云芝糖肽在ROS、p38信号通路中对巨噬细胞Raw264．7的免疫调节作用

目的：探究云芝糖hk（PSP）对静息和脂多糖（LPS）过度刺激两种不同状态的巨噬细胞Raw264．7产生活性氧簇（ROS）TLp38、p-p38的影响及其相关机制。方法：采用流式细胞术方法检测ROS的表达;Westernblot方法检测MAPK信号通路中p38磷酸化及非磷酸化的相对表达量。结果：100g／mlPSP下调1g／ml LPS单独作用于巨噬细胞时ROS及p-p38的表达量;100g／mlPSP上调正常巨噬细胞p-p38的表达量;p38表达量基本不变。结论：PSP通过ROS,p38两条通路参与巨噬细胞的免疫调节作用。     

五灵胶囊对LPS诱导鼠枯否细胞p38MAPK信号转导通路的影响

目的:探讨五灵胶囊对脂多糖(LPS)诱导的大鼠枯否细胞(Kupffer cells,KC)p38MAPK信号转导通路的影响。方法:分离纯化KCs,60ng/ml LPS刺激建立LPS的肝细胞损伤模型;40只SD大鼠药物处理后,分离制备含药血清。实验分为四组:空白血清组、LPS+空白血清组、含药血清Ⅰ组(10.0g/kg)+LPS、含药血清Ⅱ组(6.25g/kg)+LPS。KCs产生促炎因子(I125放免法测定TNF-α、IL-6、IL-8,比色法测定NO生成量),采用Western blot法检测ERK、p-ERK、p38、p-p38、TNF-α和STAT3的蛋白水平。结果:1、空白血清+LPS组,TNF-α、IL-6、IL-8和NO浓度明显高于空白血清组;2、同空白血清+LPS组比较,含药血清Ⅰ、Ⅱ组TNF-α、IL-6、IL-8和NO水平明显降低;3、与空白血清组比较,空白血清+LPS组能上调KCs对p-ERK、P38、p-P38、STAT3和TNF-α表达(p<0.01,p<0.05),对ERK表达无影响(p>0.05)4、同空白血清+LPS组比较,含药血清Ⅰ+LPS、含药血清Ⅱ+LPS组p-p38、S...     

黄芪甲苷对马兜铃酸诱导的巨噬细胞M1型极化的作用及机制研究

目的:探讨黄芪甲苷对马兜铃酸诱导的RAW264.7细胞向M1型极化的影响,并初步探索其可能的作用机制。方法:分别采用马兜铃酸和脂多糖(LPS)刺激RAW264.7细胞24 h,伴或不伴黄芪甲苷进行药物干预处理。采用细胞计数检测试剂盒-8(CCK 8)检测细胞活性变化,流式细胞仪检测巨噬细胞分型,酶联免疫吸附试验(ELISA)检测细胞上清液中白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)的分泌量。反转录实时定量PCR(RT-qPCR)技术检测RAW264.7细胞IL-6、TNF-αmRNA表达。蛋白免疫印迹法(Western blot)检测RAW264.7细胞p-p38和p38 MAPK蛋白表达水平。结果:CCK8结果提示黄芪甲苷在5~50μg/mL浓度范围对RAW264.7巨噬细胞无明显毒性,本研究选取10μg/mL作为实验干预浓度。黄芪甲苷能够显著改善马兜铃酸诱导的巨噬细胞活性(P<0.05),同时减少IL-6和TNF-α的分泌水平和mRNA表达水平(均P<0.05),抑制马兜铃酸和LPS诱导的M1/M2巨噬细胞比例(P<0.05)。黄芪甲苷可部分抑制马兜铃酸诱导的巨噬细胞p38 MAPK磷酸化水平(P<0.05)。结论:黄芪甲苷可减少巨噬细胞M1型极化,降低炎症因子IL-6和TNF-α水平,减少巨噬细胞的活性,从而起到减缓马兜铃酸肾损害的作用,其作用机制可能与部分抑制p38 MAPK信号活性有关。     

