Endotoxin-treated Mice Research Articles

Attenuation of lung inflammation by adrenergic agonists in murine acute lung injury.

Acute lung injury leading to a systemic inflammatory response greatly increases mortality in critically ill patients. Cardiovascular management of these patients frequently involves beta-adrenergic agonists. These agents may alter the inflammatory response. Therefore, the authors tested the hypothesis that beta-adrenergic agonists alter the pulmonary inflammatory response during acute lung injury in mice. Five-week-old CD-1 mice received continuous infusions of 10 microg x kg(-1) x min(-1) dobutamine, 6 microg x kg(-1) x min(-1) dopexamine, or vehicle via intraperitoneal mini osmotic pumps, followed immediately by intratracheal instillation of approximately 2 microg/kg endotoxin (or phosphate-buffered saline control). Six hours later the mice were killed, and lung lavage was performed. Interleukin-6 and -10 concentrations in lung homogenates were measured using enzyme-linked immunosorbent assay. Interleukin-6 and macrophage inflammatory protein-2 mRNA was measured using reverse-transcription polymerase chain reaction. Interleukin-6 protein and mRNA significantly increased after intratracheal endotoxin (P < 0.001), and the fraction of neutrophils in lung lavage fluid increased in endotoxin-treated (41 +/- 25%) versus control mice (2 +/- 4%, P < 0.05). Treatment of endotoxic mice with dobutamine significantly decreased interleukin-6 protein (P < 0.05) and mRNA (P < 0.05) expression. Dopexamine had similar but less pronounced effects. Dobutamine decreased interleukin-10 expression, whereas dopexamine did not. In endotoxemic mice, both dobutamine and dopexamine decreased induction of macrophage inflammatory protein-2 mRNA (P < 0.05) and reduced the fraction of neutrophils in lung lavage fluid (P < 0.05). In endotoxin-induced acute lung injury, beta-adrenergic agonists can significantly decrease proinflammatory cytokine expression, decrease induction of chemokine mRNA, and decrease the resultant neutrophil infiltrate in the lung.

Developmental effects of early immune stress on aggressive, socially reactive, and inhibited behaviors.

The origins of individual differences in social development are examined in relation to early stress (immune challenge) and social milieu (maternal behavior) in a genetic-developmental analysis using an animal model. Neonatal male mice (5 or 6 days of age) from two lines of mice selectively bred for high versus low levels of inter-male aggressive behavior received a standard immune challenge (i.p. injections of 0.05 mg/kg endotoxin or saline). Animals were reared by their line-specific biological dam or by a foster dam from a line bred without selection. Adult levels of social behaviors were assessed in a dyadic test (age 45-50 days). Mice from the high-aggressive line show more developmental sensitivity to immune challenge than mice from the low-aggressive line, and line differences persist regardless of the early maternal environment. As adults, endotoxin-treated mice from the high-aggressive line have lower levels of aggressive behavior, longer latency to attack, and higher rates of socially reactive and inhibited behaviors compared to saline controls. Developmental effects of endotoxin in the low-aggressive line are minimal: endotoxin increases socially reactive behaviors, compared to saline controls, but only for mice reared by their biological dams. Rearing by foster dams increases social exploration in the low-aggressive line. The findings raise novel questions regarding the openness of behavioral systems to effects of nonobvious but omnipresent features of the environment, such as antigenic load, how these effects are integrated to affect social development and psychopathology, and the nature of intrinsic factors that contribute to individual differences in sensitivity to early stressors.

High-dose dexamethasone accentuates nuclear factor-kappa b activation in endotoxin-treated mice.

