Escherichia coli lipopolysaccharide effects on proliferating rat liver cells in culture: a morphological and functional study.

Liver cells have been implicated in playing an important role in the pathogenesis of endotoxic shock-associated liver injury. The present study was designed to investigate Escherichia coli 0111:B4 lipopolysaccharide (LPS) effects on proliferating rat liver cells in culture. Isolated cell system can rarely serve as models of complete organisms, but with using an in vitro test-model, endogenic factors (e.g. hormonal effects, nervous influences, blood activation, etc.) or experimental stress in laboratory animals can be eliminated as variables affecting hepatocellular responses. In the proposed in vitro model, using specially proliferating liver cells, morphological cell alterations were observed after 30 min and low doses endotoxin administration (10 micrograms/ml). LPS induced an extreme fragility and a diminished adhesion capacity on cells. Cell-to-cell contacts were also disturbed. The LPS treatment produced extreme heterogeneity in liver cell size, enlargement of nuclei, nuclear and cytoplasmic protrusions, and increased the number of large nucleoli and lipid droplets, also decreasing the angiotensin action on intracellular calcium levels. The effects observed after the LPS exposition can be related with an altered metabolism of the liver cells in culture due to a destabilization of plasma membrane, a transmembrane signalling alteration, and a mitochondrial damage. The specificity of cell response to endotoxic lipopolysaccharide suggests a multiple membrane damage inducing important metabolic disturbances.     

Divergent results induced by different types of septic shock in transglutaminase 2 knockout mice

Acute sepsis can be induced by cytokines such as TNF-α and biological products such as LPS. All of these agents cause systemic inflammation, which is characterized by hemodynamic shock and liver toxicity. However, the outcomes of different septic shock models were totally opposite in transglutaminase 2 knockout (TGase 2^?/?) mice. The aim of our study was to clarify the role of TGase 2 in liver injury. Therefore, we explored the role of TGase 2 in liver damage using two different stress models: LPS-induced endotoxic shock and TNF-α/actinomycin D (ActD)-induced sepsis. TNF-α-dependent septic shock resulted in increased liver damage in TGase 2^?/? mice compared with wild-type (WT) mice, and was accompanied by increased levels of caspase 3 and cathepsin D (CTSD) in the damaged liver. Conversely, LPS-induced septic shock resulted in ablation of inflammatory endotoxic shock in TGase 2^?/? mice and decreased liver injury. We found that TGase 2 protected liver tissue from TNF-α-dependent septic shock by reducing the expression of caspase 3 and CTSD. However, TGase 2 differently participated in increased the hemodynamic shock in LPS-induced septic shock through macrophage activation rather than protecting direct liver damage. Therefore, these findings demonstrate that septic shock caused by different agents may induce different results in TGase 2^?/? mice depending on the primary target organs affected.     

脂多糖诱导小鼠脏器中胞间粘附分子-1的表达

为研究脂多糖（lipopolysaccharide,LPS)诱导的内毒素休克小鼠多种脏器中胞间粘附分子-1（intercellu-lar adhesion molecule-1,ICAM-1)表达的差异。用5mg/kgLPS腹腔注射小鼠后,分别采用Western blotting和RT-PCR法检测组织中ICAM-1蛋白和mRNA的表达情况,结果显示,在正常小鼠,ICAM-1蛋白和mRNA的表达在肺中最多,其次是脾脏,在肾脏和肠有少量表达,在肝脏和心脏中未能检出,LPS腹腔注射后6h可诱导小鼠发生内毒素休克,此时,ICAM-1蛋白表达仍以在肺中最多,在肝、脾、心、肾和肠依次减少;其中在肺,肾和脾分别比正常时增加4.5、3.0和1.5倍,而且在正常时不能检出的肝和心中呈现阳性,但在肠中则变化不大,脏器中ICAM-1mRNA亦相应显著增加,上述结果表明,在LPS诱导的内毒素休克小鼠的多种脏器中ICAM-1蛋白和mRNA表达显著增加,脏器间ICAM-1表达上调的差异可能带来内毒素休克时脏器的不同易伤性,抑制ICAM-1的表达可能对内毒素休克的防治有重要的意义。     

