LPS-challenged Rats Research Articles

Retinoic acid amplifies the host immune response to LPS through increased T lymphocytes number and LPS binding protein expression

Vitamin A deficiency is associated with increased susceptibility to infection but the effects of Vitamin A supplementation on host response to pathogens are controversial. This study investigated the mechanisms by which all-trans retinoic acid (atRA) modulates the host immune response in an experimental model of Vitamin A supplementation before and after challenge with LPS in rats. We show here that a supplementation with five daily injections of 10 mg/kg atRA increased the number of T lymphocytes in the peripheral blood. In addition, we show that atRA increased the expression of the LPS binding protein (LBP), a component of the LPS recognition system. The retinoic acid receptor (RAR)α agonist Ro 4060-55 but not the pan-retinoid X receptors (RXRs) agonist Ro 2573-86 mimicked the effects of atRA on LBP expression suggesting that atRA enhances LBP expression through a RARα-mediated pathway. In order to investigate the significance of increased LBP expression we challenged atRA-supplemented rats with the Gram-positive bacteria Listeria monocytogenes (LM) that activates the immune response independently from LBP. In sharp contrast to our previous observations that atRA supplementation enhances IFN-γ expression and NOS2 pathway activation in LPS-challenged rats [Devaux, Y., Grosjean, S., Seguin, C., David, C., Dousset, B., Zannad, F., Meistelman, C., de Talancé, N., Mertes, P.M., Ungureanu-Longrois, D., 2000. Retinoic acid and host–pathogen interactions: effects on inducible nitric oxide synthase in vivo. Am. J. Physiol. 279, E1045–E1053], atRA did not increase the LM-induced IFN-γ expression and NOS2 pathway activation. Overall, these data demonstrate that although atRA induces a “priming” of the immune system characterized by increased T lymphocytes number and LBP expression, the profile of the immune response depends on the inflammatory/infectious stimulus. These results could explain why Vitamin A supplementation could have beneficial/neutral or deleterious effects according to the identity of the infectious pathogen.

Rapid increase in serum lipid peroxide 4-hydroxynonenal (HNE) through monocyte NADPH oxidase in early endo-toxemia

We have developed a time-resolved fluoroimmunoassay (TR-FIA) for a lipid peroxide 4-hydroxynonenal (HNE), which is 100-fold more sensitive than conventional enzyme-linked immunosorbent assay (ELISA) and is an easier technique to use for a large number of samples without pre-treatment. By this assay, we found that a low dose of bacterial lipo-polysaccharide (LPS), injected intra-peritoneally (0.5 mg/kg), increased serum HNE level by 28-folds, with a peak at 20 min. LPS also increased HNE in vitro to a much higher level in the monocyte-enriched plasma than in the leukocyte-enriched plasma, with a peak at 10 min. The HNE production after LPS treatment was inhibited by apocynin, a specific NADPH oxidase inhibitor in vivo and in vitro, and to a lesser extent by dimethylsulfoxide a solvent for apocynin and a hydroxyl radical scavenger in vitro. These data suggest that monocyte NADPH oxidase is involved in the lipid peroxidation (HNE formation) in the LPS-challenged rat. This is the first clear demonstration of the link between an inflammatory stimulus and lipid peroxidation in the blood.

Lipopolysaccharide-induced c-Src expression plays a role in nitric oxide and TNFα secretion in macrophages