褪黑素对内毒素致大鼠急性肺损伤的保护作用

目的:研究内毒素(LPS)致大鼠急性肺损伤(ALI)时,p-p38蛋白激酶(p-p38MAPK)在肺组织的表达及褪黑素(MT)对肺组织的保护作用及其机制。方法:将72只健康雄性SD大鼠随机分为3组,每组24只,对照组(Control)、模型组(LPS)和褪黑素干预组(LPS+MT),采用气管内滴注LPS的方法建立大鼠ALI的模型,通过免疫组织化学染色和Western blot技术检测大鼠肺组织中p-p38蛋白激酶的表达变化,并在光镜下观察大鼠肺组织形态学变化。结果:Control组气道和肺组织可见反应极弱的p-p38蛋白激酶阳性细胞,散在分布于气道上皮细胞和肺泡上皮细胞;LPS组p-p38蛋白激酶阳性细胞较对照组明显增多(P0.05或P0.01),主要分布于浸润的炎症细胞、气道上皮细胞、肺泡上皮细胞和血管内皮细胞;LPS+MT组气道和肺组织中阳性细胞数较LPS组明显减少(P0.05或P0.01),Western blot结果与免疫组织化学一致。结论:LPS致大鼠急性肺损伤模型中,肺内炎性、非炎性细胞均有p38MAPK信号通路的激活;MT对急性肺损伤的保护机制可能与其抑制p38 MAPK信号通路的过度激活有关。     

红景天苷通过调节p38和JNK信号通路抑制BV-2细胞分泌炎性因子

红景天苷是传统中药红景天的主要成分,已有研究显示红景天苷具有一定的抗炎性作用。本研究旨在探讨红景天苷对BV-2细胞炎性反应的调节作用及可能机制。实验分为对照组、脂多糖(lipopolysaccharide,LPS)组、红景天苷组、红景天苷预处理+LPS组,用100μg/L LPS和/或10 mg/L红景天苷处理BV-2细胞,收集细胞培养基上清(条件培养基),采用real-time PCR方法检测细胞IL-6和TNF-αm RNA表达水平,ELSIA方法检测培养基IL-6和TNF-α含量,条件培养基处理PC12细胞24 h后,Hoechst 33258染色检测细胞核形态学改变,cell counting kit 8(CCK-8)试剂盒检测细胞活力,免疫印迹方法检测BV-2细胞p38和JNK的磷酸化水平。结果表明100μg/L LPS处理可显著提高BV-2细胞内IL-6和TNF-α的表达水平(P0.05)。红景天苷可显著下调LPS诱导的BV-2细胞IL-6和TNF-α表达量(P0.05),下调p38和JNK的磷酸化水平(P0.05)。SB202190和SP600125可降低LPS引起的IL-6和TNF-α表达,与红景天苷共处理后可拮抗LPS的诱导效应。与LPS组相比,红景天苷预处理+LPS组的条件培养基处理后,PC12细胞的细胞活力显著升高(P0.05),同时细胞核固缩和聚集情况减少(P0.05)。以上结果提示,红景天苷可在一定程度上抑制小胶质细胞活动,通过调节p38和JNK信号通路减少炎性因子释放,从而降低PC12细胞的损伤。     

