肝癌细胞冻融抗原负载树突状细胞瘤苗的制备与活化

目的: 探讨制备肝癌树突状细胞(DCs)瘤苗和体外诱导DCs活化的优化方法。方法: 取健康人新鲜血50mL, Ficoll密度梯度离心分离外周血单个核细胞(PBMC), 制备肝癌细胞冻融抗原, 采用不同因子组合培养PBMC, 诱导和激活DCs, A组: rhGM-CSF+IL-4; B组: rhGM-CSF+IL-4+冻融抗原负载;C组:rhGM-CSF+IL-4+TNF-α; D组: rhGM-CSF+IL-4+TNF-α+冻融抗原负载。结果: 各组均诱导出DCs, 并高表达CD11c和CD54, 冻融抗原可明显上调CD86、CD80、HLA-DR表达和IL-12 p40分泌(P<0.01), TNF-α促进CD86表达的作用更显著, 但对IL-12分泌无影响, 依次用TNF-α诱导和冻融抗原负载可进一步促进CD86表达和IL12 p40分泌(P<0.05)。CD54和HLA-DR双标记免疫细胞化学染色显示, 各组DCs大多为CD54⁺。表达HLA-DR的DCs均为CD54⁺HLA-DR⁺, 其中, A组CD54⁺HLA-DR⁺细胞数量最少, D组最多, 平均每个细胞HLA-DR的表达水平也与此对应。结论: 采用rhGM-CSF、IL-4诱导的未成熟DCs, 依次使用TNF-α刺激与肝癌细胞冻融抗原修饰可有效促进DCs的活化与成熟。     

肾病综合征患儿PMN凋亡与细胞因子、粘附分子关系的探讨

目的:观察肾病综合征（NS）患儿周围血中性粒细胞（PMN）凋亡的变化，并检测周围血中细胞因子IL-8、IL-6、TNF-α、NO和粘附分子P-选择素(P-sel)、细胞间粘附分子-1（ICAM-1）的水平，探讨细胞因子和粘附分子对PMN凋亡的影响。 方法:采用流式细胞术检测28例NS病人周围血PMN凋亡，ELISA法检测细胞因子和粘附分子水平。 结果:活动期NS患者PMN凋亡率明显低于健康人对照组和缓解期NS患者,缓解期NS患者PMN凋亡率与对照组无明显差别,不同病情活动期NS患者之间PMN凋亡有显著差异,活动期NS患者周围血中IL-8、IL-6、TNF-α、NO、P-sel 、ICAM-1水平均高于对照组和缓解组，且与PMN凋亡呈负相关，与病情呈正相关。缓解组患者IL-8 、IL-6、TNF-α、NO、P-sel和ICAM-1水平与对照组无显著差异。 结论: NS患者PMN凋亡延迟，且与病情及疗效密切相关。炎性细胞因子产生过多、免疫细胞粘附分子表达上调可能是导致PMN凋亡延迟的重要机制，适度调控PMN凋亡有可能会改善NS预后。     

肺炎大鼠心肌TNF-α、IL-6mRNA表达及腺苷对其影响

目的:观察金葡菌感染大鼠肺炎时心肌组织TNF-α、IL-6mRNA表达, 以及腺苷对其影响。方法:50只SD大鼠被随机分为5组, 为对照组、肺炎组和腺苷治疗A、B、C组。金葡菌经气管插管注入复制肺炎大鼠模型, 于注菌后第2、3、4d静脉点滴腺苷治疗, 每天90min, 剂量分别为50、100、150μg·kg^-1·min^-1。第5d处死大鼠, 立即取心脏液氮保存, 心肌组织用于病理检测和采用RT-PCR方法检测TNF-α、IL-6mRNA的表达。结果:(1)肺炎组心肌中TNF-α、IL-6mRNA的表达显著高于对照组(均P<0.01);(2)腺苷治疗B、C组心肌中TNF-α、IL-6mRNA的表达低于肺炎组(P<0.01), 且治疗C组心肌IL-6mRNA表达低于治疗B组(P<0.01);而A组心肌中TNF-α、IL-6mRNA的表达与肺炎组无明显差异(P>0.05);(3)腺苷治疗可以减轻心肌的病理损伤(P<0.01)。结论:金葡菌感染大鼠肺炎可致心肌损害, 细胞炎症因子IL-6和TNF-α参与心肌损伤的发生和发展, 外源性腺苷治疗可抑制炎症因子TNF-α、IL-6的分泌, 从而有利于减轻炎症和保护心肌。     

