Functional heterogeneity of the kupffer cell population is involved in the mechanism of gadolinium chloride in rats administered endotoxin

BACKGROUND: The purpose of this study was to determine if evidence of functional heterogeneity between subtypes of the Kupffer cell (KC) may be involved in the mechanism of the protective effect of gadolinium chloride (GdCl3) in endotoxemia. METHODS: Rats pretreated with or without GdCl3 were administered lipopolysaccharide (LPS) or vehicle. Serum and liver tissues were collected after LPS administration for cytokine measurements and pathological and immunohistochemical evaluation. RESULTS: After LPS administration, increases in expression of TNF-alpha and IL-6 mRNA in the liver were blunted significantly by GdCl3. In control liver tissue, ED2-positive cells were a predominant fraction, with a few ED1-positive cells, and GdCl3 eliminated only ED2-positive cells. Further, ED2-positive cells were larger in size than ED1-positive ones. Importantly, the number of ED1-positive cells in the liver was increased about threefold in the control group but not in the GdCl3 group after LPS injection. Intermediate or large KCs isolated by counterflow centrifugal elutriation showed greater capacity for phagocytosis and production of superoxide and TNF-alpha than small ones. In contrast, IL-6 production was increased to a greater extent in small than in intermediate or large cells. GdCl3 eliminated the intermediate or large KC subpopulation predominantly. CONCLUSION: Collectively, functional heterogeneity of the KC population was involved in the mechanism of the protective effects of GdCl3 in endotoxemia. TNF-alpha derived from activated intermediate or large KCs may activate small KCs and the latter may be recruited to other organs, such as lungs and kidneys, and produce a large amount of IL-6, leading to multiple organ failure.     

氯化钆阻断大鼠原位肝移植免疫耐受的分子机制

目的 观察氯化钆(GdCl3)阻断枯否细胞(KCs)功能对大鼠移植肝脏中KCs核因子(NF)-kB活性的影响,探讨NF-KB活性与FasL及细胞因子基因转录的关系.方法 肝移植术后1、2和4 h活杀动物,取肝脏分离KCs,分别用凝胶电迁移率改变分析法(EMSA)、Western blot蛋白印迹法和逆转录-聚合酶链反应(RT-PCR)法检测KCs中NF-KB活性、Fas/FasL基因和蛋白质的表达及细胞因子白细胞介素(IL)-4和TNF-α基因转录.结果 GdCl3组KCs中NF-KB活性受到明显抑制,术后2、4 h FasL和细胞因子IL-4基因转录和蛋白的表达也明显减少(P<0.05),但术后2h TNF-α基因转录没有显著减少(P>0.05).结论 GdCl3可能阻断KCs中NF-kB活性,使KCs中FasL及IL-4的基因和蛋白表达减少,影响移植肝脏免疫耐受的形成.     

p38丝裂原活化蛋白激酶信号转导通路在严重烧伤大鼠枯否细胞肿瘤坏死因子α和白细胞介素1β产生中的作用

目的 探讨p38丝裂原活化蛋白激酶(MAPK)信号转导通路在严重烧伤大鼠枯否细胞(KCs)促炎性细胞因子肿瘤坏死因子α(TNF-α)和白细胞介素1β(IL-1β)产生中的作用。方法 健康成年的雄性SD大鼠32只,随机分为：假烫组;假烫 SB203580组;烧伤对照组;烧伤 SB203580组,每组8只。假烫或烧伤24h后分离出肝脏KCs,培养18h后加入50ng／ml的LPS进行刺激,18h后取上清液,用酶联免疫吸附法(ELISA)测定TNF-α和IL-1β的含量,并收集KCs,实时逆转录聚合酶链反应检测KCs内TNF-α和IL-1β mRNA表达的改变,蛋白印迹(Western blot)法检测KCs中p38MAPK和JNK活性的变化。结果 烧伤大鼠分离出的KCs培养上清液中TNF-α和IL-1β含量、KCs中TNF-α和IL-1β mRNA的表达均较假烫组的明显增强,同时KCs中p38 MAPK活性和JNK活性升高,SB203580能显著抑制大鼠KCs上清液中TNF-α和IL-1β含量、KCs中TNF-α和IL-1β mRNA的表达和p38MAPK活性的升高,对JNK活性无影响。结论p38MAPK信号转导通路介导了严重烧伤大鼠KCs促炎性细胞因子TNF-α和IL-1β的产生。     

