膜上皮脱落、炎症细胞浸润等典型的哮喘病理学改变,其中B组以嗜酸粒细胞浸润为主,C组以中性粒细胞浸润为主,D、E组较之明显好转。结论本实验建立的豚鼠中性粒细胞性哮喘模型是成功的。     

中性粒细胞性支气管哮喘小鼠模型的建立及其气道高反应规律的研究

目的 通过建立中性粒细胞性哮喘(neutrophilic asthma,NA)模型,检测NA、嗜酸粒细胞性哮喘(eosinophilic asthma,EA)小鼠模型气道阻力、气道高反应的变化,探讨不同气道炎症状态下哮喘小鼠气道高反应性(airway hyperreactivity,AHR)的特点.方法 BALB/c小鼠,随机分成NA组、EA组、正常组(NS组).NA组给予卵蛋白、脂多糖气道滴入致敏,EA组给予卵蛋白腹腔注射致敏,两组均于第21天起给予卵蛋白雾化激发.NS组用生理盐水致敏与激发,方法同NA组.采用Buxco小鼠肺功能仪于雾化第1、7、14天检测气道阻力的变化.肺组织HE染色及PAS染色,观察肺组织病理改变及杯状细胞增生变化.收集支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALF),进行细胞总数及分类计数.结果 ①3.125～50 mg/ml乙酰甲胆碱(methacholine,Mch)NA组、EA组激发第1天AHR高于NS组;12.5～50 mg/ml Mch NA组AHR高于EA组(P值均＜0.05).②50 mg/ml Mch NA组、EA组激发第1天AHR高于激发第7、第14天(P＜0.05).③激发第1天NA组、EA组BALF细胞总数及分类计数与NS组无差异(P＞0.05);激发第7、第14天NA组、EA组BALF中细胞总数较NS组、激发第1天升高(P＜0.05).NA组BALF中中性粒细胞(NEU)%高于EA组、NS组和激发第1天,嗜酸粒细胞(EOS)%高于NS组、激发第1天而低于EA组(P值均＜0.05).EA组BALF中EOS%、NEU%均高于NS组和激发第1天(P值均＜0.05).④激发第7、14天NA组、EA组均可见肺泡间隔炎症细胞浸润;NA组以NEU浸润为主,EA组EOS浸润明显.⑤激发第1天NA组、EA组杯状细胞%较NS组无差异(P＞0.05);激发第7、14天NA组、EA组较NS组及激发第1天升高(P＜0.05).⑥NA组、EA组激发第1天AHR与气道炎症无相关;而持续激发NA组AHR与细胞总数、NEU %、杯状细胞 %呈正相关;EA组AHR与细胞总数、EOS%、杯状细胞%呈正相关.结论 LPS联合OVA气道致敏可成功建立NA小鼠模型,NA小鼠较EA小鼠具有更严重的气道高反应,持续变应原激发可降低NA、EA小鼠气道高反应,NA、EA小鼠早期AHR与气道炎症无相关,而晚期AHR与气道炎症呈正相关.     

中性粒细胞与白细胞介素8在哮喘发病中的作用

目的探讨中性粒细胞与白细胞介素8（IL-8）在支气管哮喘(简称哮喘)发病中的作用。方法对轻度哮喘患者行气道激发试验,对哮喘患者中性粒细胞超氧阴离子（O(*)/(2)）产生、血浆及诱导痰IL-8、丙二醛(MDA)水平进行测定分析。结果中、重度哮喘患者每106个周围血中性粒细胞O(*)/(2)产生水平为［（20.9±5.1）nmol/L］,与轻度哮喘患者［(15.2±4.2)nmol/L］比较,差异有显著性（P<0.01）,轻度哮喘组与正常人［(11.3±2.4)nmol/L］比较,差异有显著性(P<0.05);周围血中性粒细胞O(*)/(2)产生与气道反应性指标一秒钟用力呼气容积下降20%时所需的累积量(PD20FEV1)呈显著负相关(r=-0.693,P<0.05);急性发作期哮喘患者血浆及诱导痰IL-8水平［(585±75)ng/L、(791±103)ng/L］、MDA水平［(6.3±1.6)mmol/L、(21.8±6.3)mmol/L］,与缓解期哮喘患者血浆及诱导痰IL-8水平［(227±54)ng/L］、［(322±95)ng/L］、MDA水平［(5.4±1.0)mmol/L］、［(15.1±5.6)mmol/L］比较,差异有显著性（P<0.01）;缓解期哮喘患者与正常人血浆及诱导痰IL-8水平［(188±46)ng/L］、［(224±51)ng/L］及MDA水平［(4.1±0.4)mmol/L］、［(9.5±4.2)mmol/L］比较,差异有显著性（P<0.05）,诱导痰MDA水平与PD20FEV1呈显著负相关(r=-0.708,P<0.01);诱导痰IL-8水平与诱导痰中性粒细胞百分数呈显著正相关（r=0.838,P<0.01）。结论中性粒细胞产生的氧自由基可能参与哮喘气道炎症及气道高反应的形成。     