C反应蛋白对人外周血CD14单核细胞TLR4信号转导的影响

目的: 观察C反应蛋白(CRP)对CD14⁺单核细胞Toll样受体4(TLR4)表达的影响,以探讨CRP在急性冠脉综合征(ACS)致炎机制中的作用。方法: 不同浓度(5、25、50、100 mg/L)和不同作用时间(6、12、24、48 h)的CRP刺激正常人外周血CD14⁺单核细胞,或者不同浓度TLR4抑制剂预先干预CD14⁺单核细胞后,再给予CRP刺激。应用流式细胞仪检测细胞表面TLR4蛋白的表达,定量PCR方法检测TLR4 mRNA和髓样分化蛋白2(MD-2)mRNA表达,ELISA检测刺激前后细胞上清液肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)、金属蛋白酶9(MMP-9)水平。结果: CRP可剂量依赖和时间依赖地增加CD14⁺单核细胞表达TLR4和MD-2,高浓度TLR4抑制剂可完全阻断CRP对TLR4、MD-2的影响。TNF-α、IL-6、MMP-9与TLR4、MD-2也呈现相同的变化。结论: CRP可激活正常人CD14⁺单核细胞TLR4信号转导途径,并诱导产生TNF-α、IL-6、MMP-9,提示CRP可作为病原相关分子模式(PAMP),通过TLR4模式受体介导而产生炎症反应,参与动脉粥样硬化形成,促进ACS炎症的发展。     

烫伤后IL-6和IL-1α对大鼠外周血中性粒细胞凋亡的影响

目的:研究严重烫伤后血IL-6和IL-1α对中性粒细胞(PMN)凋亡的影响。方法:复制30%体表面积Ⅲ度烫伤大鼠模型;分离PMN,TUNEL荧光标记,流式细胞仪分析细胞凋亡;PMNcaspase3活性以荧光免疫吸附酶法测定;血清IL-6和IL-1α水平以酶联免疫法测定。结果:血清IL-6水平(μg/L)在伤后各组(3、6、12、24、48h依次分别为9.14±1.16、12.49±1.14、3.01±0.75、1.41±0.28和1.56±0.43)和IL-1α水平(ng/L)在伤后3、6、12h组(90.08±8.39、320.93±14.48和47.84±5.19)均分别显著高于伤前对照组IL-6(0.24±0.07)和IL-1α(27.65±4.86)水平(P<0.05);伤后各组PMN凋亡率(%)按时点依次为9.89±2.00、4.98±1.35、1.31±0.72、2.49±1.87和6.88±1.13显著少于伤前组13.66±3.88(P<0.05);PMNcaspase-3的活性测定结果与PMN的凋亡表现相一致。结论:大鼠烫伤后外周血PMN凋亡明显延迟;IL-6和IL-1α等细胞因子是重要的影响因素,减少细胞内caspase-3的激活可能是其机制之一。     

火把花根片抑制大鼠急性肺损伤黏附分子表达

目的： 探讨火把花根片对大鼠急性肺损伤（ALI）黏附分子表达的影响。方法: 经尾静脉注射油酸建立ALI模型并分为ALI组、火把花根＋ALI组和对照组。用流式细胞术和免疫组化SP法测定外周血中性粒细胞（PMN）和单核细胞黏附分子CD11a、CD11b和CD18的表达及肺组织ICAM-1活性，检测肺湿/干重比(W/D)、肺通透指数(LPI)、支气管肺泡灌洗液(BALF)中白细胞(WBC)数和活化PMN比值。结果: ALI组PMN和单核细胞表面的CD11a、CD11b、CD18和肺组织ICAM-1表达水平高于对照组(P<0.01) ，火把花根＋ALI组上述指标显著低于ALI组 (P<0.01)。肺W/D比 、LPI、BALF中WBC计数和活化PMN比值显著低于ALI组 (P<0.01)。结论: PMN和单核细胞黏附分子CD11a、CD11b、CD18和肺组织ICAM-1的表达上调，参与ALI的病理发展过程。火把花根片对ALI具有拮抗作用。     