Gadolinium chloride prevents mortality in hepatectomized rats given endotoxin

The purpose of this study was to determine if inhibition of Kupffer cells by gadolinium chloride (GdCl(3)) affects the arterial ketone body ratio (AKBR), liver injury, and mortality in hepatectomized rats administered lipopolysaccharide (LPS). Rats treated with or without GdCl(3) received a 70% hepatectomy. Either LPS (5 mg/kg) or vehicle (saline) was administered 48 h after hepatectomy. Further, hepatectomized rats were administered superoxide dismutase (CuZnSOD, 9 x 10(4) U/kg) before and every 3 h after LPS injection up to 9 h to assess involvement of superoxide in liver injury in this model. All hepatectomized rats with saline died within 24 h after LPS administration. In contrast, GdCl(3) prevented this mortality completely. Serum AST levels were about 160 IU/L in hepatectomized rats with vehicle; however, values were increased approximately 25-fold by LPS administration. In contrast, these increases were blunted significantly by about 90% by GdCl(3). Further, GdCl(3) also prevented decreases in AKBR caused by LPS. LPS caused severe liver injury, which was stopped almost completely by GdCl(3). LPS-induced increases in superoxide production by isolated Kupffer cells were stopped by about 90% by GdCl(3). Importantly, SOD administration prevented decreases in AKBR, liver injury, and mortality significantly as well as GdCl(3). These results indicated that GdCl(3) prevented liver injury and mortality caused by LPS most likely by inhibiting superoxide production by Kupffer cells. Thus, inhibition of activation of Kupffer cells could be useful for preventing liver dysfunction in postoperative endotoxemia.     

烧伤并发内毒素血症早期肝损害与TNF—αmRNA表达的实验研究

目的 观察TNF－αmRNA及其蛋白的组织分布和细胞定位,探讨烧伤并发内毒素血症早期肝损害的发生机理。方法 选用20％Ⅲ度TBSA烧伤大鼠合并腹腔内毒素（LPS）注射造成烧伤复合内毒素血症肝损害模型,采用光镜,电镜及免疫组化原位杂交染色观察产肝脏的形态功能变化、血清TNF－α含量变化、肝组织TNF－α和TNF－α mRNA的细胞定位及其分布。结果 烧伤复合内毒素血症组,光镜下主要表现肝窦反应,枯否细胞（KCs)激活与增生以及肝细胞（HCs)变性坏死等;电镜下主要表现肝窦内血小板聚集、纤维素沉积与中性粒细胞（PMNs)扣押以及KCs、肝细胞退变溶解等。血清丙氨酸氨基转氨酶（ALT）显著升高（P＜0．01）和白蛋白（ALB）轻度下降。组织TNF－α主要定位于肝窦内皮细胞（SECs)、KCs。TNF－αmRNA主要定位于KCs、PMNs、巨噬细胞（MPs)。而在单因素组主要特点为病变程度轻,肝功能损害不明显,血清TNF－α峰值滞后以及组织TNF－α和TNF－αmRNA表达相对较弱等。结论 TNF－α是参与烧伤复合内毒素血症早期肝脏损害的重要细胞因子。     

Glycine attenuates endotoxin-induced liver injury by downregulating TLR4 signaling in Kupffer cells