原儿茶酸对脂多糖诱导的急性肺损伤小鼠的保护作用

目的:探索原儿茶酸(protocatechuicacid,PCA)对脂多糖(lipopolysaccharide,LPS)诱导的急性肺损伤(acute lung injury,ALI)小鼠的保护作用,探讨其保护机制。方法:将40只昆明小鼠按随机数字表法均分为空白对照组(NC组)、LPS模型组、原儿茶酸预处理组(PCA+LPS组)、地塞米松阳性对照组(Dex+LPS组),每组10只,模型组以5mg·kg-1脂多糖腹腔内注射诱导急性肺损伤。6h后处死小鼠,HE染色观察肺组织病理学变化;BCA法检测肺泡灌洗液中总蛋白浓度;ELISA检测肺泡灌洗液炎症因子TNF-α、IL-1β含量;Western Blot检测肺组织中p38MAPK、p-p38MAPK、p-ATF2蛋白的表达水平。结果:与对照组相比,模型组小鼠肺损伤明显,肺泡内出血、水肿、炎细胞浸润,肺泡灌洗液中TNF-α、IL-1β的含量及总蛋白浓度增加,肺组织中p38MAPK/p-p38MAPK、p-ATF2表达均明显增加(均P0.01)。与模型组相比,原儿茶酸预处理组、地塞米松阳性对照组肺组织病理损伤程度明显减轻,肺泡灌洗液中TNF-α、IL-1β的含量及总蛋白浓度、肺组织中p38MAPK/p-p38MAPK、p-ATF2表达均明显降低(均P0.01)。结论:PCA对LPS诱导的急性肺损伤有保护作用,其作用机制可能与其抑制p38MAPK-p-ATF2信号通路的活化、降低肺组织炎症反应有关。     

去乙酰化酶抑制剂TSA通过激活ERK和p38抑制间充质干细胞成脂分化

本文研究了丝裂原活化蛋白激酶(mitogen activated protein kinases, MAPKs)信 号通路在组蛋白去乙酰化酶抑制剂(histone deacetylase inhibitors, HDACis)曲古抑 菌素A(trichostatin A, TSA)抑制间充质干细胞(mesenchymal stem cells, MSCs) C3H10T1/2成脂分化中的调节机制.首先利用 MTT法检测TSA对其增殖活性的影响;Western印迹法首先检测MAPKs信号通路中pERK和p-p38蛋白在间充质干细胞C3H10T1/2成 脂分化过程中的表达情况,以及不同浓度、不同时间TSA处理对pERK和p-p38蛋白差异变 化情况;其次再用Western印迹检测TSA对成脂分化过程中间充质干细胞pERK和p-p38蛋 白表达的影响.MTT结果显示,TSA浓度在1 nmol/L~100 nmol/L范围内抑制C3H10T1/2细胞 的增殖活性,且TSA浓度约为60 nmol/L时即抑制一半以上的C3H10T1/2细胞增殖活 性.Western印迹结果显示,TSA处理5 min~80 min,及浓度在1 nmol/L~100 nmol/L范围内 激活MAPK信号通路中pERK和p-p38蛋白的表达;C3H10T1/2细胞成脂分化过程中,胞内pERK和p-p38蛋白的表达呈现下调趋势;而TSA抑制了成脂分化过程中C3H10T1/2细胞内pERK和p-p38蛋白的表达变化.本研究结果提示,在C3H10T1/2细胞成脂分化过程中,MAPK信号途径分子pERK和p-p38表达下调;TSA可能是通过活化pERK和p-p38进而抑制间充质干细胞C3H10T1/2成脂分化.     

番茄红素对血管内皮细胞氧化应激损伤的作用及机制研究

目的:观察番茄红素(lycopene,LYC)对于血管内皮细胞功能的作用,探讨其作用机制。方法:人脐静脉内皮细胞(HUVECs)处理实验分组:对照组,H2O2组,H2O2+LYC组(1、2、4、8μmolL-1)。MTT法检测HUVECs存活率;免疫印迹法(Western blot)检测p38MAPK蛋白磷酸化水平、抗凋亡蛋白B淋巴细胞/白血病-2(bcl-2)及线粒体凋亡通路相关蛋白bax的表达;细胞黏附能力测定和伤口愈合实验检测HUVECs粘附率和迁移率;TUNEL法检测HUVECs凋亡率;ELASA法测定HUVECs内活性氧(ROS),超氧化物歧化酶(SOD),乳酸盐脱氢酶(LDH)释放量和caspase-3的活性。结果:H2O2损伤后HUVECs存活率显著降低(P0.01),凋亡率显著增加(P0.01),黏附和迁移能力显著降低(P0.01),bax和p-p38MAPK的表达上调,bcl-2的表达下调,并且ROS、LDH的释放和caspase-3的活性增加(P0.01),SOD的释放减少。而LYC的预处理可以明显逆转H2O2以上作用。结论:H2O2氧化应激损伤中,LYC保护内皮细胞可能与其抗过氧化损伤细胞凋亡,抑制异常的p38MAPK信号通路有关。     

