Evidence for the involvement of interleukin 10 in the differential deactivation of murine peritoneal macrophages by prostaglandin E2

Among other effects, prostaglandins (PG) of the E series are known to inhibit several acute and chronic inflammatory conditions in vivo and proinflammatory cytokine production by activated macrophages in culture. The research presented here demonstrates that the inhibitory effect of PGE2 on tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) production by lipopolysaccharide (LPS)-stimulated murine peritoneal macrophages involves IL-10. In a dose-dependent manner, PGE2 inhibits LPS-induced release of TNF-alpha and IL-6, but not of lactate or nitric oxide. The decrease in the level of these cytokines is inversely proportional to the increase in immunoreactive IL-10. This differential inhibitory effect of PGE2 is mimicked by agents that elevate intracellular levels of cAMP, but not cGMP. Neutralizing anti IL-10 antibody but not neutralizing antibodies against other macrophage secretory products (IL-6, leukemia inhibitory factor, and transforming growth factor beta [TGF-beta]), significantly reverse the potent inhibitory effect of PGE2. In vivo, the administration of PGE2 before LPS challenge significantly reduces circulating TNF-alpha and IL-6 levels. Anti-IL-10 antibody substantially enhanced the LPS-induced TNF-alpha and IL-6 levels in mice that received either LPS alone or LPS plus PGE2. These results suggest that the anti-inflammatory effect of PGE2 on mononuclear phagocytes is mediated in part by an autocrine feedback mechanism involving IL-10.     

Inhibition of lipopolysaccharide-induced prostaglandin E2 production and inflammation by the Na+/H+ exchanger inhibitors

We analyzed the effects of the Na+/H+ exchanger (NHE) inhibitor 3,5-diamino-6-chloro-N-(diaminomethylidene)pyrazine-2-carboxamide hydrochloride (amiloride) and its analogs 5-(N,N-dimethyl)-amiloride (DMA) and 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) on the lipopolysaccharide (LPS)-induced production of prostaglandin (PG) E2 in vitro and in vivo. In the mouse macrophage-like cell line RAW 264, these inhibitors suppressed the LPS (1 microg/ml)-induced production of PGE2 at 8 h in a concentration-dependent manner. They also reduced the LPS-induced release of arachidonic acid from membrane phospholipids at 4 h and the LPS-induced increase in the level of cyclooxygenase (COX)-2 protein at 6 h, but not the level of COX-2 mRNA at 3 h. The LPS-induced phosphorylation of mitogen-activated protein kinases and degradation of inhibitor of kappaB-alpha were not inhibited by these drugs. In an air pouch-type LPS-induced inflammation model in mice 30 mg/kg amiloride and 10 mg/kg EIPA as well as the COX inhibitor indomethacin (10 mg/kg), significantly reduced the level of PGE2 in the pouch fluid at 8 h and the vascular permeability from 4 to 8 h. The accumulation of pouch fluid and leukocytes in the pouch fluid at 8 h was significantly inhibited by amiloride and EIPA but not by indomethacin. These findings suggested that the NHE inhibitors suppress the production of PGE2 through inhibiting the release of arachidonic acid and the increase in COX-2 protein levels and thus induce anti-inflammatory activity.     