嗜酸性粒细胞与支气管哮喘

在１９９５年世界卫生组织（ＷＨＯ）和美国国立心肺血液研究所发表的“全球哮喘防治的创议”中将哮喘定义为一种“气道的慢性炎症性疾病”，并指出这种气道炎症是由嗜酸性粒细胞（ＥＯＳ）等炎性细胞参与形成的非特异性炎症。人们很早就发现哮喘患者体内有明显的ＥＯＳ增...     

模拟临床的中性粒细胞性激素抵抗型支气管哮喘小鼠模型的建立及意义

目的建立一种模拟临床的中性粒细胞性激素抵抗型支气管哮喘(哮喘)小鼠模型并探讨其意义。方法采用屋尘螨(house dust mite,HDM)和脂多糖(lipopolysaccharide,LPS)混合液,气管内给药建立哮喘小鼠模型。将18只雌性C57BL/6小鼠按照数字表法随机分为对照组、哮喘组(HDM+LPS)和地塞米松(dexamethasone,Dex)组(HDM+LPS+Dex)。肺功能仪测定小鼠气道阻力,HE染色观察肺组织炎症细胞浸润,过碘酸-雪夫染色(PAS)观察杯状细胞增生,瑞氏染色检测BALF炎症细胞总数及分类计数,聚合酶链式反应(PCR)和酶联免疫吸附试验(ELISA)检测肺组织和BALF炎症因子。流式细胞术检测肺组织Th17细胞分化。结果HE染色显示,哮喘组肺组织炎症较对照组显著增高(P<0.05),地塞米松组肺组织炎症较哮喘组稍减轻(P>0.05);BALF细胞分类计数显示,哮喘组炎症细胞(除外嗜酸粒细胞)浸润较对照组显著增加[炎症细胞总数分别为(2797±400)×106/L和(105±75)×106/L,中性粒细胞计数分别为(1151±395)×106/L和(12±6)×106/L,淋巴细胞计数分别为(897±135)×106/L和(11±5)×106/L,巨噬细胞计数分别为(215±51)×106/L和(34±16)×106/L,均P<0.05],地塞米松组肺组织炎症细胞总数及巨噬细胞计数较哮喘组显著下降[炎症细胞总数(1140±418)×106/L vs(2797±400)×106/L,巨噬细胞计数(117±31)×106/L vs(215±51)×106/L,均P<0.05],但淋巴细胞和中性粒细胞计数无统计学意义[淋巴细胞计数(587±208)×106/L vs(897±135)×106/L,中性粒细胞计数(294±134)×106/L vs(1151±395)×106/L,均P>0.05];免疫组织化学检测结果同样证实,地塞米松组肺组织中性粒细胞浸润较哮喘组不能被有效抑制;哮喘组和地塞米松组气道阻力较对照组均显著增高(P<0.05),但两组间气道阻力差异无统计学意义(P>0.05);与哮喘组相比,地塞米松组肺组织Th2炎症指标显著降低(均P<0.05),而Th17细胞炎症指标有升高趋势,Th1炎症指标无显著改善;流式细胞术显示,地塞米松组肺组织Th17细胞浸润较哮喘组显著增多[分别为(5.8±1.9)%和(2.3±0.8)%,P<0.01]。结论成功建立了一种模拟临床的中性粒细胞性哮喘小鼠模型。地塞米松非但不能抑制小鼠中性粒细胞气道炎症和气道高反应性,而且促进Th17细胞分化,印证该哮喘模型存在激素抵抗。     