BACKGROUND: Several experimental studies have observed better outcomes after glycine treatment in patients with endotoxin-induced liver injuries, but its molecular mechanism is not yet fully understood. The purpose of this study was to evaluate the hypothesis that glycine attenuates endotoxin-induced liver injury by affecting endotoxin signal transduction in liver macrophages. METHODS: An animal model of endotoxin-induced liver injury was established by intraperitoneally injecting mice with 10 mg/kg body weight endotoxin fed a pretreatment diet with or without 5% (w/w) glycine. Blood and liver samples were obtained for analysis of liver morphology and to determine concentrations of alanine aminotransferase, endotoxin receptor Toll-like receptor 4 (TLR4), tumor necrosis factor-alpha (TNF-alpha), and interleukin (IL)-10 at various time points after injection. To investigate the effect of glycine on liver macrophages, Kupffer cells (KCs) were isolated and challenged by LPS (100 ng/mL), with or without glycine (4 mmol/l) pretreatment, and the expressions of TLR4, IL-10, and TNF-alpha were assayed at mRNA and protein levels. DNA-binding activity of nuclear factor-kappa B (NF-kappaB) was also analyzed using enzyme-linked immunosorbent assay. RESULTS: Dietary glycine significantly improved the survival rate of endotoxemic mice (P < .05), whereas serum alanine aminotransferase and TNF-alpha levels were significantly decreased at different time points (P < .05); IL-10 levels were increased (P < .05). Concurrently, LPS-induced hepatic tissue injury was attenuated as indicated by morphologic analysis; secretion of IL-10 in liver tissue (P < .05) was enhanced; and expression of TLR4 and TNF-alpha in liver tissue was downregulated (P < .05). Consistent with these in vivo experiments, enhanced secretion of IL-10 and inhibited expression of TLR4 and TNF-alpha caused by glycine pretreatment were also observed in LPS-stimulated KCs. NF-kappaB DNA-binding activity was also significantly inhibited by glycine (P < .05, respectively). CONCLUSIONS: Dietary glycine improved survival rates and liver function in endotoxemic mice by regulating the production of proinflammatory or anti-inflammatory cytokines in liver. It attenuated liver injury by deactivating KCs through inhibiting TNF-alpha secretion and increasing IL-10 production. The downregulative effect of glycine on the endotoxin signaling pathway and TLR4/NF-kappaB/TNF-alpha may be a novel potential mechanism by which glycine inhibits KC activity.     

CD14的表达及其在库普弗细胞激活中的意义

目的　探讨LPS对库普弗细胞 (KC)CD14表达的影响及CD14在LPS激活KC中的意义。　方法　在分离培养大鼠KC的基础上 ,应用免疫组织化学染色、RT PCR等方法分别测定CD14表达的变化、培养KC中上清中TNFα、IL 6和NO浓度。　结果　 (1)不同浓度的LPS使KC中CD14mRNA的表达及其蛋白合成明显增加 ,其表达量与LPS浓度呈剂量依赖性相关 ;(2 )同一浓度的LPS可使KC中CD14mRNA的表达及其蛋白合成明显增加 ,且在 3～ 6h左右达到高峰 ;(3)LPS刺激KC后产生的活性介质能明显上调新培养KC中CD14mRNA的表达及其蛋白合成 ;(4)在血清存在的情况下加入抗CD14单抗或在无血清的情况下单独加入LPS ,可明显降低KCTNFα、IL 6和NO的释放。而后者如果同时加入LBP ,则可明显上调培养KC中的TNFα、IL 6和NO浓度。　结论　 (1)LPS及其刺激KC后产生的活性介质与CD14mRNA的表达及其蛋白合成密切相关 ,并推测在实验 1～ 3h的CD14表达的增强可能主要由LPS引起 ,而此后CD14表达的进一步增强可能与KC释放的细胞因子密切相关 ;(2 )低浓度LPS对KC的激活是CD14依赖的。     

Benefit of Kupffer cell modulation with glycine versus Kupffer cell depletion after liver transplantation in the rat: effects on postischemic reperfusion injury, apoptotic cell death graft regeneration and survival.