Effect of lipoteichoic acid on dermal vascular permeability in mice

Lipoteichoic acid (LTA), the cell wall component of Gram-positive bacteria, has been shown to cause inflammatory responses comparable to lipopolysaccharide (LPS) of Gram-negative bacteria. This study examined the activity of LTA to induce dermal microvascular permeability changes in mice. Vascular permeability was assessed by extravasation of Pontamine sky blue. Subcutaneous injection of LTA (200-400 microg/site) in mice that were preinjected i.v. with the dye increased local dye leakage in the skin at 1 to 3 h. The LTA-induced dye leakage was inhibited by indomethacin, valeryl salicylate, diphenhydramine, and a platelet-activating factor antagonist but not by inhibitors of nitric-oxide synthase, cyclooxygenase-2, or guanylate cyclase or by antibodies against tumor necrosis factor-alpha or interleukin-1alpha. LTA induced comparable increases in dye leakage in inducible nitric-oxide synthase-deficient mice and wild-type controls. Pretreatment of normal mice with i.v. LTA did not confer tolerance to LTA- or LPS-induced dye leakage. In contrast, systemic LPS administration induced tolerance against subsequent challenge with LPS but not LTA. Serum corticosterone levels, which were suggested to induce tolerance, were not increased by LTA pretreatment but were increased by LPS. Thus, LTA increases dermal microvascular permeability in mice. Among the inflammatory mediators, eicosanoids, platelet-activating factor, and histamine mediate the effect of both LTA and LPS, whereas nitric oxide, tumor necrosis factor-alpha, and interleukin-1alpha may not play a major role in LTA-induced dye leakage. The difference between LTA and LPS to stimulate corticosterone may partially explain the failure of LTA to induce tolerance against vascular dye leakage.     

Prevention of ethanol-induced liver injury in rats by an agonist of peroxisome proliferator-activated receptor-gamma,pioglitazone

Agonists of peroxisome proliferator-activated receptor (PPAR)-gamma have been shown to reduce tumor necrosis factor-alpha (TNF-alpha)-induced insulin resistance. On the other hand, sensitization of Kupffer cells to lipopolysaccharide (LPS) and their production of TNF-alpha are critical for progression of alcoholic liver injury. This study was intended to determine whether pioglitazone, a PPAR-gamma agonist, could prevent alcohol-induced liver injury. Rats were given ethanol (5 g/kg b.wt.) and pioglitazone (500 microg/kg) once every 24 h intragastrically. Ethanol for 8 weeks caused pronounced steatosis, necrosis, and inflammation in the liver. These pathological parameters were diminished greatly by pioglitazone. Kupffer cells were sensitized to LPS after ethanol for 4 weeks as evidenced by aggravation of liver pathology induced by LPS (5 mg/kg) and enhancement of LPS (100 ng/ml)-induced intracellular Ca2+ concentration elevation in Kupffer cells. The parameters were diminished by treatment with pioglitazone. LPS-induced TNF-alpha production by Kupffer cells from the 4-week ethanol group was 3 to 4 times higher than control. This increase was blunted by 70% with pioglitazone. Gut permeability was 10-fold higher in the 4-week ethanol group, and pioglitazone treatment did not change the value. Inclusion of TNF-alpha in culture media of Kupffer cells enhanced CD14 expression, LPS-induced intracellular Ca2+ concentration response, and production of TNF-alpha. These results indicate that pioglitazone prevents alcoholic liver injury through abrogation of Kupffer cell sensitization to LPS.     

Opposite effects of enhanced tumor necrosis factor-alpha production from Kupffer cells by gadolinium chloride on liver injury/mortality in endotoxemia of normal and partially hepatectomized mice

Gadolinium chloride (GdCl3) reportedly inhibits Kupffer cell function including TNF-alpha production and thereby improves organ dysfunctions after LPS challenge, particularly in partially hepatectomized (PH) mice. In addition, TNF-alpha reportedly promotes the regeneration of hepatocytes after PH. However, we have frequently seen GdCl3 treatment increase the mortality of normal mice after LPS injection. Therefore, we investigated this controversial issue in the present study. The mice treated by GdCl3 (10 mg/kg, i.v.) at 24 h before LPS challenge showed increased serum TNF-alpha and ALT levels after LPS challenge and a decreased mouse survival rate. The Kupffer cells from GdCl3-treated mice consistently produced a much larger amount of TNF-alpha following in vitro LPS stimulation than those of the control mice despite the fact that the Kupffer cells decreased in number and also demonstrated decreased superoxide production. Anti-TNF-alpha Ab before LPS-injection greatly improved GdCl3-induced mouse mortality and the degree of liver injury. In marked contrast, the increased amount of TNF-alpha induced by GdCl3 improved the survival after LPS challenge in PH mice because TNF-alpha promoted hepatocyte mitosis/regeneration in PH liver as evidenced by the fact that the inhibition of TNF-alpha before PH suppressed hepatocyte regeneration and decreased survival after LPS challenge. In conclusion, GdCl3 depletes the superoxide-producing Kupffer cells but conversely enhances the function of TNF-alpha-producing Kupffer cells, which thereby leads to LPS-induced mortality. Meanwhile, the increased TNF-alpha production induced by GdCl3 supports liver regeneration and increases the survival after LPS challenge in PH mice.     