As tyrosine kinases are indispensable in lipopolysaccharide (LPS)-induced macrophage activation, the myeloid-specific Src members (i.e. Lyn, Fgr and Hck) are speculated to play important roles in this process. However, the normal LPS responsiveness in lyn −/− fgr −/− hck −/− macrophages implicates the presence of an elusive, compensating tyrosine kinase(s). In this study, we demonstrate the upregulation of c-Src in Raw264.7 and peritoneal macrophages (PEMs) by LPS, which is inhibited by PP2 (an inhibitor for Src family kinases), pyrrolidinedithiocarbamate (PDTC; NF-κB inhibitor) and LY294002 (PI3K inhibitor). And this LPS-mediated c-Src induction is also observed in macrophages recovered from LPS-challenged rats. Intriguingly, PP2 attenuates the ability of PEMs to elicit COX-2 expression and nitric oxide production in response to LPS. Similar results are also observed when macrophages recovered from rats receiving either LPS alone or LPS and PP2 both are compared. Furthermore, administration of PP2 in Raw264.7 and animal models of sepsis greatly suppresses TNFα secretion and serum TNFα level, respectively. Therefore, we conclude that c-Src, with its LPS induction, has an unperceived role in transmitting LPS signaling in macrophages.

291 In vivo hepatocyte apoptosis associated with inhibited synthesis of NF-κB-dependent survival proteins and phosphorylation of eukaryotic initiation factor-2α (eIF2α) in livers from LPS-challenged cirrhotic rats

291 In vivo hepatocyte apoptosis associated with inhibited synthesis of NF-κB-dependent survival proteins and phosphorylation of eukaryotic initiation factor-2α (eIF2α) in livers from LPS-challenged cirrhotic rats

The role of neutrophils in LPS-induced changes in pulmonary function in conscious rats

We have previously reported on a model of lipopolysaccharide (LPS)-induced pulmonary inflammation in rats, where LPS-challenged animals develop a significant pulmonary neutrophilia and mucus hypersecretion. In the current studies, we utilized whole body plethysmography and computer assisted data acquisition to examine changes in pulmonary parameters, e.g. frequency ( f), tidal volume and Penh as a measure of bronchoconstriction, due to LPS-challenge in conscious rats. Compared to saline challenge, LPS-challenged rats displayed a significant increase in ( f), which began within 30 min, peaked by 2 h and remained elevated up to 24 h. Mirroring this increase in ( f) was a decrease in the observed tidal volume of LPS-challenged rats. Additionally, compared to saline challenge, LPS-challenge provoked a significant and spontaneous bronchoconstriction, as measured by Penh, 2 h after challenge. In order to further understand these observed LPS-induced pulmonary changes, we utilized two classes of pulmonary obstructive disease standards, namely, bronchodilators and anti-inflammatory agents, and examined their ability to affect the spontaneous bronchoconstriction and the increase in ( f) seen at two discrete time points, i.e. 2 and 24 h after LPS-challenge. While ineffective on either the 2 h increase in ( f) or the LPS-induced inflammation, animals pretreated with salbutamol (10 mg/kg, p.o.) were protected from the increase in ( f) seen at the 24 h time point after LPS-challenge. In contrast, when animals were pretreated with theophylline (10 mg/kg, p.o.) no effect on the LPS-induced pulmonary inflammation or increase in ( f) was noted. Meanwhile, in animals pretreated with either betamethasone (3 mg/kg, p.o.) or SB207499 (10 mg/kg, p.o.), a PDE4 inhibitor, doses previously shown to block the LPS-induced neutrophilic inflammation, the persistent increase in ( f) seen at 24 h was attenuated, but neither compound was able to attenuate either the increase in ( f) or the spontaneous bronchoconstriction seen at 2 h. In summary, the intra-tracheal LPS-challenge of rats results in pulmonary inflammation and dysfunction, which is similar to that seen in COPD patients. We conclude that the early increase in ( f) and bronchoconstriction are not dependent upon airway inflammation, but airway inflammation most likely contributes to the persistent increase in ( f) seen at 24 h.