姜黄素对内毒素诱导肠黏膜微血管内皮细胞COX-2表达的影响及机制研究

目的探讨姜黄素对LPS诱导的大鼠肠黏膜微血管内皮细胞表达COX-2的影响及机制。方法选用大鼠肠黏膜微血管内皮细胞,随机分组,用p38MAPK抑制剂、PPARγ拮抗剂、姜黄素进行干预。荧光定量RT-PCR检测COX-2 mRNA的表达量,WB法检测其相关蛋白。结果LPS组较正常对照组COX-2增高3.5倍,差异有统计学意义(P0.01)。姜黄素组、p38MAPK选择性抑制剂组、PPARγ拮杭剂组较LPS组,COX-2明显降低。阻断p38MAPK后,COX-2蛋白表达下降。姜黄素可以增加PPARγ、COX-2、p-p38的表达。结论姜黄素可降低COX-2表达水平,p38MAPK、PPARγ通路参与姜黄素调控COX-2的表达。     

香草乙酮改善葡聚糖硫酸钠诱发溃疡性结肠炎小鼠的炎症反应与NOXs-ROS-p38MAPK信号通路的关系

本文旨在探讨香草乙酮(apocynin)治疗葡聚糖硫酸钠(dextran sulfate sodium,DSS)诱导的溃疡性结肠炎(ulcerative colitis,UC)小鼠的分子机制。以饮用水配制5%DSS诱发UC动物模型,2%香草乙酮治疗UC小鼠,采样并应用HE染色进行结肠组织病理学评估。采用鲁米诺化学发光方法检测结肠组织活性氧自由基(reactive oxygen species,ROS)生成及DPI抑制后的NADPH消耗率分析NADPH氧化酶(NADPH oxidases,NOXs)活性,Western blot法检测p38MAPK磷酸化水平,Griess方法分析NO,酶联免疫法检测前列腺素E2(prostaglandin E2,PGE2),real time PCR及Western blot法检测i NOS、COX2的表达,酶联免疫吸附实验测定细胞因子TNF-α、IL-6、IFN-γ、IL-1β的水平。体外实验采用分离的结肠组织中性粒细胞,检测其ROS生成、NOXs活性、NO、PGE2的变化,并应用Western blot检测其NOX1、p-p38MAPK的表达。结果显示,香草乙酮治疗后UC小鼠结肠组织NOXs活性及ROS生成被抑制(P0.01),p38MAPK磷酸化水平降低,NO、PGE2及相关细胞因子生成减少(P0.01),UC炎症反应得到缓解。在炎症部位的中性粒细胞中,香草乙酮抑制ROS生成及NOX活性(P0.01),并降低NOX1的表达、p38MAPK的磷酸化及NO、PGE2的生成(P0.01)。因此,香草乙酮可能通过NOXs-ROS-p38MAPK信号通路缓解DSS诱发UC小鼠的炎症反应,中性粒细胞是参与其保护机制的主要炎症细胞。     

Mechanism of attenuation by β-hydroxy-β-methylbutyrate of muscle protein degradation induced by lipopolysaccharide