Inhibition or destruction of Kupffer cells (KC) may protect against ischemia-reperfusion (IR) induced primary graft nonfunction (PNF) in liver transplantation. Besides KC activation, PNF is characterized by microvascular perfusion failure, intrahepatic leukocyte accumulation, cell death and hepatocellular dysfunction. KCs can be inactivated by different agents including gadolinium chloride (GdCl3), methyl palmitate (MP) and glycine. The effects of three KC inactivators on IR-injury after rat liver transplantation were compared in the present study. Lewis liver donors were treated with GdCl3, MP, glycine or saline (control). Liver grafts were transplanted following 24 h storage (UW solution). KC populations and IR damage were assessed by histologic analysis, quantitative real-time polymerase chain reaction (RT-PCR) and intravital microscopy. The number of hepatic ED-1 positive macrophages was diminished after GdCl3 (114.8+/-4.4/mm2 liver tissue) and MP treatment (176.0+/-5.0), versus the glycine (263.9+/-5.5) and control (272.1+/-5.6) groups. All three treatment modalities downregulated phagocytic activity for latex particles, paralleled by reduced microvascular injury (acinar perfusion index, GdCl3: 0.75+/-0.03; MP: 0.83+/-.03; glycine: 0.84+/-0.03; 0.63+/-0.03). Quantitative RT-PCR revealed elevated myeloperoxidase mRNA after glycine versus GdCl3 and MP pretreatment (3.2- and 3.4-fold, P=0.011, respectively), without difference to controls (2.9-fold of glycine). TNFalpha-mRNA was reduced after glycine- (5.2-fold), GdCl3- (19.7-fold), MP-treatment (39.5-fold) compared with controls. However, profound prevention of intrahepatic cell death and liver graft failure was solely achieved with glycine preconditioning. Different than GdCl3 and MP, glycine modulates rather than destroys KCs. Glycine appears to preserve cell viability and to TNFalpha/leukocyte dependent organ regeneration capacity, which is related to increase graft survival following liver transplantation.     

Autoregulation by eicosanoids of human Kupffer cell secretory products. A study of interleukin-1, interleukin-6, tumor necrosis factor-alpha, transforming growth factor-beta, and nitric oxide.

OBJECTIVE: Methods employed previously to analyze the secretory behavior of rodent Kupffer cells (KC) were used to examine the human KC's secretory response to lipopolysaccharide (LPS). SUMMARY BACKGROUND DATA: As the resident hepatic macrophage, the KC resides at the interface between the portal and systemic circulations. Consequently, this cell may play an integral role in the immune response to antigens and bacteria in the sinusoid. Study of cytokine production by the KC has relied predominantly on the rat as the source of these cells. Whether human KCs respond similarly to rat KCs after LPS stimulation has been a matter of speculation. METHODS: Kupffer cells obtained from seven human livers were tested under conditions identical to those used to study rat KCs. Kupffer cells rested for 12 hours after isolation were stimulated with LPS (2.5 micrograms/mL). Arginine concentration in the culture medium varied from 0.01 to 1.2 mM. To examine the role of eicosanoids, parallel culture wells received indomethacin (10 microM). Culture supernatants were assayed for interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), transforming growth factor-beta (TGF-beta), prostaglandin E2 (PGE2), and nitric oxide. RESULTS: Similar to the rat KC, LPS-stimulated human KCs released IL-1, IL-6, TNF-alpha, TGF-beta, and PGE2. However, unlike rat KCs, nitric oxide could not be detected, regardless of whether the human KCs were exposed to LPS, interferon-gamma (INF-gamma), or LPS + IFN-gamma. Similar to rat KCs, indomethacin prevented PGE2 release while significantly upregulating TNF-alpha, IL-1, and IL-6, but not TGF-beta, consistent with an autoregulatory control of eicosanoids over proinflammatory cytokines. As has been shown in the rat, physiologic levels of L-arginine (0.01 mM) significantly enhanced LPS-induced PGE2 secretion relative to the response in medium containing standard L-arginine concentration (1.2 mM); however, unlike the rat KC, the human's cytokine response to LPS was not downregulated by this enhanced PGE2 release. CONCLUSIONS: Although many functional features are shared by rat and human KCs, significant differences do exist. Such discrepancies reinforce the need to proceed with caution when generalizing from the results obtained in other species to human physiology.     