Acetaldehyde accumulation suppresses Kupffer cell release of TNF-? and modifies acute hepatic inflammation in rats

Alcohol-related diseases have multiple and varied associations with acetaldehyde, a highly toxic product of ethanol oxidation that accumulates in the absence of active aldehyde dehydrogenase (ALDH). This study was designed to clarify the role of acetaldehyde in liver injury, specifically in vivo and in vitro effects on Kupffer cell release of the inflammatory cytokine tumor necrosis factor-alpha (TNF-Alpha). Rats pretreated overnight with the ALDH inhibitor disulfiram (or saline control) were ethanol loaded and challenged with lipopolysaccharide (LPS), and their blood and histological parameters were examined 3 h later. Similarly, isolated rat Kupffer cells were pretreated with disulfiram or cyanamide incubated in ethanol (1 h), then challenged with LPS and evaluated 2 h later for TNF-Alpha and acetaldehyde levels in the culture medium. TNF-Alpha release from Kupffer cells after LPS challenge was also evaluated following incubation in acetaldehyde and acetate for comparison with ethanol loading. Higher blood acetaldehyde concentration following disulfiram pretreatment significantly attenuated acute hepatic inflammation in the ethanol-loaded, LPS-challenged rat (18 +/- 2.9 vs 30 +/- 3.7 polymorphonuclear cells/portal area; P = 0.01). After LPS challenge, ALDH inhibitor pretreatment attenuated Kupffer cell release of TNF-Alpha in the presence of disulfiram at 5063 +/- 151 pg/ml and cyanamide at 4390 +/- 934 pg/ml, versus no inhibitor, 5869 +/- 265 pg/ml ( P < 0.01), but not in the absence of ethanol. Acetaldehyde significantly suppressed Kupffer cell TNF-Alpha release ( P < 0.05), but acetate treatment did not. Acetaldehyde accumulation suppresses macrophage function, at least suppressing TNF-Alpha release, which plays a role in modifying acute hepatic inflammation in rats.

Terlipressin inhibits in vivo aortic iNOS expression induced by lipopolysaccharide in rats with biliary cirrhosis

In cirrhosis, lipopolysaccharide (LPS, a product of Gram-negative bacteria) in the blood may cause septic shock. LPS-elicited induction of arterial inducible nitric oxide synthase (iNOS) results in nitric oxide (NO)-induced vasodilation, which causes arterial hypotension and hyporeactivity to α 1-adrenergic constrictors. In vitro studies have suggested that vasopressin inhibits iNOS expression in cultured vascular smooth muscle cells exposed to LPS. Thus, the aim of this study was to investigate the effects of terlipressin administration (a vasopressin analog) on in vivo LPS-induced aortic iNOS in rats with cirrhosis. LPS (1 mg/kg, intravenously) was administered followed by the intravenous administration of terlipressin (0.05 mg/kg, intravenously) or placebo 1 hour later. Arterial pressure was measured, and contractions to phenylephrine (an α 1-adrenoceptor agonist), iNOS activity, and iNOS expressions (mRNA and protein) were investigated in isolated aortas. LPS-induced arterial hypotension and aortic hyporeactivity to phenylephrine were abolished in rats that received terlipressin. LPS-induced aortic iNOS activity and expression were suppressed in terlipressin-treated rats. In conclusion, in LPS-challenged rats with cirrhosis, terlipressin administration inhibits in vivo LPS-induced aortic iNOS expression. Terlipressin administration may be a novel approach for the treatment of arterial hypotension and hyporeactivity to α 1-adrenergic constrictors in patients with cirrhosis and septic shock. (H EPATOLOGY 2002;36:1070-1078.)

Inflammatory response and expression of pulmonary alkaline phosphatases in rats challenged with lipopolysaccharides: Evaluation by electrophoresis.

The characteristics of alkaline phosphatase (AP) and nitric oxide synthase (NOS) in the lung were investigated using rats challenged with lipopolysaccharide (LPS). Lung homogenates and alveolar lavage fluid were prepared from rats after an intratracheal instillation of 20mg/kg body weight of LPS. AP activities in homogenates were elevated up to 2hr and had recovered to the control level by 6hr. In contrast, those in lavage fluid were significantly decreased after 30min and recovered to the control level by 6hr. Although APs in normal lavage fluid were of the membranous-type in addition to the soluble-type, APs after LPS-challenge were soluble-type only. The 75kDa AP molecule in the homogenate at 2hr after LPS-challenge was increased, in comparison with the control, as was the level of AP activity. In contrast, there was no difference in AP protein level in lavage fluid between the control and LPS-challenged rats. Inducible NOS (iNOS) was obviously induced in lung homogenates by LPS-challenge. Moreover, proteins containing nitrotyrosine as a marker of peroxynitrite were identifiable by Western blot analysis in lavage fluid from rats challenged with LPS. These results indicate that LPS-challenge directly affects the expression and secretion of APs from type II pneumocytes.