We examined the effects of dexamethasone treatment on nuclear factor (NF)-kappa B activation and lung inflammation in transgenic reporter mice expressing photinus luciferase under the control of an NF-kappa B-dependent promoter (HLL mice). In vitro studies with bone marrow and peritoneal macrophages derived from these mice showed that treatment with dexamethasone blocked luciferase induction after treatment with Escherichia coli lipopolysaccharide (LPS); however, treatment of mice with intraperitoneal injection of dexamethasone at doses of 0.3 microg/g and 1 microg/g failed to inhibit NF-kappa B-dependent luciferase activity in the lungs. Furthermore, intraperitoneal treatment with 10 microg/g of dexamethasone prior to LPS paradoxically resulted in augmented luciferase activity as compared with that of mice treated with LPS alone. NF-kappa B-dependent luciferase expression in the lungs was detected by bioluminescence imaging and by measurement of luciferase activity in homogenized lung tissue. In these studies, there was an excellent correlation between indirect measurement of luciferase activity by bioluminescence in living mice and direct measurement of luciferase activity in lung tissue. Dexamethasone treatment did not affect LPS-induced neutrophilic influx or the concentration of macrophage inflammatory protein-2 in lung lavage fluid. These findings emphasize the potential error of extrapolating in vitro findings to complex in vivo events such as regulation of inflammation.

ADAR1 is involved in the development of microvascular lung injury.

Deamination of adenosine on pre-mRNA to inosine is a recently discovered process of posttranscription modification of pre-mRNA, termed A-to-I RNA editing, which results in the production of proteins not inherent in the genome. The present study aimed to identify a role for A-to-I RNA editing in the development of microvascular lung injury. To that end, the pulmonary expression and activity of the RNA editase ADAR1 were evaluated in a mouse model of endotoxin (15 mg/kg IP)-induced microvascular lung injury (n=5) as well as in cultured alveolar macrophages stimulated with endotoxin, live bacteria, or interferon. ADAR1 expression and activity were identified in sham lungs that were upregulated in lungs from endotoxin-treated mice (at 2 hours). Expression was localized to polymorphonuclear and monocytic cells. These events preceded the development of pulmonary edema and leukocyte accumulation in lung tissue and followed the local production of interferon-gamma, a known inducer of ADAR1 in other cell systems. ADAR1 was found to be upregulated in alveolar macrophages (MH-S cells) stimulated with endotoxin (1 to 100 microg/mL), live Escherichia coli (5x10(7) colony-forming units), or interferon-gamma (1000 U/mL). Taken together, these data suggest that ADAR1 may play a role in the pathogenesis of microvascular lung injury possibly through induction by interferon.

Structures, Enzymatic Properties, and Expression of Novel Human and Mouse Secretory Phospholipase A2s

Mammalian secretory phospholipase A(2)s (sPLA(2)s) form a family of structurally related enzymes that are involved in a variety of physiological and pathological processes via the release of arachidonic acid from membrane phospholipids or the binding to specific membrane receptors. Here, we report the cloning and characterization of a novel sPLA(2) that is the sixth isoform of the sPLA(2) family found in humans. The novel human mature sPLA(2) consists of 123 amino acids (M(r) = 14,000) and is most similar to group IIA sPLA(2) (sPLA(2)-IIA) with respect to the number and positions of cysteine residues as well as overall identity (51%). Therefore, this novel sPLA(2) should be categorized into group II and called group IIE (sPLA(2)-IIE) following the recently identified group IID sPLA(2) (sPLA(2)-IID). The enzymatic properties of recombinant human sPLA(2)-IIE were almost identical to those of sPLA(2)-IIA and IID in terms of Ca(2+) requirement, optimal pH, substrate specificity, as well as high susceptibility to the sPLA(2) inhibitor indoxam. Along with the biochemical properties of proteins, genetic and evolutional similarities were also observed among these three types of group II sPLA(2)s as to the chromosomal location of the human gene (1p36) and the exon/intron organization. The expression of sPLA(2)-IIE transcripts in humans was restricted to the brain, heart, lung, and placenta in contrast to broad expression profiles for sPLA(2)-IIA and -IID. In sPLA(2)-IIA-deficient mice, the expression of sPLA(2)-IIE was markedly enhanced in the lung and small intestine upon endotoxin challenge, which contrasted with the reduced expression of sPLA(2)-IID mRNA. In situ hybridization analysis revealed elevation of sPLA(2)-IIE mRNA at alveolar macrophage-like cells in the lung of endotoxin-treated mice. These findings suggest a distinct functional role of novel sPLA(2)-IIE in the progression of inflammatory processes.