The mechanism of the effect of β-hydroxy-β-methylbutyrate (HMB) on protein degradation induced by lipopolysaccharide (LPS) has been evaluated in murine myotubes. HMB (50 μM) completely attenuated total protein degradation induced by LPS (1–100 ng/ml), formation of reactive oxygen species (ROS) and activation of caspase-3/-8. Specific inhibitors of caspase-3/-8 completely attenuated ROS production, total protein degradation and the LPS-induced autophosphorylation of dsRNA-dependent protein kinase (PKR). Protein degradation in response to LPS or ROS production was not seen in myotubes transfected with mutant PKRΔ6, suggesting that PKR was involved in ROS production, which was essential for total protein degradation. This was confirmed using the antioxidant butylated hydroxytoluene (BHT) which completely attenuated protein degradation in response to LPS. The link between PKR activation and ROS production was mediated through p38 mitogen-activated protein kinase (MAPK), which was activated by LPS in myotubes transfected with wild-type PKR, but not PKRΔ6. Both ROS production and protein degradation induced by LPS were completely attenuated by SB203580, a specific inhibitor of p38MAPK. This suggests that LPS induces protein degradation through a signalling cascade involving activation of caspase-3/-8, activation of PKR and production of ROS through p38MAPK, and that this process is attenuated by HMB.     

High-density Lipoprotein Increases the Uptake of Oxidized Low Density Lipoprotein via PPARγ/CD36 Pathway in Inflammatory Adipocytes

Aim: Previous studies have demonstrated that the dysregulated-secretion of adipokines by adipocytes may contribute to obesity-associated atherosclerosis (As) and high density lipoprotein (HDL) may protect against atherogenesis through multiple pathways. This study was to explore the effect of HDL on the oxLDL uptake in inflammatory adipocytes stimulated by endotoxin lipopolysaccharide (LPS) and the possible mechanism.Methods and Results: 3T3-L1 adipocytes were cultured and induced to differentiation and maturation. Acute inflammation in adipocytes was induced by LPS (100 ng/ml) for 6 hours. The adipocytes were pretreated with HDL in various concentrations (10, 50, 100 μg/ml) for 16 hours or with specific PPARγ antagonist (GW9662, 10 μM) or agonist (Rosiglitazone, 10 μM) for 30 min before administration of LPS. The results showed that LPS significantly increased the release of inflammation-related adipokines, such as monocyte chemoattractant protein-1 (MCP-1), plasminogen activator inhibitor 1 (PAI-1), tumor necrosis factor-alpha (TNF-α), interleukin (IL)-8 and IL-6, while decreasing the release of leptin and adiponectin. Meanwhile, LPS reduced the uptake and degradation of ¹²⁵I-oxLDL, and down-regulated the expression of PPARγ and CD36. Pretreatment with HDL dose-dependently affected the release of IL-8 and IL-6 and the reduced uptake and degradation of oxLDL of adipocytes stimulated by LPS, accompanied with marked upregulation of PPARγ and CD36 expression. Pretreatment with GW9662 markedly inhibited the upregulation of CD36 expression mediated by HDL (100 μg/ml), while the effects of Rosiglitazone were opposite to GW9662.Conclusions: HDL may increase oxLDL uptake of inflammatory adipocytes stimulated by LPS via upregulation of PPARγ/CD36 pathway, which may be a new mechanism of anti-atherosclerosis mediated by HDL.     

Differential regulation of cytokine and chemokine production in lipopolysaccharide-induced tolerance and priming