Anti-inflammatory effect of the aqueous extract from Lonicera japonica flower is related to inhibition of NF-kappaB activation through reducing I-kappaBalpha degradation in rat liver.

We investigated the anti-inflammatory effects of aqueous extract from Lonicera japonica flower (AELJ), a traditional skin rash drug, in lipopolysaccharide (LPS)-induced rat liver sepsis. Immunoblot analysis showed that the level of nuclear factor (NF)-kappaBp65 was rapidly up-regulated and inhibitory (I)-kappaBalpha was down-regulated by LPS challenge. However, AELJ inhibited the increase of NF-kappaBp65 and degradation of I-kappaBalpha in the liver of LPS-challenged rats. Immunohistochemical analysis of rat hepatocytes showed that LPS-induced inflammatory responses, involving degradation of I-kappaBalpha and induction of NF-kappaBp65, tumor necrosis factor (TNF)-alpha and inducible nitric oxide synthase (iNOS), are partially inhibited by pretreatment with AELJ. These results suggest that AELJ may act as a therapeutic agent for inflammatory disease through a selective regulation of NF-kappaB activation.

Serine/threonine phosphorylation in cellular signaling for alveolar macrophage phagocytic response to endotoxin.

Protein serine/threonine (ser/thr) phosphorylation is an early signaling event in macrophage activation. We investigated the changes in stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) activity and effects of phosphatase inhibition on alveolar macrophage (AM) function in rats challenged with intratracheal endotoxin. Animals were sacrificed 90 min post intratracheal lipopolysaccharide (LPS, 100 microg/rat) challenge. AMs were incubated with or without phosphatase inhibitors at 37 degrees C for 30 min. Phagocytosis, CD18 expression, SAPK/JNK and phosphatase activities of AMs were determined. LPS challenge activated SAPK/JNK activity and enhanced phagocytosis of AMs without altering phosphatase activity in these cells. Inhibition of phosphatase 1 and 2A activity with okadaic acid and calyculin A exerted a bi-phasic effect on AM phagocytic function. Okadaic acid at a concentration of 1 microM increased the mean channel fluorescence intensity (MCF) and the percentage of cells engaged in phagocytosis (percent phagocytosis) in AMs from saline-treated rats. This inhibitor at concentrations of 0.5 and 1 microM enhanced both the MCF and percent phagocytosis of AMs from LPS-challenged rats. Calyculin A at a concentration of 10 nM increased the MCF phagocytosis of AMs from LPS-challenged rats. At higher concentrations (20 and 30 nM), calyculin A showed a suppression on both the MCF and percent phagocytosis of AMs in both saline and LPS groups. AM CD18 expression was not altered following LPS challenge. Phosphatase inhibitors at doses that enhanced AM phagocytosis showed either no effect (okadaic acid) or inhibition (calyculin A) of AM CD18 expression. These results suggest that ser/thr phosphorylation and dephosphorylation participate in mediating the phagocytic response of AMs to LPS.

Inhibition of nitric oxide synthase prevents hyporesponsiveness to inhaled nitric oxide in lungs from endotoxin-challenged rats.