髓过氧化物酶、CD11b和IL-8在哮喘大鼠模型中表达增加

目的观察哮喘模型大鼠中性粒细胞(PMN)CD11bI、L-8和髓过氧化物酶(MPO)的表达,探讨PMN在哮喘中的作用及其可能的机制。方法复制哮喘大鼠模型,随机分成哮喘组和对照组,分离纯化血PMN;免疫组化法检测MPO表达,ELISA法测定IL-8蛋白,流式细胞术测定血PMN CD11b表达,支气管肺泡灌洗液(BALF)行细胞计数。结果哮喘组肺组织和血PMN中MPO、CD11b及血PMN和BALF中IL-8的水平显著高于对照组(P<0.01)。哮喘组BALF中PMN和嗜酸性粒细胞计数显著高于对照组(P<0.01)。结论BALF中PMN数量增加及血中PMN和肺组织中CD11bI、L-8和MPO在哮喘时表达增加,他们可能参与了哮喘的炎症过程。     

严重烧伤大鼠在体肺泡巨噬细胞CD14的表达调控对内毒素和细胞因子的影响

目的 探讨严重烧伤和内毒素(LPS)对大鼠肺泡巨噬细胞(AM)CD14膜蛋白(CD14)和mRNA基因表达变化的影响,以及抗CD14抗体拮抗前后AM产生肿瘤坏死因子α(TNF-α)和白细胞介素6(IL-6)的影响及调控作用.方法 (1)体内部分:取成年SD大鼠96只,随机分为烧伤组、烧伤对照组、LPS组和LPS对照组,烧伤组和LPS组各42只,两组各分为1 h、2 h、4 h、6 h、8 h、12 h共7个时相点,每时相点6只大鼠.烧伤对照组和LPS对照组各6只大鼠,只检测一个时相点.烧伤组和LPS组分别在大鼠20%Ⅲ度烧伤和LPS注射后各时相点抽取外周血后立即处死行全肺在体支气管肺泡灌洗(BAL)分离提取相应各组AM.外周血检测外周血LPS浓度,各时相点灌洗提取的AM分别用RT-PCR方法观察相同时相点CD14 mRNA表达、免疫组织化学方法观察蛋白含量变化;(2)体外部分:取成年SD大鼠168只,随机分为烧伤血清组、血清抗体组、LPS组、LPS抗体组,每组42只,每组再分为1 h、2 h、4 h、6 h、8 h、12 h共7个时相点,每时相点6只大鼠.另设烧伤对照组和LPS对照组,各6只成年SD大鼠.各组大鼠行全肺在体支气管肺泡灌洗分离AM,并按时相点分别进行体外培养,前四组分别加入烧伤血清、烧伤血清+抗体、LPS、LPS+抗体,在以上相同时相点终止培养,RT-PCR、免疫组化及ELISA方法分别检测四组肺泡巨噬细胞在各时相点CD14mRNA表达、蛋白表达及分泌TNF-α和IL-6的变化.结果 (1)体内部分:烧伤组及LPS组注射后大鼠各时相点外周血LPS浓度均明显高于相应对照组(P<0.01).在体大鼠AM各时相点CD14 mRNA表达与烧伤对照组比较均明显增高(P<0.01);(2)体外部分:烧伤血清组与烧伤对照组比较,LPS组和LPS对照组比较发现,烧伤血清组、LPS组大鼠AM各时相点CD14 mRNA表达、蛋白表达均明显增高,AM培养上清中TNF-α和IL-6浓度亦相应显著增高(P<0.01).以烧伤血清与AM培养1 h后,烧伤血清组培养上清中TNF-α和IL-6浓度即显著增加.与烧伤血清组比较,血清抗体组肺泡巨噬细胞在各时相点CD14 mRNA表达、蛋白表达显著降低(P<0.01),TNF-α和IL-6浓度亦显著降低(P<0.01).与LPS组比较,LPS抗体组肺泡巨噬细胞在各时相点CD14 mRNA表达、蛋白表达显著降低(P<0.01),TNF-α和IL-6浓度亦显著降低(P<0.01).结论 严重烧伤后外周血LPS浓度增加,在体肺泡巨噬细胞CD14 mRNA表达显著增加,离体肺泡巨噬细胞CD14 mRNA表达和蛋白表达均显著增加,使LPS对免疫系统的激活作用显著增大,AM分泌TNF-α和IL-6明显增加,而抗CD14抗体可以明显拮抗AM合成和分泌炎性介质.提示严重烧伤后通过调节CD14的作用而减少炎性介质的合成和分泌是可行的.     

