Effects of Lipopolysaccharides from Gram-Negative Bacteria on the Level of Thiols in Blood Platelets

Lipopolysaccharide (endotoxin, LPS) activates blood platelets and stimulates generation of free radicals in these cells. The mechanism of platelet activation induced by LPS is not known. The aim of the present study was to examine how glutathione (GSH) and other thiol-containing compounds are involved in the oxidative stress in blood platelets caused by LPS. The HPLC technique has been used on the analysis of non-protein thiols from human blood platelets treated with lipopolysaccharides of different Gram-negative bacteria (Proteus mirabilis, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa). Our results show that LPSs caused an increase (about 10%) of the level of reduced glutathione (GSH) and other nonprotein thiols such as cysteine (CSH) and cysteinylglycine (CGSH), whereas the total pool of these compounds was almost unchanged. LPS may react directly with thiols, since after incubation of LPSs with glutathione alone (in reduced form) we observed a distinct decrease of the level of platelet GSH.     

The production of inflammation mediators by mouse peritoneal cells under conditions of combined exposure to staphylococcal enterotoxin and lipopolysaccharide

The influence of staphylococcal enterotoxin of type A (SEA) and enterobacterial lipopolysaccharide (LPS) on the production of tumor necrosis factor alpha (TNF alpha), gamma-interferon and active forms of oxygen by mouse peritoneal cells was studied. Both SEA and LPS, when injected to animals, produced stimulating influence on the oxygen metabolism of phagocytizing cells. The highest toxic doses of LPS induced the maximal generation of oxygen radicals. Under the conditions of the development of lethal toxic shock, i.e. after the combined injection of SEA and LPS, the synergic activation of oxygen metabolism was observed, which was also manifested by the pronounced production of TNF alpha and the increased synthesis of gamma-interferon.     

Induction of interleukin-1 in human monocytes by the superantigen staphylococcal enterotoxin A requires the participation of T cells

Nanogram quantities of the bacterial superantigen Staphylococcal Enterotoxin A (SEA) induced significant amounts of extracellular IL-1 alpha and IL-1 beta in human peripheral blood mononuclear cells. Induction of maximal IL-1 alpha and IL-1 beta levels by lipopolysaccharide (LPS) required microgram quantities. LPS induced detectable extracellular IL-1 content within 3-6 hr and maximal levels were detected already after 12 hr. Induction of IL-1 production by SEA showed a delayed release with peak values after 24-48 hr. IL-1 beta was the major species of IL-1 seen in both SEA- and LPS-stimulated culture supernatants. SEA was in general a relatively stronger inducer of extracellular IL-1 alpha than LPS. SEA-induced extracellular IL-1 production in human monocytes was entirely dependent on the presence of T cells, whereas addition of T cells to LPS-stimulated purified human monocytes only marginally enhanced the extracellular IL-1 production. The capacity to induce extracellular IL-1 production in monocytes in response to SEA was high in the CD4+ 45RO+ memory T cell subset, whereas CD4+ 45RA+ naive T cells and CD8+ T cells had lower IL-1-inducing capacity. The T cell help for IL-1 production could not be replaced by a panel of T cell-derived recombinant lymphokines added to SEA-stimulated monocytes, including IFN-gamma and TNF, indicating the participation of cell membrane-bound ligands or hitherto unidentified soluble mediators.     

Lowering of bactericidal activity of mouse peritoneal macrophages under combined use of staphylococcal enterotoxin type A and endotoxin

The present study investigated the effect of staphylococcal enterotoxin type A (SEA) and endotoxin Serratia marcescens (LPS) on the phagocytosis and killing of Staphylococcus aureus by mouse peritoneal macrophages. Two hours after enterotoxin intraperitoneal injection phagocytic and bactericidal activity were depressed. 24 hours later there was increased functional activity of macrophages by SEA and LPS, apart. But when two toxins were administered together (LPS four hours later enterotoxin) marked inhibition of bacterial killing was observed. When peritoneal macrophages were treated in vitro for 24 hours with the same toxins they were also markedly suppressed in bactericidal activity.     