甘氨酸抑制脂多糖介导的鼠枯否细胞激活的时相选择及机制探讨

目的探讨甘氨酸抑制脂多糖介导的鼠枯否细胞激活效应相关机制和甘氨酸的最佳用药时机。方法将40只BALB/c小鼠分为内毒素组、预防组、早期治疗组和后期治疗组,每组10只。分离培养枯否细胞后,内毒素组加入脂多糖(10mg/L),预防组、早期治疗组和后期治疗组分别在加入脂多糖前24h、加入后0和4h加入甘氨酸(1mmol/L),分别在加入脂多糖后0、1、2、6和12h,采用逆转录聚合酶链反应及蛋白印迹法测定枯否细胞的白细胞介素1受体相关激酶4(IRAK4)mRNA和蛋白表达水平,用酶联免疫吸附法检测枯否细胞的核因子κB(NFκB)活性和培养上清液的肿瘤坏死因子α(TNFα)含量。结果脂多糖刺激后,预防组的IRAK4mRNA和蛋白表达、NFκB活性的相对峰值分别为3.64±1.13、34.54±10.31、0.47±0.10,TNFα峰值为(1780.70±210.17)pg/ml,与早期治疗组比较,差异均无统计学意义,但与内毒素组和后期治疗组比较,各峰值均明显降低,差异有统计学意义。结论提前或者在脂多糖刺激的同时应用甘氨酸,能有效抑制脂多糖介导的枯否细胞激活效应,其作用机制之一可能为抑制IRAK4的表达。     

Organ-specific regulation of pro-inflammatory molecules in heart, lung, and kidney following brain death

BACKGROUND: Nonspecific inflammatory events following brain death may increase the intensity of the immunological host response. The present study investigated the course of pro-inflammatory molecules in heart, lung, kidney, and plasma after brain death induction. MATERIALS AND METHODS: Brain death was induced in five pigs by inflation of an intracranial Foley catheter and five pigs were sham-operated as controls. Each experiment was terminated 6 h after brain death/sham operation and the organs were harvested. We measured the mRNA and protein levels for TNF-alpha, IL-1beta, and IL-6 in heart, lung, kidney, and plasma. Additionally, the mRNA expression for IL-6R, ICAM-1, MCP-1, and TGF-beta was determined in each organ. RESULTS: After 6 h, the plasma cytokine levels were higher in the brain-dead animals than in the sham-operated. In heart, lung, and kidney there was an increase in IL-6 and IL-1beta following brain death, while TNF-alpha was up-regulated in lung only (P < 0.05). MCP-1 and TGF-beta were significantly higher in heart and lung and IL-6R increased in heart after brain death (P < 0.05). CONCLUSIONS: Brain death was associated with non-uniform cytokine expression patterns in the investigated organs. These expression patterns may cause variable pro-inflammatory priming resulting in different degrees of damage and explain the organ-specific variation in outcomes after transplantations.     

Regulation of pro-inflammatory and anti-inflammatory cytokine responses by Kupffer cells in endotoxin-enhanced reperfusion injury after total hepatic ischemia

The effects of Kupffer cells on cytokine responses in endotoxin-enhanced reperfusion injury after total hepatic ischemia were investigated in this study. Male rats pretreated with either normal saline solution (NS group) or gadolinium chloride (GdCl(3)) to inhibit Kupffer cell function (GC group) were subjected to 60 min of hepatic ischemia. These animals received either normal saline solution or sublethal doses of endotoxin (1 mg/kg) at reperfusion. In the NS group, endotoxin administration induced an enhanced tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 production 1 h after reperfusion with a subsequent peak of macrophage inflammatory protein-2 (MIP-2) levels, which resulted in a 7-day survival rate of 30%. Despite endotoxin administration, GdCl(3) pretreatment significantly suppressed TNF-alpha and increased interleukin-10 production 1 h after reperfusion, which led to a decline in MIP-2 production and amelioration of functional and structural liver damage with a 7-day survival rate of 80%. Augmented pro-inflammatory and anti-inflammatory cytokine responses by Kupffer cells were associated with endotoxin-enhanced reperfusion injury after hepatic ischemia. Kupffer cell blockade has a potential to attenuate the insult via modulation of cytokine responses.     