干扰素-γ、肿瘤坏死因子-α和白细胞介素-1β在体外对人气管平滑肌细胞凋亡的作用

目的:研究是否干扰素-γ(IFN-γ)、肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)在体外能够诱导人气管平滑肌细胞(ASMCs)凋亡。方法:分离人ASMCs并在含有10%胎牛血清的DMEM培养基中培养。用4-6代的细胞作实验。IFN-γ、TNF-α和IL-1β,每种细胞因子单独或一起用于处理人ASMCs。用MTT法在0、24、48、72h检测IFN-γ、TNFα和IL-1β对细胞生长的作用。用光镜和电镜观察形态学的改变。用琼脂糖电泳分析DNA片断。用SP-免疫组化染色方法检测p53、bcl-2、bax基因表达的改变。通过碎片DNA原位末端标记技术(TUNEL)检测凋亡细胞的百分率。结果①IFN-γ以时间依赖的方式单独或/和TNF-α和IL-1β一起减低存活的细胞数;②光镜和电镜检查显示人ASMCs细胞皱缩、膜出泡,核缩小,染色质浓聚和核破碎;③琼脂糖电泳显示在用上述细胞因子联合处理的人ASMCs,有代表寡核苷酸片断整倍数的特征性的DNA梯状条带(大约180-200bp);④在细胞因子联合处理组p53和bax基因表达显著高于对照组,但bcl-2基因表达低于对照组(P＜0.01);⑤用IFN-γ(4×10⁵U/L)、TNF-α(4×10⁵U/L)和/或IL-1β(10×10⁴U/L)同时刺激诱导人ASMCs凋亡。在用细胞因子联合处理组的人ASMCs的凋亡指数显著高于对照组(P＜0.01)。结论:用IFN-γ、TNF-α和/或IL-1β联合处理诱导人ASMCs凋亡。这些免疫性细胞因子在哮喘和慢性阻塞性肺疾病的气道重建中也许起了重要的作用。     

白藜芦醇对氧糖剥夺/再复氧损伤后小胶质细胞系N9活化的影响

目的 探讨白藜芦醇对氧糖剥夺/再复氧损伤（OGD/R）后小胶质细胞系N9活化的影响。 方法 体外培养的N9小胶质细胞行氧糖剥夺150 min,复氧培养24 h。实验分为正常组（Nor）、对照组(Ctrl)和白藜芦醇预处理组(Res)。细胞计数盒-8（CCK-8）法测细胞活力,流式细胞仪检测细胞凋亡,免疫荧光法检测Iba1蛋白表达,Western blotting检测钙离子接头蛋白1（Iba1）、CD11b、Caspase-3、Bax、白细胞介素-10(IL-10)、肿瘤坏死因子α（TNF-α）和IL-1β蛋白表达,ELISA法检测培养细胞上清液中IL-10、TNF-α和IL-1β蛋白含量。 结果 CCK-8 法检测结果显示,OGD/R损伤后对照组、1、5、20、40和80 μmol/L白藜芦醇组细胞活力均较正常组降低（P＜0.05,n=3）,但5、20和 40 μmol/L白藜芦醇组细胞活力较对照组显著增强（P＜0.05,n=3）,以20 μmol/L组最强。流式细胞术、免疫荧光、Western blotting和ELISA法显示,对照组和白藜芦醇组CD11b、Iba1、Caspase-3、Bax、IL-10、TNF-α、IL-1β蛋白表达或含量或凋亡细胞百分比均显著高于正常组（P＜0.05,n=3）,但白藜芦醇组除IL-10蛋白表达或含量较对照组增高外,其余均 低于对照组（P＜0.05,n=3）。结论 白藜芦醇预处理可抑制OGD/R后小胶质细胞的活化及凋亡,减轻炎症反应。     