Altered glutathione homeostasis in animals prenatally exposed to lipopolysaccharide

We previously reported that injection of bacterial lipopolysaccharide (LPS) into gravid female rats at embryonic day 10.5 resulted in a birth of offspring with fewer than normal dopamine (DA) neurons along with innate immunity dysfunction and many characteristics seen in Parkinson's disease (PD) patients. The LPS-exposed animals were also more susceptible to secondary toxin exposure as indicated by an accelerated DA neuron loss. Glutathione (GSH) is an important antioxidant in the brain. A disturbance in glutathione homeostasis has been proposed for the pathogenesis of PD. In this study, animals prenatally exposed to LPS were studied along with an acute intranigral LPS injection model for the status of glutathione homeostasis, lipid peroxidation, and related enzyme activities. Both prenatal LPS exposure and acute LPS injection produced a significant GSH reduction and increase in oxidized GSH (GSSG) and lipid peroxide (LPO) production. Activity of gamma-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in de novo GSH synthesis, was up-regulated in acute supranigral LPS model but was reduced in the prenatal LPS model. The GCS light subunit protein expression was also down-regulated in prenatal LPS model. GSH redox recycling enzyme activities (glutathione peroxidase, GPx and glutathione reducdase, GR) and glutathione-S-transferase (GST), gamma-glutamyl transpeptidase (gamma-GT) activities were all increased in prenatal LPS model. Prenatal LPS exposure and aging synergized in GSH level and GSH-related enzyme activities except for those (GR, GST, and gamma-GT) with significant regional variations. Additionally, prenatal LPS exposure produced a reduction of DA neuron count in the substantia nigra (SN). These results suggest that prenatal LPS exposure may cause glutathione homeostasis disturbance in offspring brain and render DA neurons susceptible to the secondary neurotoxin insult.     

alpha-Phenyl-n-tert-butyl-nitrone reduces lipopolysaccharide-induced white matter injury in the neonatal rat brain

Lipopolysaccharide (LPS)-induced white matter injury in the neonatal rat brain is at least partially associated with oxidative stress. alpha-Phenyl-n-tert-butyl-nitrone (PBN) (100 mg/kg) significantly attenuated LPS (1 mg/kg)-induced brain injury, as indicated by the reduction in bilateral ventricular enlargement, apoptotic cell death of oligodendrocytes (OLs), and the loss of OL immunoreactivity in the neonatal rat brain. Protection of PBN was linked with the attenuated oxidative stress induced by LPS, as indicated by the decreased elevation of 8-isoprostane content and by the reduced number of 4-hydroxynonenal or malondialdehyde positive OLs following LPS exposure. Interestingly, while LPS exposure elevated, rather than depleted, levels of the reduced glutathione (GSH) and the GSH/GSSG (oxidized form) ratio, LPS exposure significantly suppressed glutathione peroxidase activity in the rat brain. PBN attenuated LPS-induced alterations in glutathione homeostasis in the rat brain. Additionally, the inflammatory responses were also reduced in the PBN-treated brain, as indicated by the decreased number of activated microglia following LPS exposure and by the consequently decreased elevation of interleukin1-beta and tumor necrosis factor-alpha contents in the rat brain. The overall results suggest that antioxidant PBN, more than a straightforward free radical scavenger, may also involve anti-inflammatory and anti-apoptotic properties in protection of the neonatal rat brain from LPS-induced injury.     

Diethylmaleate and buthionine sulfoximine, glutathione-depleting agents, differentially inhibit expression of inducible nitric oxide synthase in endotoxemic mice.

Diethylmaleate (DEM) and buthionine sulfoximine (BSO), glutathione (GSH)-depleting agents, reduced the metabolic activity and the protein level of iNOS in both macrophages and hepatocytes activated by lipopolysaccharide (LPS). In this study, we examined the effects of DEM and BSO on iNOS expression in LPS-treated mice under the assumption that the level of GSH may alter the expression of nitric oxide synthase. Serum levels of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) were also determined. DEM markedly decreased the levels of hepatic GSH in response to LPS. Treatment of mice with DEM significantly reduced serum nitrite/nitrate levels and hepatic iNOS protein and mRNA induction by LPS. Although BSO inhibited the level of hepatic GSH in LPS-treated mice, the agent did not alter serum nitrite/nitrate levels and hepatic iNOS expression. DEM completely inhibited an increase of serum IL-1beta level by LPS, whereas BSO failed to inhibit it. Neither DEM nor BSO significantly affected the induction of serum TNF-alpha level by LPS. These results showed that DEM and BSO differentially affect the expression of iNOS in endotoxemic mice, suggesting the possibility that suppression of iNOS expression by DEM may be associated with the inhibition of IL-1beta but not of TNF-alpha.     