Inhalation of nitric oxide (NO) selectively dilates the pulmonary circulation and improves arterial oxygenation in patients with adult respiratory distress syndrome (ARDS). In approximately 60% of patients with septic ARDS, minimal or no response to inhaled NO is observed. Because sepsis is associated with increased NO production by inducible NO synthase (NOS2), the authors investigated whether NOS inhibition alters NO responsiveness in rats exposed to gram-negative lipopolysaccharide (LPS). Sprague-Dawley rats were treated with 0.4 mg/kg Escherichia coli O111:B4 LPS with or without dexamethasone (inhibits NOS2 gene expression; 5 mg/kg), L-NAME (a nonselective NOS inhibitor; 7 mg/kg), or aminoguanidine (selective NOS2 inhibitor; 30 mg/kg). Sixteen hours after LPS treatment, lungs were isolated-perfused; a thromboxane-analog U46619 was added to increase pulmonary artery pressure (PAP) by 5 mmHg, and the pulmonary vasodilator response to inhaled NO was measured. Ventilation with 0.4, 4, and 40 ppm NO decreased the PAP less than in lungs of LPS-treated rats (0.75+/-0.25, 1.25+/-0.25, 1.75+/-0.25 mmHg) than in lungs of control rats (3+/-0.5, 4.25+/-0.25, 4.5+/-0.25 mmHg; P < 0.01). Dexamethasone treatment preserved pulmonary vascular responsiveness to NO in LPS-treated rats (3.75+/-0.25, 4.5+/-0.25, 4.5+/-0.5 mmHg, respectively; P < 0.01 vs. LPS, alone). Responsiveness to NO in LPS-challenged rats was also preserved by treatment with L-NAME (3.0+/-1.0, 4.0+/-1.0, 4.0+/-0.75 mmHg, respectively; P < 0.05 vs. LPS, alone) or aminoguanidine (1.75+/-0.25, 2.25+/-0.5, 2.75+/-0.5 mmHg, respectively; P < 0.05 vs. LPS, alone). In control rats, treatment with dexamethasone, L-NAME, and aminoguanidine had no effect on inhaled NO responsiveness. These observations demonstrate that LPS-mediated increases in pulmonary NOS2 are involved in decreasing responsiveness to inhaled NO.

Inhibition of inducible nitric oxide synthase ameliorates endotoxin- induced gut mucosal barrier dysfunction in rats

BACKGROUND & AIMS: The permeability of intestinal epithelial monolayers increases after exposure to nitric oxide. The aim of this study was to investigate the role of excessive NO production on intestinal barrier function in rats injected with lipopolysaccharide (LPS).METHODS: Rats were injected with saline or LPS (5 mg/ kg). Bacterial translocation to mesenteric lymph nodes, liver, and spleen was assessed 24 hours after LPS injection. Mucosal permeability was determined by loading fluorescein-labeled dextran (mol wt, 4000 daltons) into an intestinal segment and measuring its appearance in plasma. Intestinal mucosal mitochondrial respiration was assessed using 3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyltetrazolium bromide.RESULTS: Intestinal tissue from LPS-challenged rats showed upregulation of inducible NO synthase (iNOS) messenger RNA expression and subsequent up-regulation of iNOS enzymatic activity. Plasma concentrations of nitrite plus nitrate (NO2-/NO3-) were increased for at least 24 hours after injection of LPS. Treatment with the selective iNOS inhibitor, aminoguanidine, inhibited iNOS enzymatic activity and overproduction of NO2-/NO3-. LPS-induced bacterial translocation was reduced by aminoguanidine. LPS-induced intestinal hyperpermeability was ameliorated by both aminoguanidine and another selective iNOS inhibitor, S-methylisothiourea. LPS depressed intestinal mucosal mitochondrial function, and this effect was ameliorated by aminoguanidine.CONCLUSIONS: Overproduction of NO may contribute to intestinal barrier dysfunction in LPS challenged rats, possibly by interfering with mitochondrial oxidative metabolism.(Gastroenterology 1997 Oct;113(4):1246-57)

Nitric oxide inhibits LPS-induced tumor necrosis factor synthesis in vitro and in vivo