Mycobacterium tuberculosis-induced activation accelerates apoptosis in peripheral blood neutrophils from patients with active tuberculosis

The activation of circulating polymorphonuclear neutrophils (PMN) from patients with active tuberculosis (TB-PMN) may be associated with induction of apoptosis. Spontaneous or Mycobacterium tuberculosis (MTB)-induced apoptosis of PMN were evaluated by microscopy, DNA content, and their binding to Annexin V at 0, 3, and 18 h. In addition, the expression of CD11b and of CD16 were evaluated as parameters of activation and apoptosis, respectively. Recently isolated TB-PMN showed a higher CD11b expression than normal PMN (N-PMN), but there were no features of apoptosis, even though an enhancement of Fas expression was observed. Spontaneous apoptosis was accelerated in TB-PMN at 3 h, but no differences were observed in TB- and N-PMN at 18 h of culture. When stimulated with MTB, both TB- and N-PMN steadily increased CD11b expression along the culture period. MTB induced apoptosis of N-PMN at 3 h with loss of CD16 expression. By contrast, MTB delayed the apoptotic rate of TB-PMN, preserving the CD16 receptor at 3 h, whereas it accelerated apoptosis at 18 h, increasing at the same time the expression of CD11b. Taken together, these data suggest that the acceleration of apoptosis observed in TB-PMN could be associated with the MTB-induced activation.     

Three different neutrophil subsets exhibited in mice with different susceptibilities to infection by methicillin-resistant Staphylococcus aureus

Neutrophils (PMN) have been described as critical effector cells in the host's antibacterial innate immunities. However, the classification of murine PMNs remains unclear. Here, we show that in addition to normal PMN (PMN-N), there are at least two distinct subsets of PMNs (PMN-I and PMN-II) distinguished as follows: (1) cytokine and chemokine production (PMN-I, IL-12/CCL3; PMN-II, IL-10/CCL2; PMN-N, no cytokine/chemokine production), (2) macrophage activation (PMN-I, classically activated macrophages; PMN-II, alternatively activated macrophages; PMN-N, no effect on macrophage activation), (3) Toll-like receptor (TLR) expression (PMN-I, TLR2/TLR4/TLR5/TLR8; PMN-II, TLR2/TLR4/TLR7/TLR9; PMN-N, TLR2/TLR4/TLR9), and (4) surface antigen expression (PMN-I, CD49d(+)CD11b-; PMN-II, CD49d(-)CD11b+; PMN-N, CD49d(-)CD11b-). PMN-I was obtained from MRSA (methicillin-resistant Staphylococcus aureus)-resistant hosts, while MRSA-sensitive hosts were a source of PMN-II. PMN-N was obtained from naive mice. Anti-MRSA innate immunities might be influenced differently by these biochemically and physically distinguished PMNs. PMN-N may convert to PMN-I or PMN-II in response to host circumstance.     

Fas Ligand-dependent and -independent mechanisms of toxicity induced by T cell lymphomas in lymphoid organs and in the liver