Differential effects of dietary casein and soybean protein isolate on lipopolysaccharide-induced hepatitis in D-galactosamine-sensitized rats

Effects of dietary protein type on lipopolysaccharide (LPS)-induced hepatitis, as assessed by plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, were investigated in D-galactosamine (GalN)-sensitized rats. The plasma ALT and AST activities in rats fed on 25% soybean protein isolate (SPI) diet were significantly suppressed to about 1/4 and 1/5 of the values in rats fed on 25% casein diet, respectively, 8 h after the injection of LPS + GalN. Although hepatic ALT and AST activities of normal rats were also lower in the SPI group than in the casein group, this could not explain the differences in plasma enzyme activities between the two groups. The hepatic glutathione concentration of normal rats was lower in the SPI group than in the casein group, but it was reversed in rats injected with drugs. The results suggest that SPI can protect animals from LPS + GalN-induced hepatitis, and that the hepatic glutathione level may participate in the effects of SPI.     

Localization of an immune functional site on staphylococcal enterotoxin A using the synthetic peptide approach

Using the synthetic peptide approach, we have identified a part of the staphylococcal enterotoxin A (SEA) molecule that is responsible for stimulation of T cell proliferation and induction of the lymphokine IFN-gamma. Peptides were synthesized corresponding to amino acids 1 to 27, SEA(1-27), and 28 to 45, SEA(28-45). Both peptides were tested for direct competition with SEA for blockage of SEA induced proliferation and production of IFN-gamma by T cells. Further, antibodies were produced to the peptides and tested for their ability to bind to SEA and block SEA function. SEA (1-27), but not SEA (28-45), blocked proliferation of human peripheral T cells and induction of IFN-gamma by the T cell line, L12-R4. The inhibitory effects were specific, because SEA (1-27) did not inhibit the induction of T cell proliferation by the mitogen PHA. Consistent with the direct inhibition of function, antibodies to SEA (1-27), but not SEA (28-45), neutralized the mitogenic activity of SEA on human PBL. The data suggest that a functional site on SEA that is responsible for its modulation of T cell function involves the N-terminal 27 amino acids. Residues 1 to 27 of SEA could potentially interact at either the level of the TCR or may block the proposed binding of SEA to class II MHC Ag, based on recent data showing that these molecules are involved in SEA-induced proliferation.     

Streptozotocin may provide protection against subsequent oxidative stress of endotoxin or streptozotocin in rats

Endotoxin lipopolysaccharide (LPS) and streptozotocin-induced diabetes are known to cause oxidative stress in vivo. There is some evidence that a sublethal dose of LPS provides protection against subsequent oxidative stress. Because of its wide use as a diabetogenic agent, this study was undertaken to determine if streptozotocin can likewise provide a protective effect against further oxidative stress in rats. Female Sprague–Dawley rats were given streptozotocin (50 mg/kg intraperitoneally once) prior to exposure to either bacterial endotoxin from Salmonella abortus equii (5 mg/kg intraperitoneally) or three additional daily doses of streptozotocin (50 mg/kg intraperitoneally). One week after LPS or streptozotocin treatments, oxidative stress was determined by measuring changes in antioxidant activity (glutathione peroxidase, glutathione reductase, superoxide dismutase, catalase, glutathione S-transferase, and γ-glutamyltranspeptidase) and in concentrations of glutathione, nitrite, and thiobarbituric acid reactants in liver, kidney, intestine, and spleen. High levels of some antioxidants in the LPS-control and streptozotocin-control rats, in contrast to normal levels found in diabetes + LPS and multidose-streptozotocin rats, suggest that streptozotocin, like LPS, may confer a protective effect against subsequent oxidative stress. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 12: 143–149, 1998     