Increased in-vivo intestinal protein synthesis and unchanged protein breakdown in acute endotoxintreated mice

Increased in-vivo intestinal protein synthesis and unchanged protein breakdown in acute endotoxintreated mice

Extrahepatic Expression and Regulation of Protein C in the Mouse

Activated protein C (APC) acts as an anticoagulant by inhibiting coagulation factors Va and VIIIa. Although the liver appears to be the primary site of protein C (PC) synthesis, the demonstration that other components of this system are produced extrahepatically raises the possibility that PC itself is synthesized in other tissues. We therefore used quantitative reverse transcription-polymerase chain reaction, in situ hybridization, and immunohistochemistry to screen various murine tissues for PC expression. Relatively high levels of PC mRNA were detected in the kidney (35% of liver) and testis (22% of liver). PC mRNA and antigen were demonstrated in tubular epithelial cells in the renal cortex, in spermatogenic cells in the testis, and in epithelial cells in the epididymis. Low but significant levels of PC mRNA were detected in the epididymis (1.7% of the level in liver), brain (1.1% of liver), and lung (0.8% of liver). PC antigen was demonstrated in bronchial epithelial cells in the lung, in pyramidal neurons in the cerebrum, and in Purkinje cells in the cerebellum. The extrahepatic expression of PC mRNA (ie, in the kidney) was significantly decreased in mice with renal disease (eg, in MRL lpr/lpr mice with autoimmune lupus nephritis, in db/db mice with diabetic nephropathy, and in endotoxin-treated mice with acute renal injury). The decreased renal expression of PC may contribute to the increased procoagulant potential of the kidney during septic and inflammatory processes and to the progression of kidney disease associated with these conditions.

Renal expression of fibrinolytic genes and tissue factor in a murine model of renal disease as a function of age.

Abnormal expression of fibrinolytic genes [e.g., tissue-type and urokinase-type plasminogen activators (t-PA and u-PA) and their specific inhibitor (PAI-1)] and of the procoagulant molecule tissue factor (TF), has been reported in various types of renal diseases. In this review, the expression pattern of these genes was demonstrated in two murine models of renal disease. One is acute renal failure due to microthrombosis under septic conditions, using endotoxin-treated mice, and the other one is lupus nephritis observed in female MRL lpr/lpr mice. Quantitative reverse transcription-polymerase chain reaction analysis and in situ hybridization were employed to investigate the expression of their mRNAs in tissues from endotoxin-treated mice or from MRL lpr/lpr mice. A dramatic increase in PAI-1 activity in plasma and in PAI-1 mRNA in the kidneys was observed in both models, and this increase appeared to correlate with fibrin deposition in the renal microvasculature and with the progression of lupus nephritis. In addition to these changes in PAI-1, decreases in u-PA mRNA and increases in TF mRNA were demonstrated in the kidneys from lupus-prone mice as a function of age. Similar changes were also observed in the kidneys from endotoxin-treated mice. The induction of PAI-1 and TF, and the decrease in u-PA expression in the kidneys of lupus-prone or of endotoxemic mice may promote the formation of renal microthrombi and thus contribute to the progression of renal damage in these models.

Iron attenuates nitric oxide level and iNOS expression in endotoxin-treated mice

The effect of exogenous Fe-citrate complex (Fe doses of 120 and 240 μmol/kg) on nitric oxide (NO) production in vivo has been studied in blood and liver tissue of endotoxin-treated mice. Fe-citrate complex was administered to mice subcutaneously at the same time with intravenous injection of Escherichia coli lipopolysaccharide (LPS). Iron-dependent decrease in NO − 2/NO − 3 and nitrosyl hemoglobin levels in blood of animals was detected at 6 h after LPS administration, suggesting systemic attenuation of NO generation. NO production in the liver tissue of LPS-treated mice was decreased after Fe administration judging from the amount of mononitrosyl-iron complexes formed in the tissue by diethyldithiocarbamate. The iNOS protein determination in the liver tissue of LPS-treated mice demonstrated iron-dependent inhibition of iNOS expression. We have found previously that exogenous iron does not affect systemic NO level when it is given at 6 h after LPS injection, i.e. after iNOS expression. This is a first report demonstrating iron-dependent iNOS down-regulation in endotoxin-treated mice.