LPS pretreatment of human pro-monocytic THP-1 cells induces tolerance to secondary LPS stimulation with reduced TNFalpha production. However, secondary stimulation with heat-killed Staphylococcus aureus (HKSa) induces priming as evidenced by augmented TNFalpha production. The pro-inflammatory cytokine, IFNgamma, also abolishes suppression of TNFalpha in LPS tolerance. The effect of LPS tolerance on HKSa and IFNgamma-induced inflammatory mediator production is not well defined. We hypothesized that LPS, HKSa and IFNgamma differentially regulate pro-inflammatory mediators and chemokine production in LPS-induced tolerance. THP-1 cells were pretreated for 24 h with LPS (100 ng/ml) or LPS (100 ng/ml) + IFNgamma (1 microg/ml). Cells were subsequently stimulated with LPS or HKSa (10 microg/ml) for 24 h. The production of the cytokines TNFalpha, IL-6, IL-1beta, and GMCSF and the chemokine IL-8 were measured in supernatants. LPS and HKSa stimulated TNFalpha (3070 +/- 711 pg/ml and 217 +/- 9 pg/ml, respectively) and IL-6 (237 +/- 8.9 pg/ml and 56.2 +/- 2.9 pg/ml, p < 0.05, n = 3, respectively) in control cells compared to basal levels (< 25 pg/ml). LPS induced tolerance to secondary LPS stimulation as evidenced by a 90% (p < 0.05, n = 3) reduction in TNFalpha. However, LPS pretreatment induced priming to HKSa as demonstrated by increased TNFalpha (2.7 fold, from 217 to 580 pg/ml, p < 0.05, n = 3 ). In contrast to suppressed TNFalpha, IL-6 production was augmented to secondary LPS stimulation (9 fold, from 237 to 2076 pg/ml, p < 0.01, n = 3) and also primed to HKSa stimulation (62 fold, from 56 to 3470 pg/ml, p < 0.01, n = 3). LPS induced IL-8 production and to a lesser extent IL-1beta and GMCSF. LPS pretreatment did not affect secondary LPS stimulated IL-8 or IL-1beta, although HKSa stimulation augmented both mediators. In addition, IFNgamma pretreatment reversed LPS tolerance as evidenced by increased TNFalpha levels while IL-6, IL-1beta, and GMCSF levels were further augmented. However, IL-8 production was not affected by IFNgamma. These data support our hypothesis of differential regulation of cytokines and chemokines in gram-negative- and gram-positive-induced inflammatory events. Such changes may have implications in the pathogenesis of polymicrobial sepsis.     

NADPH oxidase-2 derived ROS dictates murine DC cytokine-mediated cell fate decisions during CD4 T helper-cell commitment

NADPH oxidase-2 (Nox2)/gp91(phox) and p47(phox) deficient mice are prone to hyper-inflammatory responses suggesting a paradoxical role for Nox2-derived reactive oxygen species (ROS) as anti-inflammatory mediators. The molecular basis for this mode of control remains unclear. Here we demonstrate that IFNγ/LPS matured p47(phox-/-)-ROS deficient mouse dendritic cells (DC) secrete more IL-12p70 than similarly treated wild type DC, and in an in vitro co-culture model IFNγ/LPS matured p47(phox-/-) DC bias more ovalbumin-specific CD4(+) T lymphocytes toward a Th1 phenotype than wild type (WT) DC through a ROS-dependent mechanism linking IL-12p70 expression to regulation of p38-MAPK activation. The Nox2-dependent ROS production in DC negatively regulates proinflammatory IL-12 expression in DC by constraining p38-MAPK activity. Increasing endogenous H(2)O(2) attenuates p38-MAPK activity in IFNγ/LPS stimulated WT and p47(phox-/-) DC, which suggests that endogenous Nox 2-derived ROS functions as a secondary messenger in the activated p38-MAPK signaling pathway during IL-12 expression. These findings indicate that ROS, generated endogenously by innate and adaptive immune cells, can function as important secondary messengers that can regulate cytokine production and immune cell cross-talk to control during the inflammatory response.     

The effect of resveratrol on the platelet secretory process induced by endotoxin and thrombin

The effect of resveratrol (trans-3,4',5-trihydroxystilbene) on the release of adenine nucleotides and proteins from blood platelets activated by lipopolysaccharide (LPS), from Proteus mirabilis and by thrombin, were studied. Thrombin stimulated the release of adenine nucleotides from dense granules and proteins from alpha-granules. The LPS (0.3 microg/10(8) platelets, 5 min, 37 degrees C), like thrombin (2.5 U/10(8) platelets, 5 min, 37 degrees C) was found to cause a release of adenine nucleotides and proteins (p <0.05). Resveratrol (6.25-100 microg/ml, 30 min, 37 degrees C) had a different effect on the platelet release reaction caused by either LPS or thrombin. The results indicated that resveratrol inhibited, in dose-dependent manner, the secretory process (release of adenine nucleotides and proteins) induced by thrombin (p <0.05), but it significantly stimulated the liberation of proteins from blood platelets activated by LPS (p <0.05).     