盐酸戊乙奎醚对内毒素性急性肺损伤大鼠肺组织Toll样受体4mRNA和Toll样受体2mRNA表达的影响

目的 探讨盐酸戊乙奎醚对内毒索性急性肺损伤大鼠肺组织Toll样受体4(TLR4)mRNA和Toll样受体2(TLR2)mRNA表达的影响.方法 健康SD大鼠60只,雌雄不拘,体重200～220g,采用随机数字表法,将大鼠随机分为5组(n=12),对照组(C组)、LPS组和低、中、高剂量盐酸戊乙奎醚组(P1组～P3组).C组腹腔注射生理盐水2ml;LPS组腹腔注射LPS 8mg/kg;P1组～P3组分别腹腔注射LPS 8 mg/kg和盐酸戊乙奎醚0.3、1.0和3.0 mg/kg.给药结束后6 h时开胸,心室取血,并取肺组织,采用ELISA法测定血清TNF-α和Ib-6的浓度,RT-PCR法测定肺组织TLR4 mRNA和TLR2 mRNA 的表达水平,并观察肺组织病理学结果.结果 与C组比较,LPS组、P1组～P3组血清TNF-α、IL-6浓度和肺组织TLR4 mRNA、TLR2 mRNA表达均升高(P<0.05);与LPS组比较,P2组和P3组血清TNF-α、IL-6浓度和肺组织TLR4 mRNA、TLR2 mRNA表达均降低(P<0.05),P1组上述指标差异无统计学意义(P>0.05);与P1组比较,P2组和P1组血清TNF-α、IL-6浓度和肺组织TLR4 mRNA、TLR2 mRNA表达均降低(P<0.05);P2组和P3组血清TNF-α、IL-6浓度和肺组织TLR4 mRNA、TLR2 mRNA表达比较差异无统计学意义(P>0.05).P2组和P3组肺组织病理学损伤程度明显轻于LPS组.结论 盐酸戊乙奎醚可通过下调肺组织TLR4 mRNA和耵JR2 mRNA的表达,降低炎性反应,从而减轻大鼠内毒素性急性肺损伤.

Abstract：

Objective To investigate the effect of penehyclidine (PHCD) on Toll-like receptor 4 (TLR4)mRNA and Toll-like receptor 2 (TLR2) mRNA expression in the lung tissue in rats with acute lung injury induced by lipopolysaccharide (LPS) .Methods Sixty healthy SD rats of both sexes weighing 200-220 g were randomly divided into 5 groups ( n = 12 each) :control group (group C) , LPS group and P1-3 groups. Acute lung injury was induced by intraperitoneal (IP) LPS 8 mg/kg in LPS and P1-3 groups. PHCD 0.3, 1.0 and 3.0 mg/kg were given IP after LPS administration in P1-3 groups. The animals were anesthetized at 6 h after IP LPS. Blood samples were collected for determination of serum TNF-α and IL-6 concentrations ( by ELISA) and then sacrificed, the lungs were immediately removed for determination of TLR4 mRNA and TLR2 mRNA expression (by RT-PCR), and microscopic examination. Results LPS significantly increased TLR4 mRNA and TLR2 mRNA expression in the lung tissue and serum TNF-α and IL-6 concentrations. PHCD 1.0 or 3.0 mg/kg significantly inhibited LPS-induced increase in TLR4 mRNA and TLR2 mRNA expression in the lung tissue and serum TNF-α and ILr6 concentrations.The lung histopathologic damage was significantly ameliorated in P2 and P3 groups as compared with group LPS.Conclusion PHCD can protect the lungs against LPS-induced acute lung injury through inhibiting TLR4 mRNA and TLR2 mRNA expression in the lung tissue and reducing the inflammatory response.     

Role of p38 mitogen-activated protein kinase in Kupffer cell secretion of the proinflammatory cytokines after burn trauma

This study was designed to investigate the role of p38 mitogen-activated protein (MAP) kinase on Kupffer cells (KCs) secretion of proinflammatory cytokines such as tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta and hepatic injury following burn trauma. Sprague-Dawley rats were randomized into four groups: (1) sham burn rats given vehicle, (2) sham burn rats given the p38 MAP kinase inhibitor SB203580 (10mg/kg i.v., 15min and 12h after sham burn), (3) rats given a 30% total body surface area (TBSA) full-thickness burn and fluid resuscitation plus vehicle, and (4) burn rats given injury and fluid resuscitation plus SB203580. Rats from each group were killed at 24h post-burn to examine plasma aspartate transaminase (AST) and alanine transaminase (ALT) and KCs were isolated. The KCs secretion of TNF-alpha and IL-1beta and p38 MAP kinase activity (by Western blot analysis) were also examined. These studies showed by more significant activation of p38 MAP kinase in KCs harvested from burn rats than from shams. Burn trauma resulted in hepatic dysfunction and promoted KCs secretion of TNF-alpha and IL-1beta. SB203580 inhibited p38 MAP kinase activity, reduced KCs secretion of proinflammatory cytokines, and alleviated burn-mediated hepatic dysfunction. These data suggest p38 MAP kinase activation is one important aspect of the signaling event that may mediate the KCs secretion of proinflammatory cytokines TNF-alpha and IL-1beta following burn trauma.     