The effect of nitric oxide (NO) on LPS-stimulated TNF-α synthesis has been studied in vitro and in vivo. The synthesis of TNF-α in J774 macrophages stimulated with LPS (0.l μg/ml) was increased in concentration-related fashion by NO synthase inhibitor L-NMMA (3-30-300 μM) and reduced by either L-arginine (3-30-300 μM) or the NO donor SIN-1 (1-10-1OOμM). The level of TNF-α in the serum of LPS-challenged rats (6mg/kg/i.p.) was increased in animals pre-treated s.c. with L-NMMA (10 and 50mg/kg) and reduced in those given L- arginine (100 and 300mg/kg). These results show a negative feedback mechanism exhibited by NO on TNF-α synthesis suggesting an important regulatory link between NO and TNF-α in pathological processes.

Effect of TNFα production inhibitors BRL 61063 and pentoxifylline on the response of rats to poly I:C

BRL 61063 is a novel xanthine phosphodiesterase (PDE) type IV inhibitor with selective inhibitory activity for tumor necrosis factor (TNF) α production. This compound inhibits TNFa production by activated human blood monocytes in vitro and in animal models of endotoxemia and influenza infection. Inhibition of TNFa may be beneficial in many diseases; however, little is known about potential adverse effects of such inhibition on host defense. In an ex vivo study, we examined the effect of BRL 61 063 on the microbicidal and tumoricidal activity of pulmonary lavage cells during a local inflammatory response in rats challenged with Poly I:C. Pentoxifylline, a PDE inhibitor which also blocks TNFa production, was used for comparison. Treatment with BRL 61063 or pentoxifylline did not block the inflammatory response to Poly I:C or the activation of bronchoalveolar lavage (BAL) cells but reduced the level of tumoricidal activity attained. At the dosages used, pentoxifylline was more inhibitory than BRL 61063. Drug treatment did not prevent further stimulation of tumoricidal activity by LPS in vitro. LPS-stimulated cells from BRL 61063-treated rats reached a level of activation similar to the control group while the LPS-stimulated activity of BAL cells from pentoxifylline treated rats remained lower than control. Although pentoxifylline was more inhibitory for tumoricidal activity than BRL 61063, the latter was a more potent inhibitor of TNFα release as measured in vivo in LPS-challenged rats. This finding indicates that TNFα is not the main mediator involved in the activation of pulmonary macrophage tumoricidal function. Treatment with either BRL 61063 or pentoxifylline had little or no effect on the Poly I:C-induced candidacidal activity of BAL cells indicating that these compounds are unlikely to compromise non-specific host defense against infection.

STAUROSPORINE ENCAPSULATED INTO pH-SENSITIVE LIPOSOMES REDUCES TNF PRODUCTION AND INCREASES SURVIVAL IN RAT ENDOTOXIN SHOCK

Bacterial lipopolysaccharide (LPS) can elicit septic shock; however, there is growing evidence that most of its pathophysiological effects are mediated by the release of tumor necrosis factor (TNF) and other cytokines. In turn, LPS-induced TNF production is thought to implicate the activation of intracellular protein kinase C (PKC). In this study, we examined whether pH-sensitive liposomes containing staurosporine (STP), a potent inhibitor of PKC, when injected intravenously would suppress TNF production and reduce mortality in an endotoxin rat model. We found that pretreatment of rats with pH-sensitive STP-liposomes by intravenous administration 1.5 h prior to LPS injection decreased lethality from 80% to approximately 30%. Importantly, this improvement in outcome was associated with significant reductions in TNF serum levels; 1 h after LPS injection serum TNF was 73% lower than in a saline control group, and at 2 h TNF levels were 84% lower. STP-liposome pretreatment also ameliorated the severe reduction in body temperature, characteristic for a hypodynamic shock, that was observed in LPS-challenged rats, but had relatively little effect on the transient leukopenia. We conclude that STP-liposomes can suppress LPS-induced TNF production by the mononuclear phagocytic system, can reduce the symptoms of septic shock, and can increase survival.