Reactive oxygen species are involved in the IFN‐γ‐stimulated production of Th2 chemokines in HaCaT keratinocytes

The increased generation of reactive oxygen species (ROS) induces inflammation in different cell types. However, it is unclear whether ROS play an essential role in the production of thymus and activation‐regulated chemokine (TARC/CCL17) and macrophage‐derived chemokine (MDC/CCL22) in keratinocytes. Here, we investigated the function of ROS in the production of these two Th2 chemokines in interferon‐gamma (IFN‐γ)‐treated HaCaT keratinocytes. We found that IFN‐γ‐induced production of both chemokines in parallel with the increased generation of intracellular ROS. A ROS scavenger, N‐acetyl cysteine (NAC), significantly inhibited the IFN‐γ‐induced production of chemokines as well as the activation of I kappa‐B (IκB)–nuclear factor‐kappa B (NF‐κB). Inhibitors of Janus family kinases (JAKs), p38 mitogen‐activated kinase (MAPK), and NF‐κB suppressed IFN‐γ‐induced production of TARC and MDC. NF‐κB activation was inhibited by both inhibitors of JAKs and p38 MAPK. Importantly, IFN‐γ‐stimulated phosphorylation of p38 MAPK was significantly suppressed by JAKs inhibitors, but not significantly affected by NAC or L ‐buthionine sulfoximine (L‐BSO). However, IFN‐γ‐stimulated activation of IκB and NF‐κB was suppressed by NAC but enhanced by BSO. Furthermore, inhibition of p38 MAPK and JAKs did not affect ROS generation in IFN‐γ‐stimulated HaCaT cells. These results indicate that intracellular ROS and JAKs/p38 MAPK both contribute independently to IFN‐γ‐stimulated production of TARC and MDC in HaCaT keratinocytes, by increasing NF‐κB activation. J. Cell. Physiol. 226: 58–65, 2010. © 2010 Wiley‐Liss, Inc.     

Differential regulation of cytokine release and leukocyte migration by lipopolysaccharide-stimulated primary human lung alveolar type II epithelial cells and macrophages

Bacterial colonization is a secondary feature of many lung disorders associated with elevated cytokine levels and increased leukocyte recruitment. We hypothesized that, alongside macrophages, the epithelium would be an important source of these mediators. We investigated the effect of LPS (0, 10, 100, and 1000 ng/ml LPS, up to 24 h) on primary human lung macrophages and alveolar type II epithelial cells (ATII; isolated from resected lung tissue). Although macrophages produced higher levels of the cytokines TNF-alpha and IL-1beta (p < 0.0001), ATII cells produced higher levels of chemokines MCP-1, IL-8, and growth-related oncogene alpha (p < 0.001), in a time- and concentration-dependent manner. Macrophage (but not ATII cell) responses to LPS required activation of ERK1/2 and p38 MAPK signaling cascades; phosphorylated ERK1/2 was constitutively up-regulated in ATII cells. Blocking Abs to TNF-alpha and IL-1beta during LPS exposure showed that ATII cell (not macrophage) MCP-1 release depended on the autocrine effects of IL-1beta and TNF-alpha (p < 0.003, 24 h). ATII cell release of IL-6 depended on autocrine effects of TNF-alpha (p < 0.006, 24 h). Macrophage IL-6 release was most effectively inhibited when both TNF-alpha and IL-1beta were blocked (p < 0.03, 24 h). Conditioned media from ATII cells stimulated more leukocyte migration in vitro than conditioned media from macrophages (p < 0.0002). These results show differential activation of cytokine and chemokine release by ATII cells and macrophages following LPS exposure. Activated alveolar epithelium is an important source of chemokines that orchestrate leukocyte migration to the peripheral lung; early release of TNF-alpha and IL-1beta by stimulated macrophages may contribute to alveolar epithelial cell activation and chemokine production.