Oral administration of geranylgeranylacetone improves survival rate in a rat endotoxin shock model: administration timing and heat shock protein 70 induction

The present study was performed to determine whether oral pretreatment with geranylgeranylacetone (GGA) inhibits proinflammatory cytokine liberation and nitric oxide (NO) production in lipopolysaccharide (LPS)-treated rats and protects rats against death from LPS-induced endotoxin shock, and whether such protection by GGA is related to heat shock protein (HSP) 70 induction in multiple organs of rats. GGA (200 mg/kg) was given orally to rats. LPS (20 mg/kg) was administered intraperitoneally 4, 8, 16, or 24 h after GGA administration. The survival of rats was monitored over 24 h after LPS administration. GGA treatment at 8 or 16 h before LPS dramatically improved the survival rate of LPS-treated rats. Plasma levels of proinflammatory cytokines (tumor necrosis factor-alpha and interleukin-6) and NO 6 h after LPS administration in these GGA-pretreated rats were less than one-half of those in rats treated with LPS alone. A GGA challenge 8 or 16 h before LPS administration enhanced HSP70 expression in rat organs after LPS. Treatment with GGA 8 h before LPS minimized hepatic and renal damage. Furthermore, the protective effect of GGA on mortality in LPS-treated rats was inhibited with quercetin, known as an HSP70 inhibitor. These results suggest that oral administration of GGA at an optimal time before LPS injection induces and enhances HSP70 expression in several organs, inhibits proinflammatory cytokine and NO production, and prevents organ damage, resulting in an improved survival rate.     

Calcitonin gene-related peptide inhibits local acute inflammation and protects mice against lethal endotoxemia

Calcitonin gene-related peptide (CGRP), a potent vasodilatory peptide present in central and peripheral neurons, is released at inflammatory sites and inhibits several macrophage, dendritic cell, and lymphocyte functions. In the present study, we investigated the role of CGRP in models of local and systemic acute inflammation and on macrophage activation induced by lipopolysaccharide (LPS). Intraperitoneal pretreatment with synthetic CGRP reduces in approximately 50% the number of neutrophils in the blood and into the peritoneal cavity 4 h after LPS injection. CGRP failed to inhibit neutrophil recruitment induced by the direct chemoattractant platelet-activating factor, whereas it significantly inhibited LPS-induced KC generation, suggesting that the effect of CGRP on neutrophil recruitment is indirect, acting on chemokine production by resident cells. Pretreatment of mice with 1 mug of CGRP protects against a lethal dose of LPS. The CGRP-induced protection is receptor mediated because it is completely reverted by the CGRP receptor antagonist, CGRP 8-37. The protective effect of CGRP correlates with an inhibition of TNF-alpha and an induction of IL-6 and IL-10 in mice sera 90 min after LPS challenge. Finally, CGRP significantly inhibits LPS-induced TNF-alpha released from mouse peritoneal macrophages. These results suggest that activation of the CGRP receptor on macrophages during acute inflammation could be part of the negative feedback mechanism controlling the extension of acute inflammatory responses.     

Mechanisms of increased survival after lipopolysaccharide-induced endotoxic shock in mice consuming olive oil-enriched diet