Rat glomerulosa cells in primary culture and E. coli lipopolysaccharide action

During endotoxic shock there is a dysfunction of the adrenal gland; both corticosterone and aldosterone secretion are altered. The aim of the present study is to use glomerulosa cells in primary culture as a target of lipopolysaccharide (LPS) action. Glomerulosa cells cultured in basal conditions are able to proliferate; bFGF and ACTH have antagonic effects, bFGF increases proliferation whereas ACTH is antimitogenic. LPS has a biphasic effect, in the short term it is antimitogenic and in the long term increases the proliferation rate. LPS inhibits ACTH-induced corticosterone secretion in a dose-dependent manner in glomerulosa cells in culture similar to that in fasciculata cells, but it does not exert an important direct effect on aldosterone secretion. These results show that LPS exerts different effects in ACTH and ANG II signal transduction pathways and in the two enzymes which catalyze the late step in the steroidogenesis, 11beta-hydroxylase and aldosterone synthase, which could be in agreement with the existence of both enzymes, regulated independently, in rat zona glomerulosa cells.     

Proteome analysis of rat liver mitochondria reveals a possible compensatory response to endotoxic shock

Organ failure induced by endotoxic shock has recently been associated with affected mitochondrial function. In this study, effects of in vivo lipopolysaccharide-challenge on protein patterns of rat liver mitochondria in treated animals versus controls were studied by two-dimensional electrophoresis (differential image gel electrophoresis). Significant upregulation was found for ATP-synthase alpha chain and superoxide dismutase [Mn]. Our data suggest that endotoxic shock mediated changes in the mitochondrial proteome contribute to a compensatory reaction (adaptation to endotoxic shock) rather than to a mechanism of cell damage.     

Regulation of the host response to bacterial lipopolysaccharides

Bacterial endotoxins or lipopolysaccharides (LPS) are unique glycolipids present in the outer cell membrane of all gram-negative bacteria. It is now generally recognized that LPS is of primary importance in initiating the pathophysiological changes that often accompany gram-negative bacillary infections in humans including hypotensive shock, disseminated intravascular coagulation, and metabolic abnormalities. Although the biochemical mechanisms of these changes are not well understood, increasing emphasis has been placed on defining the biochemical response of the macrophage (M phi) to LPS. In this paper we describe two M phi-derived factors induced by LPS that may be important in the expression of endotoxic activity in the host. These are a procoagulant activity, which is present on the cell membrane of LPS-treated rabbit liver M phi and acts by directly activating coagulation factor X, and a factor released into the supernatant by LPS-treated peritoneal exudate M phi, which suppresses steroidogenesis in explanted adrenocortical cells. The potential role of the M phi in regulating the binding of LPS to high-density lipoproteins through the induction of acute phase proteins is also considered.     

Organisation of microtubules and actin filaments in the cortex of differentiating Selaginella guard cells

Summary Using fluorescent probes and confocal laser scanning microscopy we have examined the organisation of the microtubule and actin components of the cytoskeleton in kidney-shaped guard cells of six species of Selaginella. The stomata of Selaginella exhibit novel cytoskeletal arrangements, and at different developmental stages, display similarities in microtubule organisation to the two major types of stomata: grass (dumbbell-shaped) and non-grass (kidney-shaped). Initially, cortical microtubules and F-actin radiate from the stomatal pore and extend across the external and internal periclinal cell surfaces of the guard cells. As the stomata differentiate, the cytoskeleton reorients only along the internal periclinal walls. Reorganisation is synchronous in guard cells of the same stoma. Microtubules on the inner periclinal walls of the guard cells now emanate from areas of the ventral wall on either side of the pore and form concentric circles around the pore. The rearrangement of F-actin is similar to that of microtubules although F-actin is less well organised. Radial arrays of both microtubules and F-actin are maintained adjacent to the external surfaces. Subsequently, in two of the six species of Selaginella examined, microtubules on both the internal and external walls become oriented longitudinally and exhibit no association with the ventral wall. In the other four species, microtubules adjacent to the internal walls revert to the initial radial alignment. These findings may have implications in the development and evolution of the stomatal complex.Abbreviations GC guard cell - MT microtubule     