In the current study, we investigated the effect of growth of FasL⁺ tumors in vivo on the functions of peripheral lymphoid organs and the liver. Injection of FasL⁺ LSA tumor cells into syngeneic C57BL/6 wild-type mice but not C57BL/6 lpr/lpr (Fas-deficient) mice caused apoptosis in splenocytes. Spleen cells expressing CD3, CD4, CD8, CD19, Mac-3, and CD44 were all susceptible to tumor-induced apoptosis. Also, activated T cells were more sensitive to apoptosis induced by LSA tumor cell lysate when compared to naïve T cells. In contrast, anti-Fas Abs (Jo2) induced apoptosis in only activated but not naïve T cells. When the LSA tumor-bearing mice were injected with a superantigen (SEA), these mice showed a significant decrease in the expansion of SEA-reactive Vβ3⁺ and Vβ11⁺ T cells. When injected into syngeneic mice, the FasL⁺ LSA tumor cells caused hepatotoxicity, as indicated by an increase in serum aspartate aminotransferase (AST) levels. Interestingly, Fas-deficient C57BL/6 lpr/lpr mice also showed significant AST levels in the serum following LSA tumor growth. Moreover, hepatocytes isolated from C57BL/6 wild-type and C57BL/6 lpr/lpr mice were equally susceptible to apoptosis induced by LSA tumor cell lysate in vitro. Using cDNA array, LSA tumor cells were found to express several cytokine genes including IL-2, IL-7, IL-11, IL-13, IL-16, lymphotoxin β, and tumor necrosis factor β. Together, these data suggested that, in mice bearing FasL⁺ LSA tumor, the immunotoxicity is FasL-based, whereas the hepatotoxicity, at least in part, may be FasL-independent.     

Increased serum IL-10 in lupus patients promotes apoptosis of T cell subsets via the caspase 8 pathway initiated by Fas signaling

We sought to investigate the expression of Fas and FasL on T cell surface and caspase 8 involvement in T cell apoptosis promoted by serum IL-10 in systemic lupus erythematosus(SLE) patients.Cells and sera were obtained from 35 SLE patients.Apoptosis of T cells in patients with SLE was increased and associated with the SLE disease activity index(SLEDAI).Elevated expression of Fas and FasL on T cell surface contributed to increased apoptosis of T cells.Increased IL-10 in the sera of SLE patients was capable of inducing Fas and FasL expression on CD4~+T cell surface,promoting apoptosis of this cell subset.Decreased IL-10 serum levels and low expression of Fas were found in 5 patients of the first follow-up group after 2-month treatment.In another group with one-year treatment,the SLEDAI declined to inactive scores.Serum IL-10 was decreased significantly,and expression of Fas and FasL on T cells was also reduced.Declined apoptosis was predominant only in CD4~+T cell subset.When sera with high level of IL-10 were used to culture PBMCs from healthy controls,activated caspase 8 was elevated in CD3~+T,CD4~+T and CD8~+T cells.The study showed that serum IL-10 induced apoptosis of T cell subsets via the caspase8 pathway initiated by Fas signaling.Increased apoptosis of T cells contributes to autoantigen burden,which is pathogenic in the development of SLE.     

Changes in neutrophil CD11b/CD18 andl-selectin expression and release of interleukin 8 and elastase in paediatric cardiopulmonary bypass

Children undergoing cardiopulmonary bypass (CPB) surgery for congenital heart defects develop an acute post-operative capillary leak which may be due to endothelial injury inflicted by adherent neutrophils (PMN). Direct immunofluorescence and flow cytometry were used to measure CD11a/CD18, CD11b/CD18 andl-selectin (l-s) expression on circulating PMN in CPB circuits containing human blood and in children undergoing CPB.In vitro, a general rise in CD11b/CD18 expression over 2h contrasted with complete loss ofl-s in a small but progressively increasing proportion of PMN. Marked but inconsistent changes in CD11b/CD18 andl-s were observedin vivo, in conjunction with fluctuations in circulating PMN count. Circulating IL-8 was detected starting at rewarming from hypothermia and reperfusion of the heart and lungs with a simultaneous, closely correlated rise in both PMN count and circulating elastase. IL-1 and TNF were not detected. These studies demonstrate changes in the pathways of PMN-endothelial interaction during and after CPB.     