Administration to mouse of endotoxin from gram-negative bacteria leads to activation and apoptosis of T lymphocytes.

Lipopolysaccharide (LPS) from gramnegative bacteria is a well-known T cell-independent B lymphocyte mitogen and macrophage/monocyte activator. While the conventional view holds that LPS is ignored by T cells, we report here that administration of LPS to mice activates all B cells, but also engages most CD4 and CD8 T cells, as measured by the expression of the activation markers CD69 and CD25 and by size increase. T cells recruited in endotoxin-treated mice showed, following in vitro stimulation by concanavalin A, altered patterns of cytokine production. In vivo, massive T cell apoptosis was evidenced in the days following LPS exposure. The present observation may contribute novel insights into the mechanisms of endotoxin shock and of the immunological consequences of gram-negative infections.

PRO-INFLAMMATORY CYTOKINES AND INTERLEUKIN 6 IN THE RENAL RESPONSE TO BACTERIAL ENDOTOXIN

Pro-inflammatory cytokines, including tumour necrosis factor α (TNF-α), interleukin (IL)-1 and IL-6 are thought to play important roles in the pathophysiology of chronic kidney disorders, including glomerulonephritis. In particular, IL-6 has received considerable attention as it appears at high concentrations to promote the progression of renal disease while at lower levels may be involved in regulating repair mechanisms. As such, cytokine profiles have been examined in the kidney by either examining secretion from isolated kidney cells or quantitating plasma and urinary levels in experimental models of glomerulonephritis. To examine the cytokine responses within the kidney, without the contribution of other organ systems, we used semi-quantitative polymerase chain reaction (RT-PCR) analysis and a recently developed kidney slice culture model from tissues of mice treated with combinations of endotoxin and neutralizing antibodies against TNF-α. The expression of IL-6, in addition to other pro-inflammatory cytokine genes, was increased by endotoxin treatment and reduced by pretreatment with neutralizing antibodies to TNF-α. Immunohistochemical staining revealed that IL-6 was expressed primarily in mesangial cells. Urinary IL-6 was also increased in endotoxin-treated mice and was inhibited by treatment with neutralizing TNF-α antibodies. Kinetics of the kidney-specific cytokine responses indicated that increase in TNF-α occurred initially, followed by IL-1β and finally IL-6. Furthermore, addition of TNF-α to glomerular mesangial cells induces IL-6 secretion. Taken together, these studies indicate that, like in the liver, a cytokine response occurs in the kidney from bacterial endotoxin and that TNF-α acts as a primary cytokine capable of stimulating additional cytokines, including IL-6.

Fibrin deposition in tissues from endotoxin-treated mice correlates with decreases in the expression of urokinase-type but not tissue-type plasminogen activator.

The primary hypothesis of this report is that the formation and subsequent removal of fibrin in specific tissues during pathologic processes reflects temporal changes in the local expression of key procoagulant and fibrinolytic genes. To begin to test this hypothesis, we have used quantitative PCR assays and in situ hybridization analysis to examine the effects of endotoxin on the expression of specific genes in murine tissues, and to relate these changes to fibrin deposition/dissolution using immunohistochemical approaches. Endotoxin caused large increases in plasminogen activator inhibitor-1 mRNA and modest increases in tissue factor mRNA in most tissues examined. However, fibrin was only detected in the kidneys and adrenals of endotoxin-treated mice, and it was transient. Unexpectedly, changes in urokinase-type plasminogen activator mRNA but not tissue-type plasminogen activator mRNA correlated with fibrin deposition/dissolution in these tissues. Pretreatment of mice with the fibrinolytic inhibitor epsilon-aminocaproic acid before endotoxin increased both the number of fibrin-positive tissues and the duration of fibrin deposition in the kidneys and adrenals. These results suggest that the absence of fibrin in some tissues reflects ongoing local fibrinolysis, and that increases in plasminogen activator inhibitory and tissue fac- tor gene expression and decreases in urokinase-type plasminogen activator expression are necessary for tissue-specific fibrin deposition. Changes in tissue-type plasminogen activator gene expression do not appear to be essential for fibrin deposition/dissolution in this murine model of sepsis.