诱导型一氧化氮合酶对内毒素休克小肠微循环的影响

采用静脉注射脂多糖(1ipopolysaccharide,LPS)的方法建立小鼠内毒素休克模型,探讨内毒素休克时小肠微循环的变化以及诱导型一氧化氮合酶(iNOS)对小肠微循环的影响。实验过程中连续监测小鼠平均动脉血压(mean afterial pressure,MAP)变化情况。利用FTTC标记红细胞和活体显微镜方法直接观察并计算小鼠小肠绒毛尖端小动脉和毛细血管内红细胞的流速和流量,并观察敲除小鼠iNOS基因和选择性iNOS抑制剂S-methylthiourea sulfate(SMT)对实验过程中小肠微循环的影响。结果显示,对于野生型小鼠,应用SMT处理和敲除iNOS基因对基线的MAP、小肠绒毛尖端小动脉和毛细血管的红细胞流速和流量没有显著性差别。给予LPS后,小鼠的MAP进行性下降。给予LPS前,应用SMT和敲除小鼠iNOS基因可以显著提高MAP：给予LPS后,小鼠小肠绒毛尖端小动脉和毛细血管内红细胞流速和流量显著下降。给予LPS前,应用SMT和敲除小鼠iNOS基因可以显著提高小肠绒毛尖端小动脉和毛细血管的红细胞流速和流量。结果表明,iNOS在内毒素休克小肠微循环衰竭的过程中发挥重要作用。一能性     

Heat shock induces simultaneous rearrangements of all known cytoskeletal filaments in normal interphase fibroblasts

Hyperthermia (heat shock (HS)) induces changes in morphology of nucleoli, cytoplasmic organelles, and cytoskeleton. Responses to hyperthermia are, as a rule, similar in all types of eukaryote cells. However, there is no information on the uniformity of the cytoskeleton heat shock response (CHSR) in different cell types. This has led to the conclusion that the eukaryote CHSR depends on the cell type. We studied CHSR only in one cell type-in normal embryonic mouse fibroblasts (NEMFs) and in normal embryonic rat fibroblasts (NERFs), as well as in normal postnatal rat fibroblasts (NPRFs), by using the method of fluorescence microscopy. Incubation of the cells at 43°C led to a rearrangement of cytoskeleton. Responses of cytoskeleton to HS in NEMF, NERF, and NPRF were similar. Heat shock resulted in disassembly of bundles of actin filaments (AFs), marked changes in microtubule (MT) morphology, and collapse of intermediate filaments (IFs) around the nucleus. Rearrangements of different cytoskeleton filament types occurred simultaneously and were seen as soon as after 2–4 min. After 30–120 min of incubation at 43°C, the cells were still capable of rebuilding the actin cytoskeleton after the temperature had returned to normal (37°C). We believe that the cytoskeleton rearrangement under the action of HS can be of vital importance in cell protection against temperature stress. Data are discussed on possible coupling of the CHSR process with rearrangement of the protein synthesizing system, which leads to initiation and/or stimulation of synthesis of HS proteins.     