RyR channel-mediated increase of cytosolic free calcium level signals cyclin B1 degradation during abortive spontaneous egg activation in rat

In few mammalian species including rat, post-ovulatory aging induces abortive spontaneous egg activation (SEA), which is morphologically characterized by exit from metaphase-II (M-II) arrest. A possibility exists that the RyR channel-mediated insufficient increase of cytosolic free Ca²⁺ level could be one of the causes for post-ovulatory aging-induced abortive SEA. To test this possibility, eggs collected after 17 h post-hCG surge were cultured with or without various concentrations of nifedipine (NF), ruthenium red (RR), and KN-93 for 3 h in vitro. Morphological changes characteristic of abortive SEA, cytosolic free Ca²⁺ level, cyclin B1 level, and meiotic status were analyzed. Data of the present study indicate that NF and RR inhibited post-ovulatory aging-induced abortive SEA in a concentration-dependent manner. Further, RR protected against RyR channel as well as caffeine-mediated increase of cytosolic free Ca²⁺ level. In addition, KN-93 inhibited post-ovulatory aging-induced abortive SEA in a concentration-dependent manner. An increase of cytosolic free Ca²⁺ level was associated with a reduction of cyclin B1 level during post-ovulatory aging-induced abortive SEA. These data indirectly suggest the involvement of RyR channels in the increase of cytosolic free Ca²⁺ level. The increased cytosolic free Ca²⁺ level triggers cyclin B1 degradation possibly through CaMK-II activity during post-ovulatory aging-induced abortive SEA in rat eggs cultured in vitro.     

Blockade of P2X7 receptor-mediated purinergic signaling with A438079 protects against LPS-induced liver injury in rats

The study aimed to investigate the hepatoprotective effects of purinergic receptor (P2X7R) antagonism by A438079 in liver damage. An experimental model of inflammation was performed by intraperitoneal (i.p.) lipopolysaccharide (LPS) administration in the rat. The groups were Control, A438079, dimethyl sulfoxide (DMSO), LPS, LPS + DMSO, and LPS + A438079. Following LPS (8 mg/kg) injection, A438079 (15 mg/kg) and DMSO (0.1 mL) were administrated (i.p) in the study groups. The blood and the liver tissues were removed for histological, biochemical, and western blot analyses. In the biochemical analysis, serum aspartate transaminase (AST) and alanine transaminase (ALT) concentrations, the tissue glutathione (GSH) level, and superoxide dismutase (SOD) activity dramatically decreased and malondialdehyde (MDA) level increased in the LPS and LPS + DMSO groups compared to the LPS + A438079 group. In the histological analysis, severe sinusoidal dilatation, necrotic hepatocytes, and inflammatory cell infiltration were observed in the LPS and LPS + DMSO groups while these effects were lessened in the LPS + A438079 group. The relative protein expression levels of P2X7R, Nf-kB-p65, IL-6, and Caspase-3 were significantly higher in the LPS and the LPS + DMSO groups than in the LPS + A438079 group. On the other hand, these protein expressions were considerably lower in the Control, A438079, and DMSO groups compared to the LPS + A438079 group. In addition, Bcl-2 protein expression was significantly lower in the LPS and the LPS + DMSO groups and higher in the LPS + A438079 group compared to the other groups. The protective effect of A438079 against LPS-induced hepatic inflammation may be related to P2X7R antagonism, inflammatory mediators, and apoptotic cell death.     