We examined the impact of dietary fatty acid intake on lipopolysaccharide (LPS)-induced endotoxic shock. C57Bl/6J mice were fed for 6 weeks with a commercial laboratory chow (CC) or with test chows containing 7% (w/w) canola oil (CO), sesame oil (SeO), soybean oil (SO), or virgin olive oil (OO). The increase in body weight and energy consumption were similar for all diets tested. In the sixth week, mice were injected intraperitoneally with 400 microg of bacterial LPS to induce endotoxic shock. LPS induced a massive neutrophil infiltration into the peritoneal cavity and an increase in lipid body (LB) formation in leukocytes recovered from the peritoneal fluid of mice fed with CC, CO, SeO, or SO. In addition, there were increases in prostaglandin E(2) (PGE(2)), leukotriene B4 (LTB(4)), and cytokines IL-6, IL-10, and MCP-1 in peritoneal lavage, as well as in plasma TNF-alpha. In contrast, mice fed with OO exhibited reduced neutrophil accumulation and LB formation, and also had lower levels of PGE(2), LTB(4), MCP-1, and TNF-alpha. All mice fed with CC, CO, SeO, or SO died within 48 to 72 h after LPS injection. Interestingly, mice fed with the OO diet were resistant to endotoxic shock, with 60% survival at 168 h. These data indicate that intake of OO may have a beneficial role, reducing the magnitude of the inflammatory process triggered by endotoxic shock through modulation of LB formation and of the production of inflammatory mediators.     

Endotoxin stimulates in vivo expression of inflammatory cytokines tumor necrosis factor alpha,interleukin-1beta, -6, and high-mobility-group protein-1 in skeletal muscle

The presence of increased levels of proinflammatory cytokines in the blood is associated with decreased muscle protein synthesis and the erosion of lean body mass in many catabolic conditions. However, little is known regarding the role of endogenous cytokine synthesis in muscle per se. The purpose of the present study was to characterize the cytokine expression profile of skeletal muscle in response to an in vivo injection of endotoxin (lipopolysaccharide, LPS). Intraperitoneal injection of a nonlethal dose of LPS (1,000 microg/kg Escherichia coli) into male rats increased the mRNA content of tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-1beta in gastrocnemius muscle as early as 1 h; IL-6 mRNA was not increased until 2 h post-LPS. Expression of TNF-alpha and IL-1beta peaked at 2 h (10- and 80-fold, respectively), whereas the increased IL-6 mRNA content (150-fold) peaked later at 4 h. The abundance of all measured cytokine mRNAs in skeletal muscle declined thereafter. The LPS-induced increase in muscle mRNA content for TNF-alpha, IL-6, and IL-1beta was dose-dependent with elevations being seen with as little as 10 microg/kg of LPS (2.5-, 8-, and 9-fold, respectively). In general, pretreatment of rats with dexamethasone attenuated but did not completely prevent the LPS-induced increase in muscle cytokine mRNA. LPS increased muscle TNF-alpha protein content approximately 2-fold and this increase was prevented by pretreatment with dexamethasone. LPS-induced increases in muscle IL-1beta and IL-6 protein were not detected. LPS also produced a 2-fold increase in the mRNA content of the high-mobility-group protein-1, a late-phase cytokine, in muscle at 12-24 h. Finally, although skeletal muscle was found to contain both the toll-like receptor (TLR)-2 and TLR4, LPS did not alter the mRNA content of TLR4 and produced a small (50%) but significant increase in TLR2 mRNA. These changes in TLRs were less dramatic than those observed for liver, spleen or cardiac muscle. Collectively these data indicate that skeletal muscle possesses many of the components of the innate immune system, including increases in both early- and late-phase cytokines and the presence of toll-like receptors.     

LPS-induced vascular endothelial growth factor expression in rat lung pericytes

Vascular endothelial growth factor (VEGF) is a potent angiogenic and vascular permeability factor. Recent studies have shown that the VEGF levels increase in several cell types, for example, macrophages and smooth muscle cells after LPS stimulation, suggesting that it is important in the initiation and development of sepsis. In particular, LPS-regulated contractility in lung pericytes may play an important role in mediating pulmonary microvascular fluid hemodynamics during sepsis. This study investigated the production of VEGF by rat lung pericytes in response to LPS. LPS was found to enhance VEGF mRNA expression in a concentration-dependent manner peaking 2 h after stimulation in pericytes. Vascular endothelial growth factor protein levels in conditioned medium and in cell lysate also increased on increasing LPS and peaked after 24 to 48 h. LPS also significantly augmented iNOS expression in lung pericytes within 6 h. However, iNOS mRNA induction occurred later than LPS-induced VEGF mRNA increases. Interestingly, attempted inhibition with nuclear factor-kappaB or tyrosine kinase did not suppress LPS-induced augmented VEGF mRNA expression in lung pericytes, although both inhibitors markedly inhibited LPS-induced iNOS mRNA expression. SB203580, a p38 MAP kinase inhibitor, repressed LPS-induced VEGF mRNA expression. Furthermore, LPS stimulated a rapid and sustained phosphorylation of p38 MAP kinase. These results show that pericytes produce VEGF in response to LPS stimulation, and that this may be partly mediated by the p38 MAP kinase pathway. More research should be done to establish the regulation of capillary hemodynamics and identify mechanisms of their regulation.     