'Endotoxin tolerance': TNF-alpha hyper-reactivity and tubular cytoresistance in a renal cholesterol loading state

The term 'endotoxin tolerance' defines a state in which prior endotoxin (lipopolysaccharide (LPS)) exposure induces resistance to subsequent LPS attack. However, its characteristics within kidney have not been well defined. Hence, this study tested the impact of LPS 'preconditioning' (LPS-PC; 18 or 72 h earlier) on: (i) selected renal inflammatory mediators (tumor necrosis factor (TNF)-alpha, interleukin-10 (IL-10), monocyte chemotactic protein-1 (MCP-1), inducible nitric oxide synthase (iNOS), Toll-like receptor 4 (TLR4); protein or mRNA); (ii) cholesterol homeostasis (a stress reactant); and (iii) isolated proximal tubule (PT) vulnerability to hypoxia or membrane cholesterol (cholesterol oxidase/esterase) attack. Two hours post LPS injection, LPS-PC mice manifested reduced plasma TNF-alpha levels, consistent with systemic LPS tolerance. However, in kidney, paradoxical TNF-alpha hyper-reactivity (protein/mRNA) to LPS existed, despite normal TLR4 protein levels. PT TNF-alpha levels paralleled renal cortical results, implying that PTs were involved. LPS-PC also induced: (i) renal cortical iNOS, IL-10 (but not MCP-1) mRNA hyper-reactivity; (ii), PT cholesterol loading, and (iii) cytoresistance to hypoxia and plasma membrane cholesterol attack. A link between cholesterol homeostasis and cell LPS responsiveness was suggested by observations that cholesterol reductions in HK-2 cells (methylcyclodextrin), or reductions in HK-2 membrane fluidity (A2C), blunted LPS-mediated TNF-alpha/MCP-1 mRNA increases. In sum: (i) systemic LPS tolerance can be associated with renal hyper-responsiveness of selected components within the LPS signaling cascade (e.g., TNF-alpha, iNOS, IL-10); (ii) PT cytoresistance against hypoxic/membrane injury coexists; and (iii) LPS-induced renal/PT cholesterol accumulation may mechanistically contribute to each of these results.     

核因子κB诱骗寡核苷酸对大鼠肝脏的Kupffer细胞共刺激分子表达的影响

目的 探讨核因子κB(NF-κB)诱骗寡核苷酸(ODN)对大鼠肝脏Kupffer细胞(KC)膜表面共刺激分子表达的影响.方法 取雄性SD大鼠,麻醉后经门静脉插管,以0.5 g/L Ⅳ型胶原酶体外循环灌注消化肝脏,不连续Percoll密度梯度离心分离KC.以人工合成的NF-κB诱骗ODN转染KC,再以终浓度为1 mg/L的脂多糖(LPS)刺激(NF-κB诱骗组),培养8 h后,采用逆转录聚合酶链反应测定KC膜表面CD80、CD40和CD54 mRNA的表达.以不转染NF-κB诱骗ODN、但接受LPS刺激的KC为对照(LPS刺激组),正常大鼠KC为对照组.每组每份标本含KC 1×105个.结果 每个大鼠肝脏可以分离得到KC(3～4)×106个,NF-κB诱骗ODN可以在KC内维持12 d而不被降解.对照组KC膜表面CD80、CD40、CD54 mRNA呈低表达;LPS刺激组KC表面3种共刺激分子表达均较对照组明显升高,分别是对照组的2.53、2.51和2.74倍(P<0.01);NF-κB诱骗组CD80和CD40 mRNA表达明显低于LPS组,仅为LPS组的0.52和0.55倍(P<0.01),而CD54 mRNA表达的差异无统计学意义(P>0.05).结论 在体外,NF-κB诱骗ODN可以高效地抑制KC表面共刺激分子的表达,为活体内应用NF-κB诱骗ODN抑制肝移植后急性排斥反应提供了实验依据.     