Modulation of polymorphonuclear leukocyte function by cetiedil

Cetiedil citrate monohydrate inhibits sickling of red cells and aggregation of platelets. We assessed its ability to attenuate polymorphonuclear leukocyte (PMN) function. PMN aggregation in response to 2 X 10(-7) M formyl-met-leu-phe (FMLP) was inhibited in a dose- dependent fashion by cetiedil concentrations ranging from 60 to 250 microM. Additionally, 125 microM cetiedil inhibited PMN aggregation in response to 2 X 10(-7) M FMLP, 20 ng/ml phorbol myristate acetate (PMA), and 1 X 10(-6) M A23187 by 69% +/- 18%, 72% +/- 20%, and 65% +/- 4%, respectively. Inhibition of FMLP-induced aggregation was provided by only 5 min of incubation of the drug with the cells and was partially reversible. Cell viability was unaffected by exposure of PMN to the drug. Correspondingly, 125 microM cetiedil prevented the translocation of calcium from the PMN membrane as assessed by chlorotetracycline fluorescence. Paralleling the effect of the drug on PMN aggregation, 125 microM cetiedil inhibited release of superoxide by 55% and decreased the number of available 3H-FMLP receptors. However, its effect on release of the primary granule constituent, myeloperoxidase, was minimal (4.5% inhibition), while the effect on release of the specific granule product, lactoferrin (27% inhibition), was modest. These studies indicate that cetiedil affects PMN aggregation and superoxide release to a much greater extent than PMN degranulation. Thus, cetiedil may have potential uses in modulating inflammatory response in vivo.     

Defective polymorphonuclear leukocyte metabolism and function in canine cyclic neutropenia.

Humans and grey collie dogs with cyclic neutropenia are known to suffer from an increased rate of bacterial infection. Because of the previously described microanatomic abnormalities of lysosome formation found in the polymorphonuclear leukocytes (PMNs) of dogs with canine cyclic neutropenia, studies of these cells were undertaken. PMNs from grey collie dogs were found to have significant metabolic and functional abnormalities when compared with normal collie PMNs. These included abnormally increased postphagocytic C1-glucose oxidation, decreased iodination of trichloroacetic acid-precipitable protein in the resting and phagocytizing state, decreased levels of intracellular myeloperoxidase,and a bactericidal defect against a variety of bacteria. Phagocytosis was normal. These abnormalities appear to differ from those previously described in the PMNs of patients with chronic granulomatous disease of childhood and the Chediak-Higashi syndrome and more closely resemble those seen in hereditary myeloperoxidase deficiency. Thus, the studies reported here demonstrate defective PMN function in a disease state previously believed to be a model only of periodic hematopoiesis.     

Increased polymorphonuclear leukocyte respiratory burst function in type 2 diabetes

The predisposition to infection and chronic inflammation in diabetes may in part be related to the effects of hyperglycemia or other metabolic abnormality on polymorphonuclear leukocytes (PMN). We evaluated oxidative respiratory burst activity (superoxide production) in non-stimulated and stimulated PMN from 70 stable type 2 Hispanic diabetic patients, as compared to 70 healthy Hispanic individuals without diabetes. The influences of protein kinase C (PKC) inhibitors and certain antibiotics on superoxide production were examined. Both resting and stimulated (PMA, zymosan) PMN from diabetic individuals produced more superoxide than PMN from controls. Inhibitors of PKC, a possible mediator of the augmented respiratory burst activity, decreased superoxide production in all (resting and stimulated) diabetic and control PMN. Azithromycin, which is markedly concentrated by PMN, profoundly inhibited superoxide generation in all groups of diabetic and control cells. PMN from Hispanic diabetic patients produced greater quantities of superoxide than non-diabetic controls. This increased oxidative respiratory burst activity may predispose to infection and chronic inflammation in diabetes. PKC inhibitors and azithromycin inhibited this respiratory burst response. The possible role of PKC (especially PKC beta) as the mediator of this augmented respiratory burst response requires further evaluation, and may lead to therapeutic studies with appropriate inhibitors.     

Platelet-dependent modulation of polymorphonuclear leukocyte chemiluminescence

Human blood platelets decreased luminol-enhanced chemiluminescence of human polymorphonuclear leukocytes (PMNL) stimulated with FMLP or Ca2+-ionophore A23187 by 56 or 47%, respectively. Horseradish peroxidase potentiated the decreasing effect of platelets on A23187-stimulated PMNL (92% inhibition) or reversed inhibition of FMLP-induced chemiluminescence to 94% potentiation, indicating dependence of platelet activity on availability of extracellular peroxidase. Moreover, platelet activity may depend also on the extent of platelet activation, as non-activated platelets (in the presence of FMLP) were found to potentiate PMNL-generated chemiluminescence, while platelets activated with A23187 displayed the opposite effect. Interference of platelets with formation and liberation of superoxide anion was indicated by platelet-modified isoluminol chemiluminescence. Superoxide dismutase with catalase and sodium azide were used, respectively, to differentiate the intracellular and the extracellular part of the chemiluminescence signal. Platelets were found to be capable of modifying both components of chemiluminescence, i.e., oxygen metabolites produced on the plasma membrane as well as on membranes of intracellular granules.     