Local stimulation of alpha7 cholinergic receptors inhibits LPS-induced TNF-alpha release in the mouse lung

The cholinergic nervous system can inhibit the release of proinflammatory cytokines such as TNF-alpha from LPS-stimulated macrophages. Acetylcholine, the principal neurotransmitter of the vagus nerve, is the key mediator of this so-called cholinergic anti-inflammatory pathway, specifically interacting with alpha7 cholinergic receptors expressed by macrophages and other cell types to inhibit TNF-alpha production. The aim of the current study was to determine the capacity of the selective alpha7 cholinergic receptor agonist 3-(2,4-dimethoxybenzylidene) anabaseine (GTS-21), administered locally into the airways, to inhibit LPS-induced inflammatory responses in the mouse lung in vivo. GTS-21 dose-dependently inhibited LPS-induced TNF-alpha release by MH-S mouse alveolar macrophages in vitro. Intranasal inoculation with GTS-21 also dose-dependently inhibited TNF-alpha release into the lung compartment after intrapulmonary delivery of LPS in mice in vivo, whereas IL-6 concentrations were not affected. However, GTS-21 did not influence the influx of neutrophils into bronchoalveolar lavage fluid elicited by LPS and increased the concentrations of the neutrophil-attracting chemokines cytokine-induced neutrophil chemoattractant and macrophage inflammatory protein 2. These data indicate that local administration of GTS-21 inhibits TNF-alpha release in the lung during LPS-induced inflammation.     

The tumor necrosis factor alpha -308 G/A polymorphism does not influence inflammation and coagulation response in human endotoxemia

Tumor necrosis factor (TNF)-alpha plays a major role in the immune system. Release of proinflammatory cytokines, such as TNF-alpha and interleukin 6, by macrophages and other cells occurs in response to bacterial products. It has been reported that the TNF-alpha -308 G/A polymorphism in the TNF-alpha gene determines basal TNF-alpha levels. We hypothesized that it may also be associated with the degree of inflammatory response in a well-standardized model of systemic inflammation. Eighty-seven young men (age range, 19-35 years) received 2 ng/kg i.v. endotoxin (lipopolysaccharide [LPS]). The TNF-alpha promoter genotype was analyzed on a TaqMan genomic analyzer. Inflammation markers (interleukin 6, TNF-alpha), temperature, and coagulation markers (prothrombin fragment F1+2, D-dimer) were measured at 0, 2, 6, and 24 h after LPS infusion. Tumor necrosis factor-alpha plasma concentrations increased from a baseline 1.3 ng/L (range, 0.8-3.1 ng/L) before LPS infusion to a peak of 57.5 ng/L (range, 10.8-131.4 ng/L) at 2 h after LPS and then decreased continually to 10.8 ng/L (range, 4.7-16.5 ng/L) after 6 h and returned to baseline values after 24 h (1.9 ng/L [range, 1.1-3.9 ng/L]). We observed no significant differences in TNF-alpha baseline levels or in response to LPS after stratification of the data according to TNF-alpha genotype. Basal and peak values of selected inflammatory and coagulation markers were not different between wild-type TNF-alpha -308 individuals (GG) and carriers of the TNF-alpha -308 mutant allele (GA and AA). The TNF-alpha -308 G/A polymorphism does not contribute significantly to the individual variability of systemic TNF-alpha plasma concentrations after endotoxin challenge. Thus, if any, the impact of the TNF-alpha -308 G/A polymorphism on systemic endotoxin-triggered inflammation seems to be limited.     