73 Fibrin deposition in tissues from endotoxin-treated mice correlates with decreases in the expression of urokinase-type but not tissue-type plasminogen activator

73 Fibrin deposition in tissues from endotoxin-treated mice correlates with decreases in the expression of urokinase-type but not tissue-type plasminogen activator

Antisense DNAs selectively suppress cyclooxygenase-2 gene expression in the uterus of pregnant endotoxin-treated mice.

Antisense DNAs selectively suppress cyclooxygenase-2 gene expression in the uterus of pregnant endotoxin-treated mice.

Cyclooxygenase-2 and inducible nitric oxide synthase genes in pregnant endotoxin-treated mice are associated with preterm birth.

Cyclooxygenase-2 and inducible nitric oxide synthase genes in pregnant endotoxin-treated mice are associated with preterm birth.

Cellular expression of ceruloplasmin in baboon and mouse lung during development and inflammation.

Ceruloplasmin (CP) is an important extracellular antioxidant and free radical scavenger. Although CP is expressed mainly in the liver, recent studies have identified the lung as another major site of CP synthesis. The sites and cell types that are responsible for CP expression in baboon and mouse lung are described. CP mRNA is detected in primordial bronchial epithelium in baboon fetuses by 60 days of gestation. At 140 days of gestation and thereafter, CP mRNA is found in airway epithelium and in the ductal cells of the submucosal glands. In developing and mature mice, CP mRNA is present in epithelial cells throughout the airway. In endotoxin-treated mice, the amount of CP mRNA increases several-fold in large airways but increases only moderately in small airways. This suggests that the high concentration of CP in the mucus lining of the upper airway, which serves to filter harmful substances, is particularly important during stressful conditions. Endotoxin treatment in mice also results in the induction of high levels of CP mRNA in a subset of alveolar wall cells. The data suggest that the airway epithelial cells are the major source of CP in the lung fluid and support ceruloplasmin's critical role in host defense against oxidative damage and infection in the lung.

Role of Ca2+ on Endotoxin‐Sensitivity by Galactosamine Challenge: Lipid Peroxide Formation and Hepatotoxicity in Zymosan‐Primed Mice

This study was investigated to clarify the role of intracellular Ca2+ following endotoxin treatment (1 mg/kg, intraperitoneally) to D-galactosamine-sensitized mice (400 mg/kg, intraperitoneally), and to observe lipid peroxide levels, an index of hepatotoxicity, in endotoxin/galactosamine (Ga1N)-challenged mice under activation of macrophages, especially Kupffer cells, by zymosan. The liver lipid peroxide level and serum glutamic pyruvic transminase activity in mice 18 hr after administration of endotoxin/Ga1N were markedly higher than those in mice treated only with endotoxin. In spite of an increase in lipid peroxide formation, there was little or no effect of Ga1N administration on xanthine oxidase and superoxide dismutase activities in mice given endotoxin. However, the injection of verapamil (10 mg/kg, subcutaneously) markedly decreased lipid peroxide levels in liver of endotoxin/Ga1N-injected mice. In the mice given a Ca(2+)-deficient diet, lipid peroxide level in liver after endotoxin/Ga1N injection was markedly decreased compared to that in mice fed a normal diet. Administration of dexamethasone (200 micrograms/kg, intraperitoneally) in mice 1 hr before treatment with endotoxin/Ga1N did not induce lipid peroxide formation. Administration of endotoxin to Ga1N-treated mice resulted in a higher level of liver cytosolic free Ca2+ ([Ca2+]i) than that in endotoxin-treated mice. On the other hand, Ca(2+)-ATPase activity in liver plasma membrane in the endotoxin/Ga1N-treated mice was markedly decreased as compared with endotoxin alone. On the contrary, the Ca(2+)-ATPase activity in liver mitochondria was higher in endotoxaemic mice treated with GA1N than in mice given endotoxin alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of glyceraldehyde-3-phosphate dehydrogenase in Salmonella abortus equi lipopolysaccharide-treated mice