Epidermal cells of a symbiosis-defective mutant of Lotus japonicus show altered cytoskeleton organisation in the presence of a mycorrhizal fungus

The influence of the mycorrhizal fungus Gigaspora margarita on cytoskeleton organisation in epidermal cells of Lotus japonicus roots was compared between plants of the wild type Gifu and the mutant Ljsym4-2, in which the fungus is confined to the epidermal cells. Immunofluorescence labelling of plant microtubules and microfilaments showed only limited alterations in the peripheral cytoskeleton of epidermal cells during early stages of fungal interaction with the wild type. Later, microtubules and microfilaments enveloped the growing hypha, while the host cell nucleus moved close to the fungus. In contrast, epidermal cells of the mutant responded with disorganisation and disassembly of microtubules and microfilaments before and during fungal penetration attempts. The fungus penetrated only as far as to epidermal cells, whose cytoplasm became devoid of tubulin and actin, suggesting cell death. The close relationship between host cytoskeleton organisation and compatibility with the fungus suggests that a functional Ljsym4 gene is necessary for correct reorganisation of the epidermal cell cytoskeleton in the presence of the fungus and for avoiding hypersensitivity-like reactions.     

Characterization of the simultaneous binding of Escherichia coli endotoxin to Kupffer and endothelial liver cells by flow cytometry.

BACKGROUND: The triggering of cellular responses during endotoxic shock is initiated for the binding of endotoxin (lipopolysaccharide; LPS) to the cell surface. Kupffer and endothelial liver cells, involved in the removal of endotoxin from blood circulation, show in vitro a rapid response to LPS in the absence of serum. METHODS: A double-labeling fluorescent assay was designed to evaluate the binding properties of Escherichia coli O111:B4 LPS to individual endothelial and Kupffer cells in suspension, where both populations occurred in the same relative proportion as in liver. After immunolabeling of the Kupffer cell population with the monoclonal antibody ED1 conjugated to R. phycoerythrin, the binding characteristics of LPS labeled with fluorescein to both endothelial and Kupffer cells were simultaneously studied by flow cytometry in serum-free conditions. RESULTS: Specific and saturable binding of endotoxin was observed with both populations, showing properties of a receptor-mediated process. The Kupffer cell population showed a faster capacity and a higher affinity for LPS binding. The Hill coefficients indicated positive cooperativity in the LPS interaction with both populations. CONCLUSIONS: Specific endotoxin binding to liver sinusoidal cells occurs in a serum-independent manner, particularly at high LPS concentrations. Flow cytometry is a fast, precise, and efficient technique to evaluate the simultaneous interaction of a ligand with two different cell types.     

Changes in the ascorbic acid levels of peritoneal lymphocytes and macrophages of mice with endotoxin-induced oxidative stress.

Ascorbic acid (AA) is an important cytoplasmic antioxidant that mice synthesize in the liver, the intracellular levels of which decrease in an oxidative stress situation such as endotoxic shock. The present work deals with the changes in AA levels, that modulate the immune function, in the two main immune cells, namely macrophages and lymphocytes, from female BALB/c mice suffering endotoxic shock caused by intraperitoneal injection of Escherichia coli lipopolysaccharide (LPS) (100 mg/kg). The intake by cells of this antioxidant present in vitro at different concentrations was also studied. The animals show an oxidative stress, standardized in previous studies, that causes mortality at 30 h after LPS injection. The cells were obtained from the peritoneum at 2, 4, 12 and 24 h after LPS or PBS (control) injections and were incubated without or with AA at 0.01, 0.1 and 1 mM for 10, 30, 60, 120 or 180 min. The hepatic AA levels were also studied at 0, 2, 4, 12 and 24 h after LPS injection. The peritoneal cells obtained from animals injected with LPS showed increased AA levels in relation to the control cells at all times after LPS injection, with maximal effect at 12h. The AA levels decreased after this time, in agreement with changes in the AA hepatic levels. The increase was due to the AA of lymphocytes since macrophages showed a decrease in AA at different times after LPS injection. Both cells showed an increase in the intracellular levels of AA when this antioxidant was added in vitro. This takes place mainly at 30-60 min of incubation in cells from controls and at 10 min in cells from treated mice 12-24 h after LPS injection. The incorporation decreased at these times of endotoxic shock, a few hours before death. In all cases AA levels were higher in lymphocytes than in macrophages, and 1 mM was the most effective concentration. These results suggest that the immune cells need appropriate levels of antioxidants, such as AA, under oxidative stress conditions, and that while lymphocytes take and accumulate AA, macrophages use it.     