Helminth antigens modulate TLR-initiated dendritic cell activation

There is increasing awareness that helminth infections can ameliorate proinflammatory conditions. In part, this is due to their inherent ability to induce Th2 and, perhaps, regulatory T cell responses. However, recent evidence indicates that helminths also have direct anti-inflammatory effects on innate immune responses. In this study, we address this issue and show that soluble molecules from the eggs of the helminth parasite Schistosoma mansoni (SEA) suppress LPS-induced activation of immature murine dendritic cells, including MHC class II, costimulatory molecule expression, and IL-12 production. SEA-augmented LPS-induced production of IL-10 is in part responsible for the observed reduction in LPS-induced IL-12 production. However, analyses of IL-10(-/-) DC revealed distinct IL-10-independent suppressive effects of SEA. IL-10-independent mechanisms are evident in the suppression of TLR ligand-induced MAPK and NF-kappaB signaling pathways. Microarray analyses demonstrate that SEA alone uniquely alters the expression of a small subset of genes that are not up-regulated during conventional TLR-induced DC maturation. In contrast, the effects of SEA on TLR ligand-induced DC activation were striking: when mixed with LPS, SEA significantly affects the expression of >100 LPS-regulated genes. These findings indicate that SEA exerts potent anti-inflammatory effects by directly regulating the ability of DC to respond to TLR ligands.     

Alpha-lipoic acid rebalances redox and immune-testicular milieu in septic rats

In the present study, lipopolysaccharide (LPS), as an immune modulator in male adult rats and alpha-lipoic acid (ALA), as a powerful biological antioxidant and anti-inflammatory, are examined to help understanding the role of the immune and redox perturbation in testicular dysfunction with a possible protection. A total of 60 male Swiss albino rats were divided into 5 groups (10/group) respectively as follows Saline, ALA-vehicle, ALA (200 mg/kg), LPS (5 mg/kg) started with 20 rats and LPS + ALA. Obtained data from previously reported study, in our laboratory, and from the present one revealed that LPS induced marked reductions in sperm's count, motility and resulted in deterioration of the testicular histological features. In addition, LPS decreased testicular reduced glutathione (GSH) level and lactate dehydrogenase isoenzyme-x (LDH-x) activity. However, it increased testicular levels of malondialdehyde (MDA), nitric oxide (NO) and 8-hydroxydeoxyguanosine (8-HDG) in testicular DNA, along with increased serum IL-2 level. In contrast, rats pretreated with ALA showed almost complete normalization of all the tested parameters. In conclusion, LPS induced perturbation of the immune-testicular barrier as a result of redox imbalance with a subsequent testicular dysfunction. Pretreatment with ALA ameliorated all these effects by its immune-modulator and antioxidant mechanisms suggesting a protective role against male infertility in septic or severely infected patients.     

Effects of caffeic acid phenethyl ester on endotoxin-induced cardiac stress in rats: a possible mechanism of protection

Endotoxins (lipopolysaccharides; LPS) are known to cause multiple organ failure, including myocardial dysfunction. The present study aimed to investigate the mechanism of caffeic acid phenethyl ester (CAPE) protection against LPS-induced cardiac stress. Rats were allocated into three groups; group 1 served as a normal control group, group 2 (LPS) received a single intraperitoneal injection of LPS (10 mg/kg), group 3 (LPS + CAPE) was injected intraperitoneally with CAPE (10 mg/kg/day; solubilized in saline containing 20% tween 20) throughout a period of 10 days prior to LPS injection. Rats were maintained 4 h before sacrifice. Caffeic acid phenethyl ester pretreatment normalized LPS-enhanced activities of serum creatine kinase (CK) and lactate dehydrogenase (LDH) as well as glutathione peroxidase (GPx), and myeloperoxidase (MPO) in cardiac tissue. A significant reduction of the elevated levels of serum tumor necrosis factor-alpha (TNF-α) as well as serum and cardiac nitrite/nitrate (NOx) ) was achieved after CAPE pretreatment. CAPE also restored malondialdelyde (MDA), reduced glutathione (GSH), and cytosolic calcium (Ca2+ ) levels in the heart. A marked induction of cardiac heme oxygenase-1 (HO-1) protein level was detected in CAPE-pretreated group. Whereas, LPS-induced reduction of adenosine triphosphate (ATP) and phosphocreatine (PCr) levels was insignificantly changed. Conclusively, the early treatment with CAPE maintained antioxidant defences, reduced oxidative injury, cytokine damage, and inflammation but did not markedly improve energy status in cardiac tissue. The beneficial effect of CAPE might be mediated, at least in part, by the superinduction of HO-1.     

Induction of glutathione peroxidase in response to inactivation by nitric oxide.