Local expression of tumor necrosis factor-alpha and interleukin-10 on wound healing of intestinal anastomosis during endotoxemia in mice

BACKGROUND: This study aimed to evaluate the integrity of anastomotic wound healing after digestive surgery under septic conditions and define the participation of local expression of tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) around the anastomotic segment. MATERIALS AND METHODS: Experimental animals were divided into lipopolysaccharide (LPS) and control groups, which had either LPS or normal saline solution injected into the peritoneal cavity 24 h before transection and anastomosis of the colon. Anastomotic bursting pressure (ABP) and tissue hydroxyproline concentration (HP) were measured as indicators of wound healing. Immunohistochemical staining for TNF-alpha and IL-10 on tissue samples obtained from the anastomotic segment were examined 1, 6, and 24 h after the operation. The reactive cells were counted under light microscopy. RESULTS: ABP and HP were significantly lower in the LPS group than in the control group 7 days after surgery. In the LPS group, TNF-alpha expression increased about threefold over that in the control group 1 h after the operation. TNF-alpha-reactive cells were observed until 24 h after the operation in the LPS group, but not in the control group. On the other hand, IL-10 was not expressed in the control group during the observed period, whereas IL-10 was observed 24 h after the operation in the LPS group. CONCLUSIONS: It is suggested that anastomotic wound healing was impaired after the digestive surgery in animals treated with intraperitoneal LPS, and that local expression of TNF-alpha and IL-10 at the anastomotic site acts as an inhibitory factor in the wound healing process.     

beta(2)-adrenoceptor agonist suppresses renal tumour necrosis factor and enhances interleukin-6 gene expression induced by endotoxin.

BACKGROUND: beta(2)-Adrenoceptor activation regulates tumour necrosis factor (TNF)-alpha and interleukin-6 (IL-6) production in cultured renal cells. However, it remains uncertain whether, in vivo, the administration of beta(2)-adrenoceptor agonists regulate renal TNF-alpha and IL-6 mRNA following lipopolysaccharide (LPS) stimulation to cause endotoxaemia. This study was performed in order to evaluate the effect of beta(2)-adrenoceptor agonist on renal TNF-alpha and IL-6 production. METHODS: Four-week-old Wistar rats pre-treated with the beta(2)-adrenoceptor agonist terbutaline or formoterol, and/or the beta- and beta(2)-adrenoceptor antagonists (propanolol, ICI118,551), were injected with LPS (1 mg i.p.), and then 2, 4 or 6 h later, kidneys (cortex, medulla), spleen, thymus and plasma were collected to assay TNF-alpha and IL-6 mRNA levels and their respective protein release. RESULTS: Administration of beta(2)-adrenoceptor agonists suppressed TNF-alpha mRNA expression in the whole kidney, by 61% (P<0.05), as well as plasma, spleen and thymus TNF-alpha protein and mRNA expression 2 hours after injection of LPS. On the other hand, although IL-6 levels in plasma, spleen and thymus mRNA expression were suppressed significantly by administration of beta(2)-adrenoceptor agonists, the basal- and LPS-induced IL-6 mRNA levels in the whole kidney were increased 1.6- and 1.2-fold (P<0.05), respectively, by treatment with beta(2)-adrenoceptor agonists. beta(2)-Adrenoceptor agonist suppressed LPS-induced TNF-alpha mRNA expression by 35% (P<0.05) and stimulated LPS-induced IL-6 mRNA expression by 1.5-fold (P<0.05) in the medullary region of kidney. CONCLUSIONS: beta(2)-Adrenoceptor agonists down-regulate renal TNF-alpha mRNA expression following LPS-induced endotoxaemia. This effect was particularly apparent in the renal medulla. IL-6 mRNA expression in the renal medulla was up-regulated by the agonists whereas plasma, spleen and thymus IL-6 levels were completely inhibited by the agonist, which suggests the existence of tissue specific regulation of IL-6 production in the kidney by beta(2)-adrenoceptor activation.