Opposite effects of endotoxin on mitochondrial and endoplasmic reticulum functions

In this study, we determined functional integrity and reactive oxygen species generation in mitochondria and endoplasmic reticulum in liver of rats subjected to endotoxic shock to clarify whether intracellular reactive oxygen species (ROS) destabilize cellular integrity causing necrosis in rats challenged with lipopolysaccharide (LPS). LPS caused drastically increased plasma levels of alanine aminotransferase, suggesting damage to plasma membranes of liver cells. Liver necrosis was confirmed by histological examination. LPS induced a significant increase in ROS production in rat liver mitochondria (RLM), but did not impair mitochondrial function. In contrast to mitochondria, enzymatic activity and ROS production of cytochrome P450 were lower in microsomal fraction obtained from LPS-treated animals, suggesting the dysfunction of endoplasmic reticulum. Protein patterns obtained from RLM by two-dimensional electrophoresis showed significant upregulation of mitochondrial superoxide dismutase by LPS. We hypothesize that upregulation of this enzyme protects mitochondria against mitochondrial ROS, but does not protect other cellular compartments such as endoplasmic reticulum and plasma membrane causing necrosis.     

Different effects of endotoxic shock on the respiratory function of liver and heart mitochondria in rats

This study was designed to clarify whether mitochondrial function/dysfunction and reactive oxygen species (ROS) production have a temporal relationship with organ failure during endotoxic shock. Adult male Sprague-Dawley rats were divided into three groups receiving 1) isotonic saline (control group, n = 16); 2) 8 mg/kg lipopolysaccharide (LPS; n = 8); or 3) 20 mg/kg LPS (n = 8) intraperitoneally under short anesthesia with 3.5% of isoflurane. After 16 h, animals were killed to analyze plasma, rat liver mitochondria (RLM), and rat heart mitochondria (RHM). In accordance with plasma analysis, LPS-treated rats were divided into "responders" and "nonresponders" with high and low levels of alanine aminotransferase and creatine, respectively. RHM from responders had significantly lower respiratory activity in state 3, suggesting a decreased rate of ATP synthesis. In contrast, RLM from responders had significantly higher respiratory activity in state 3 than both nonresponders and the control group. This increase was accompanied by a decrease in phosphate-to-oxygen ratio values, which was not observed in RHM. ROS generation determined with a spin probe, 1-hydroxy-3-carboxypyrrolidine, neither revealed a difference in RHM between LPS and control groups nor between responders and nonresponders. In contrast, RLM isolated from responders showed a marked increase in ROS production compared with both the control group and nonresponders. Our data demonstrate that 1) RHM and RLM respond to endotoxic shock in a different manner, decreasing and increasing respiratory activity, respectively, and 2) there is a temporal relationship between ROS production in RLM (but not in RHM) and tissue damage in rats subjected to LPS shock.     

Demonstration of microtubules in the terminal web of mature absorptive cells from the small intestine of the rat

Summary The terminal web (TW) region of mature absorptive cells in the small intestine of the rat contains an elaborate cytoskeleton which supports the apical microvillus membrane. In studies regarding the structural organization of the cytoskeleton and associated proteins in the small intestine, microtubules have not been mentioned as components of the TW. By transmission electron microscopy of conventional resin-embedded sections of rat small intestine, we observe many microtubule profiles in the TW of mature absorptive cells. These microtubules are found in various orientations, although most course parallel to the long axis of the cell, and many microtubule profiles are seen in close association with smooth-surfaced vesicles.     