To determine effect of nitric oxide (NO) on cellular glutathione peroxidase (GPX) level in living cells, we measured the activity, protein and mRNA of GPX in rat kidney (KNRK) cells under a high NO condition. Combined treatment of lipopolysaccharide (LPS, 1 microgram/ml) and tumor necrosis factor-alpha (TNF-alpha, 50 ng/ml) synergistically enhanced (23-folds) nitrite production from KNRK cells. This was suppressed by an inducible NO synthase (iNOS) inhibitor (aminoguanidine, N-nitro-L-arginine methylester hydrochloride) and arginase. iNOS expression was detected by RT-PCR in the treated cells. GPX was inactivated irreversibly when the cells had been homogenized before exposure to a NO donor, S-nitroso-N-acetylpenicillamine (SNAP). In living KNRK cells, SNAP and LPS + TNF-alpha exerted a transient effect on the GPX activity. The treatment with SNAP (200 microM) or sodium nitroprusside (200 microM) enhanced GPX gene expression, which was blocked by a NO scavenger, 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide. GPX mRNA was markedly increased by the treatment with LPS + TNF-alpha, and aminoguanidine blocked the effect. In cells metabolically labeled with 75Se, LPS + TNF-alpha accelerated the incorporation of radioactivity into GPX molecule by 2.1-fold. These results suggest that inactivation of GPX by NO triggers a signal for inducing GPX gene expression in KNRK cells, thereby restoring the intracellular level of this indispensable enzyme.     

Role for glutathione in the hyposensitivity of LPS-pretreated mice to LPS anorexia

To study the role of the redox state regulator glutathione (GSH) in bacterial lipopolysaccharide (LPS)-induced anorexia we measured total reduced GSH (trGSH) in liver, serum and brain in response to intraperitoneal (ip) lipopolysaccharide (LPS, 4 microg/mouse) injection in LPS-na?ve and LPS-pretreated (4 microg/mouse given 3 days earlier) mice. LPS reduced food intake in LPS-na?ve mice and LPS pretreatment attenuated this effect. LPS decreased trGSH at 24 hours after injection in LPS-na?ve mice but 4 days later trGSH levels were upregulated in brain and liver, and this was associated with a significant attenuation of LPS-induced anorexia. In addition, LPS increased mitochondrial GSH levels in brain and liver at 4 days after injection. Pharmacological GSH depletion with diethylmaleate and L-buthionine sulfoximine in LPS-pretreated mice ablated the hyposensitivity to the anorexic effect of LPS. Together, these findings suggest a prominent role for GSH and its intracellular repartition in LPS anorexia.     

Role for nerve growth factor in the in vivo regulation of glutathione in response to LPS in mice

Since the redox state regulator glutathione (GSH), which influences lipopolysaccharide (LPS) anorexia, may be controlled by cytokines, we studied the roles of tumour necrosis factor-alpha (TNFalpha) and nerve growth factor (NGF) in the GSH response to intraperitoneal (ip) LPS injection in mice. Basal NGF and total reduced GSH (trGSH) levels were up-regulated in brain and liver of TNFalpha-knock-out (KO) mice, and this was associated with attenuated LPS anorexia. The increases in NGF and trGSH presumably contributed to the attenuated anorexia in response to LPS because transgenic mice over-expressing NGF (NGF-tg mice) also had increased trGSH levels and displayed attenuated anorexia compared to the corresponding wild type (WT) mice. Attenuated LPS anorexia in NGF-tg mice was accompanied by reduced serum TNFalpha and IFNgamma levels compared to WT mice. In response to a second injection of LPS, NGF and trGSH levels, but not TNFalpha levels changed. This suggests that in vivo tissue trGSH changes following LPS in LPS-na?ve or LPS-pretreated mice are regulated by NGF rather than TNFalpha. The finding that genetic TNFalpha deficiency did not inhibit the acute trGSH response to LPS supports this interpretation. In sum, the results indicate i) that a decrease or increase in NGF is accompanied by a decrease or increase in trGSH levels and ii) that elevated NGF and/or trGSH levels attenuate some of the responses to LPS such as anorexia and cytokine production.