Platelet-dependent modulation of polymorphonuclear leukocyte chemiluminescence

Human blood platelets decreased luminol-enhanced chemiluminescence of human polymorphonuclear leukocytes (PMNL) stimulated with FMLP or Ca2+-ionophore A23187 by 56 or 47%, respectively. Horseradish peroxidase potentiated the decreasing effect of platelets on A23187-stimulated PMNL (92% inhibition) or reversed inhibition of FMLP-induced chemiluminescence to 94% potentiation, indicating dependence of platelet activity on availability of extracellular peroxidase. Moreover, platelet activity may depend also on the extent of platelet activation, as non-activated platelets (in the presence of FMLP) were found to potentiate PMNL-generated chemiluminescence, while platelets activated with A23187 displayed the opposite effect. Interference of platelets with formation and liberation of superoxide anion was indicated by platelet-modified isoluminol chemiluminescence. Superoxide dismutase with catalase and sodium azide were used, respectively, to differentiate the intracellular and the extracellular part of the chemiluminescence signal. Platelets were found to be capable of modifying both components of chemiluminescence, i.e., oxygen metabolites produced on the plasma membrane as well as on membranes of intracellular granules.     

Simvastatin treatment modifies polymorphonuclear leukocyte function in high-risk individuals: a longitudinal study

BACKGROUND: Although extensive experimental evidence supports a primary role of polymorphonuclear leukocytes (PMNs) in atherosclerosis, few data exist concerning the functional properties of these cells and their pharmacological modulation in high-risk individuals. OBJECTIVE: The production of the proinflammatory chemokine interleukin-8 (IL-8), migration and chemotaxis, and reactive oxygen species (ROS) generation were investigated in a longitudinal study in PMNs obtained from high-risk individuals during statin treatment. As a secondary endpoint we compared PMN function of high-risk patients with that of controls. METHODS AND RESULTS: PMNs were isolated from 21 high-risk individuals before treatment and 3 and 30 days after the beginning of simvastatin treatment, and from healthy controls. During treatment a significant reduction was observed both in resting (P = 0.009) and N-formyl-Met-Leu-Phe (fMLP)-stimulated (P = 0.008) IL-8 production, and in the chemotactic index (P = 0.038), whereas ROS generation did not significantly change. In comparison with cells from controls, PMNs obtained from patients before starting simvastatin treatment showed higher resting and fMLP-stimulated IL-8 release (P = 0.007 and P = 0.002, respectively) and ROS generation (resting, P = 0.009; and fMLP-stimulated, P = 0.046), whereas migration and the chemotactic index did not significantly differ. CONCLUSIONS: An activation of neutrophils is present in high-risk individuals, shown by the enhanced production of IL-8, and increased ROS generation. The 4-week statin treatment is able to reduce the cell capability to produce IL-8, and to decrease chemotaxis, thus affecting the proinflammatory properties of PMNs.     

Enhancement of polymorphonuclear leukocyte function against gram-positive aerobic organisms grown in the presence of lincomycin

Summary The effect of pre-incubatingStaphylococcus aureus, Streptococcus pneumoniae andStreptococcus pyogenes with subinhibitory concentrations of lincomycin was studied with respect to polymorphonuclear leukocyte function against these organisms. Culturing the above organisms in the presence of lincomycin (1/4 MIC) resulted in a significant enhancement of polymorphonuclear leukocyte chemotaxis, phagocytosis and bactericidal activity against these organisms.

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Verstärkung der Funktion polymorphkerniger Leukozyten gegen in Anwesenheit von Lincomycin kultivierte grampositive aerobe Keime

Zusammenfassung Staphylococcus aureus, Streptococcus pneumoniae undStreptococcus pyogenes wurden mit subinhibitorischen Konzentrationen von Lincomycin präinkubiert, und es wurde untersucht, welche Veränderungen sich daraus für die Funktion polymorphkerniger Leukozyten gegenüber diesen Erregern ergeben. Wenn die genannten Keime in Gegenwart von Lincomycin (bei 1/4 MHK) kultiviert wurden, nahmen Chemotaxis, Phagozytose und bakterizide Aktivität polymorphkerniger Leukozyten gegen diese Keime signifikant zu.

     

Effect of high-dose methylprednisolone infusion on polymorphonuclear leukocyte function in patients with systemic lupus erythematosus

We have studied the effect of high-dose (1 gm) methylprednisolone infusion on polymorphonuclear leukocyte (PMN) function in 11 patients with active systemic lupus erythematosus (SLE). The only alteration of polymorphonuclear leukocyte function produced consistently by methylprednisolone was decreased adherence to plastic surfaces when tested 2 hours after infusion. This steroid-induced abnormality, however, was transient. Cells obtained from patients 24 hours after a single dose of drug exhibited normal adhesiveness. These results indicate that single, large doses of methylprednisolone do not produce long-lasting abnormalities of PMN function in patients with lupus.