Tim-3 Negatively Mediates Natural Killer Cell Function in LPS-Induced Endotoxic Shock

Sepsis is an exaggerated inflammatory condition response to different microorganisms with high mortality rates and extremely poor prognosis. Natural killer (NK) cells have been reported to be the major producers of IFN-γ and key players in promoting systematic inflammation in lipopolysaccharide (LPS)-induced endotoxic shock. T-cell immunoglobulin and mucin domain (Tim)-3 pathway has been demonstrated to play an important role in the process of sepsis, however, the effect of Tim-3 on NK cell function remains largely unknown. In this study, we observed a dynamic inverse correlation between Tim-3 expression and IFN-γ production in NK cells from LPS-induced septic mice. Blockade of the Tim-3 pathway could increase IFN-γ production and decrease apoptosis of NK cells in vitro, but had no effect on the expression of CD107a. Furthermore, NK cell cytotoxicity against K562 target cells was enhanced after blocking Tim-3 pathway. In conclusion, our results suggest that Tim-3 pathway plays an inhibitory role in NK cell function, which might be a potential target in modulating the excessive inflammatory response of LPS-induced endotoxic shock.     

Differential effects of calcium deprivation on the cytoskeleton of non-tumorigenic and tumorigenic rat liver cells in culture

Normal rat liver T51B epithelial cells and Morris no. 7795 hepatoma cells growing exponentially were exposed for 24 h to standard medium containing low (0.02 mM) calcium, a concentration which drastically reduces the proliferation of normal but not tumour cells. Cell surface morphology was examined by scanning electron microscopy (SEM); and the distribution and organization of microtubules, cytokeratin and vimentin filaments, and microfilaments were analysed by indirect immunofluorescence microscopy using specific antibodies. Calcium deprivation caused the loss of intercellular cohesion in both cell types and the appearance of some microvilli and blebs, particularly on tumour cells. However, marked differential (normal vs tumour cells) effects on the organizational integrity of the cytoskeleton fibrillar network were observed. Extracellular calcium deprivation led to a particular rearrangement of microtubules, and a perinuclear accumulation of cytokeratin and vimentin filaments in normal, but not in tumour cells. A massive concentration of actin-containing microfilaments was observed in the cell periphery and blebs of hepatoma cells. In the light of the possible involvement of calcium in controlling cytoskeleton assembly, the differing cytoskeletal changes of the two cell types may be linked to their diffferent proliferative capabilities in low-calcium medium.     

Comparative study of several lymphocyte functions in two strains of mice with different models of endotoxic shock

Previously, the changes in phagocyte functions such as adherence, chemotaxis or TNFalpha production were found to be associated with oxidative stress in endotoxin-induced septic shock. However, in this type of oxidative stress the lymphocyte involvement has rarely been studied. In the present report, we analyzed the above functions in peritoneal lymphocytes from male and female BALB/c mice with a lethal endotoxic shock caused by intraperitoneal injection of E. coli lipopolysaccharide (LPS) (100 mg/kg), male and female Swiss mice with lethal endotoxic shock caused by intraperitoneal injection of LPS (150 and 250 mg/kg, respectively) or non-lethal endotoxic shock (100 mg/kg). In peritoneal lymphocytes obtained at 0, 2, 4, 12 or 24 h after LPS injection, the first two functions of these cells in the immune response, i.e. adherence to tissues and directed migration (chemotaxis), were studied. At 0, 0.5, 1, 1.5, 2, 4, 12 and 24 h after LPS injection, TNFalpha released by lymphocytes was also analyzed. The results show that endotoxic shock increases the adherence and TNFalpha release, and decreases the chemotaxis of peritoneal lymphocytes. These changes were more significant in mice with lethal than with non-lethal endotoxic shock, a fact that confirms the important role of lymphocytes during endotoxic shock.