Nitric oxide (NO) is known to inhibit glyceraldehyde-3-phosphate dehydrogenase enzyme activity caused by an NAD +-dependent posttranslational protein modification mechanism. In order to study a possible similar protein modification under in vivo conditions, mice were injected with bacterial endotoxin known to endogenously generate NO. In endotoxin-treated mice glyceraldehyde-3-phosphate dehydrogenase enzyme activity was significantly reduced in cytosolic fractions of heart and spleen, compared to 100,000 × g supernatants of untreated control animals. Enzyme activity was unaffected in lung and kidney cytosol of the endotoxin-treated group. Employing the differential NAD +-dependent labelling method, glyceraldehyde-3-phosphate dehydrogenase in heart and spleen cytosol of the endotoxin-treated groups, versus the control group, had been endogenously modified. These changes were not observed in lung and kidney cytosol of endotoxin-challenged animals. Using Western blot analysis no significant changes in the amount of protein (glyceraldehyde-3-phosphate dehydrogenase) in control versus endotoxin-treated animals was detectable. Since an endogenously NAD +-modified glyceraldehyde-3-phosphate dehydrogenase occurred in endotoxin-treated mice, at least in some organs, this NO-stimulated posttranslational protein modification mechanism seems to function under in vivo conditions. A covalent protein modification mechanism, rather than differences in the amount of the protein is likely to cause changes in enzyme activity.

Defense effects of a traditional Chinese medicine (sho-saiko-to) against metabolic disorders during endotoxemia; approached from the behavior of the calcium ion.

The present study was carried out as an approach from intracellular Ca2+ to clarify the preventive effects of a traditional Chinese medicine, Sho-saiko-to (Kampo prescription, TJ-9), against various metabolic disorders during endotoxemia. In this experiment, we estimated the cytosolic-free Ca2+ concentration ([Ca2+]i) in liver single cells using a photonic microscope system. The [Ca2+]i in a single liver cell of endotoxin (6 mg/kg, i.p.)-injected mice was greater at 18 h post-intoxication than that in the control, whereas the administration of endotoxinin to TJ-9 (500 mg/kg/d, p.o.)-pretreated mice resulted in a markedly lower level of [Ca2+]i than that in endotoxin-treated mice. In the mice pretreated with TJ-9, the CA(2+)-ATPase activity in liver plasma membrane 18 h after endotoxin injection was markedly increased as compared to that in the endotoxin-treated mice. By contrast, the Ca(2+)-ATPase activity in liver mitochondria was lower in endotoxemic mice pretreated with TJ-9 than in mice given endotoxin alone. State 3 respiration and the respiratory control index (RCI), which are the parameters of mitochondrial function, were 41% and 35% lower, respectively, in the liver of mice given endotoxin than those levels in the control. However, the levels of state 3 and RCI in endotoxin-TJ-9-treated mice were markedly increased as compared to those of the endotoxin-treated mice. These findings suggest the protective effect of TJ-9 in the damage of liver mitochondrial function in endotoxin-poisoned mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Preventive effects of a traditional Chinese medicine (sho-saiko-to) against oxygen toxicity and membrane damage during endotoxemia.

The preventive effects of a traditional Chinese medicine Sho-saiko-to (Kampo prescription, TJ-9) were determined from oxygen toxicity and membrane damage in liver during endotoxemia. The liver lipid peroxide level and xanthine oxidase activity 18 h after administration of endotoxin (6 mg/kg, i.p.) to TJ-9 (500 mg/kg/d, p.o.)-pretreated mice were markedly lower than that in endotoxin-treated mice, whereas the administration of TJ-9 significantly increased superoxide dismutase and glutathione peroxide activities in liver of endotoxin-injected mice. In the mice pretreated with a TJ-9, the levels of alpha-tocopherol and nonprotein SH in liver tissue 18 h after endotoxin injection were markedly increased as compared to those in endotoxin-treated mice. Leakages of acid phosphatase and lactate dehydrogenase isozyme in serum were markedly lower in endotoxin-TJ-9-treated mice than those in mice given endotoxin. The administration of TJ-9 clearly prevented the membrane protein damage arising from endotoxin challenge. Kampo prescription Sho-saiko-to thus appears to protect the liver plasma membrane from injury by free radicals which occur in a tissue ischemic state during endotoxemia.