Direct stimulatory effects of the TLR2/6 ligand bacterial lipopeptide MALP-2 on neutrophil granulocytes

Bacterial lipopeptides represent a group of bacterial compounds able to trigger the functions of cells of the innate immune response. Whereas diacylated lipopeptides are recognized by TLR2/6 dimers, triacylated lipopeptides were shown to act via TLR2/1 dimers. Although several previous studies dealt with the effect of the TLR2/1 ligand Pam₃CysSK₄ on neutrophil granulocytes (PMN), it is still not clear whether TLR2/6 ligand lipopeptides can directly influence PMN functions. In the present study we used highly purified human neutrophils to investigate the direct effects of the diacylated mycoplasmal macrophage activating lipopeptide-2 (MALP-2) on the function of neutrophil granulocytes. After exposure to 10 ng/ml MALP-2 neutrophils acquired activated cell shape, secreted IL-8 and MIP-1β and their phagocytic capacity was enhanced. Analysis of cell surface activation markers confirmed the activating effect of MALP-2, the expression of CD62L was downregulated whereas CD11b was upregulated on PMN after exposure to MALP-2. The constitutive apoptosis of PMN was inhibited after exposure to MALP-2. However, MALP-2 exerted only a short-term effect on the apoptosis of resting neutrophils, a longer lasting effect was observed after transendothelial migration. MALP-2 did not directly induce the production of reactive oxygen intermediates but primed PMN for a fMLP-induced oxidative burst. The migration of neutrophils was enhanced after treatment with MALP-2. This was due, however, to a chemokinetic rather than to a chemotactic effect. Pam₃CysSK₄ also activated PMN, but in comparison to MALP-2, at higher concentrations. These findings suggest that diacylated lipopeptides are important microbial structures recognized by and acting on neutrophil granulocytes.     

Apoptosis is associated with reduced expression of complement regulatory molecules, adhesion molecules and other receptors on polymorphonuclear leucocytes: functional relevance and role in inflammation.

Human polymorphonuclear leucocytes (PMN) express proteins that protect them from damage by homologous complement. Protection may be particularly important when these cells migrate to inflammatory sites where complement activation is taking place. Resolution of inflammation involves removal of these PMN. The major mechanism of removal is likely to involve PMN apoptosis followed by recognition and engulfment by macrophages. However, little attention has been paid to the possible relevance of apoptosis to PMN susceptibility to immune effectors. Here we describe a reduction in cell surface expression of two complement regulatory proteins, CD59, an inhibitor of the membrane attack complex and CD55 (decay accelerating factor), an inhibitor of the C3/C5 convertase, on a subpopulation of PMN aged in culture. Loss of these proteins, both attached to the membrane by glycosyl phosphatidylinositol (GPI) anchors, correlated closely with the appearance of apoptotic morphology. We also observed a marked reduction in expression of the GPI-anchored molecule CD16 on apoptotic PMN. Reduced expression of membrane proteins was not confined to those anchored through GPI--several transmembrane molecules including CD11a CD11b and CD18 were also reduced on apoptotic PMN, whilst other were little changed (CD35, CD46). The precipitous fall in CD16 surface expression on PMN was not specific for apoptosis--in vitro incubation of PMN with lipopolysaccharide-inhibited apoptosis but caused a reduction in CD16 expression to 'apoptotic' levels.     

Ketamine inhibits polymorphonuclear leucocyte CD11b expression and respiratory burst activity in endotoxemic rats

Objective and design: To observe the effect of ketamine on polymorphonuclear leucocytes (PMN) adhesion and respiratory burst activity in endotoxemia rats. Materials: 30 rats were randomly allocated to five groups: rats challenged with intraperitoneal injection of saline (saline group); challenged with intraperitoneal injection of LPS 10 mg/kg (LPS group); challenged with intraperitoneal injection of LPS 10 mg/kg and treated by intraperitoneal injection of ketamine 5, 25, 50 mg/kg at 0, 1, 2, 3, 4, 5 h after the injection of LPS, respectively (three ketamine treatment groups). Methods: PMN respiratory burst and CD11b expression were measured with flow cytometry at the end of 1 h, 4 h, and 6 h. Results: LPS challenge significantly increased PMN respiratory burst activity and CD11b expression when compared with the saline group (p < 0.01). There was a significant decrease in LPS-induced PMN respiratory burst activity and CD11b expression in three ketamine treatment groups when compared with LPS group (p < 0.01). Conclusions: Ketamine significantly inhibits PMN CD11b expression and respiratory burst activity in endotoxemic rats. Received 31 May 2006; returned for revision 21 July 2006; returned for final revision 10 October 2006; accepted by M. Katori 5 November 2006