P2X7 receptor activation amplifies lipopolysaccharide-induced vascular hyporeactivity via interleukin-1 beta release

Lipopolysaccharide (LPS) stimulates cytoplasmic accumulation of pro-interleukin (IL)-1beta. Activation of P2X(7) receptors stimulates conversion of pro-IL-1beta into mature IL-1beta, which is then secreted. Because both LPS (in vivo) and IL-1beta (in vitro) decrease vascular reactivity to contractile agents, we hypothesized the following: 1) P2X(7) receptor activation contributes to LPS-induced vascular hyporeactivity, and 2) IL-1beta mediates this change. Thoracic aortas were obtained from 12-week-old male C57BL/6 mice. The aortic rings were incubated for 24 h in Dulbecco's modified Eagle's medium, LPS, benzoylbenzoyl-ATP (BzATP; P2X(7) receptor agonist), LPS plus BzATP, oxidized ATP (oATP; P2X(7) receptor antagonist), or oATP plus LPS plus BzATP. After the treatment, the rings were either mounted in a myograph for evaluation of contractile activity or homogenized for IL-1beta and inducible nitric-oxide synthase (iNOS) protein measurement. In endothelium-intact aortic rings, phenylephrine (PE)-induced contractions were not altered by incubation with LPS or BzATP, but they significantly decreased in aortic rings incubated with LPS plus BzATP. Treatment with oATP or IL-1ra (IL-1beta receptor antagonist) reversed LPS plus BzATP-induced hyporeactivity to PE. In the presence of N(G)-nitro-l-arginine methyl ester or N-([3-(aminomethyl)phenyl]methyl)ethanimidamide (selective iNOS inhibitor), the vascular hyporeactivity induced by LPS plus BzATP on PE responses was not observed. BzATP augmented LPS-induced IL-1beta release and iNOS protein expression, and these effects were also inhibited by oATP. Moreover, incubation of endothelium-intact aortic rings with IL-1beta induced iNOS protein expression. Thus, activation of P2X(7) receptor amplifies LPS-induced hyporeactivity in mouse endothelium-intact aorta, which is associated with IL-1beta-mediated release of nitric oxide by iNOS.     

Transgenic expression of human complement regulators reduces skeletal muscle ischaemia/reperfusion injury in mice

This study aimed to explore the hypothesis that activated complement components contribute significantly to I/R (ischaemia/reperfusion) injury in skeletal muscle. After 50, 70 and 90 min of tourniquet ischaemia and 24 h of reperfusion, viability of the medial gastrocnemius muscle in CBA-C57BL/6 wild-type mice, assessed histochemically by reduction of NBT (Nitro Blue Tetrazolium) dye, was 60, 21 and 8% respectively. Skeletal muscle viability after 70 min of ischaemia and 24 h of reperfusion in transgenic mice expressing a combination of human CD46, CD55 and CD59, all inhibitors of complement activation, was 45% compared with 24% in ischaemic reperfused wild-type mice (P=0.008; n=6 per group). Muscle from sham-treated transgenic mice and wild-type littermates had no significant loss of viability relative to normal contralateral gastrocnemius muscle. A significant reduction in myeloperoxidase activity (a measure of neutrophil infiltration), xanthine oxidase activity (a source of free radicals) and water content (a measure of oedema) was observed in ischaemic reperfused muscle from transgenic mice compared with ischaemic reperfused wild-type muscle (P<0.05). Haematoxylin and eosin-stained histological sections also showed less damage and less apparent leucocyte infiltration in muscles from ischaemic reperfused transgenic mice than those from wild-type animals given the same degree of injury. Muscles from sham-treated transgenic and wild-type controls were almost identical with normal muscle. It is concluded that complement activation contributes to the pathogenesis of I/R injury in murine skeletal muscle, resulting in increased neutrophil infiltration into the injured muscle, increased free radical production and vascular permeability during reperfusion, and a net detrimental effect on muscle viability.