Lipopolysaccharide (LPS) and zymosan-resistant mutant isolated from a macrophage-like cell line, WEHI-3, with a defective response to LPS under serum-free conditions

A LPS-resistant mutant, W3SF-1, was isolated from a murine macrophage-like cell line, WEHI-3. The W3SF-1 mutant did not produce a significant amount of nitric oxide (NO) or TNF-alpha even with high concentrations of LPS in the presence or absence of FCS, whereas the parental WEHI-3 cells produced them in response to LPS. The parental cells expressed a significant level of TNF-alpha mRNA after LPS stimulation, whereas the mutant cells did not. This defective response of the mutant cells to LPS was neither dependent on the concentration or chemical structure of LPS, nor on the time of LPS treatment. The mutant cells also showed a defective response to zymosan, suggesting that the defect in the mutant cells is common to LPS and zymosan in the signal transduction pathways. The parental and mutant cells showed similar levels of Mac1, F4/80 and CD14, suggesting that these surface markers of macrophages are not linked directly to the defective responses of the mutant to LPS. The treatment of mutant cells with IFN-gamma did not restore the defect of NO or TNF-alpha production on LPS treatment. Binding experiments with 125I-labelled LPS showed a similar binding affinity for LPS in the parental and the mutant cells. These results suggest that the defect in the W3SF-1 mutant cells may not reside in the LPS binding but rather in the early step of signal transduction pathways in the cells after LPS binding.     

The enhancing action of D-galactosamine on lipopolysaccharide-induced nitric oxide production in RAW 264.7 macrophage cells

The effect of D-galactosamine (D-GalN) on nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells was examined. D-GalN augmented the production of NO, but not tumor necrosis factor (TNF)-alpha in LPS-stimulated RAW 264.7 cells. Pretreatment of D-GalN augmented the NO production whereas its post-treatment did not. D-GalN augmented the NO production in RAW 264.7 cells stimulated with either TNF-alpha and interferon-gamma. The augmentation of LPS-induced NO production by D-GalN was due to enhanced expressions of an inducible type of NO synthase mRNA and proteins. Intracellular reactive oxygen species (ROS) were exclusively generated in RAW 264.7 cells stimulated with D-GalN and LPS. Scavenging of intracellular ROS abrogated the augmentation of NO production. It was therefore suggested that D-GalN might augment LPS-induced NO production through the generation of intracellular ROS.     

Methylenedioxymethamphetamine (MDMA; Ecstasy) suppresses IL-1beta and TNF-alpha secretion following an in vivo lipopolysaccharide challenge

In this study we examined the effects of methylenedioxymethamphetamine (MDMA) administration on responsiveness to an in vivo immune challenge with lipopolysaccharide (LPS; 100 microg/kg; i.p.). LPS produced an increase in circulating IL-1beta and TNF-alpha in control animals. MDMA (20 mg/kg; i.p.) significantly impaired LPS-induced IL-1beta and TNF-alpha secretion. The suppressive effect of MDMA on IL-1beta secretion was transient and returned to control levels within 3 hours of administration. In contrast, the MDMA-induced suppression of TNF-alpha secretion was evident for up to 12 hours following administration. In a second study we examined the effect of co-administration of MDMA (5, 10 and 20 mg/kg; i.p.) on LPS-induced IL-1beta and TNF-alpha secretion, and demonstrated that all three doses potently suppressed LPS-induced TNF-alpha secretion, but only MDMA 10 and 20 mg/kg suppressed LPS-induced IL-1beta secretion. In addition, serum MDMA concentrations displayed a dose-dependent increase, with the concentrations achieved following administration of 5 and 10 mg/kg being in the range reported in human MDMA abusers. In order to examine the possibility that the suppressive effect of MDMA on IL-1beta and TNF-alpha could be due to a direct effect of the drug on immune cells, the effect of in vitro exposure to MDMA on IL-1beta and TNF-alpha production in LPS-stimulated diluted whole blood was evaluated. However IL-1beta or TNF-alpha production were not altered by in vitro exposure to MDMA. In conclusion, these data demonstrate that acute MDMA administration impairs IL-1beta and TNF-alpha secretion following an in vivo LPS challenge, and that TNF-alpha is more sensitive to the suppressive effects of MDMA than is IL-1beta. However the suppressive effect of MDMA on IL-1beta and TNF-alpha could not be attributed to a direct effect on immune cells. The relevance of these findings to MDMA-induced immunomodulation is discussed.     

Endothelial nitric-oxide synthase enhances lipopolysaccharide-stimulated tumor necrosis factor-alpha expression via cAMP-mediated p38 MAPK pathway in cardiomyocytes

The purpose of this study was to investigate the role of endothelial nitric-oxide synthase (eNOS), cAMP, and p38 MAPK in tumor necrosis factor-alpha (TNF-alpha) expression induced by lipopolysaccharide (LPS). LPS dose- and time-dependently induced phosphorylation of p38 MAPK and TNF-alpha expression in neonatal mouse cardiomyocytes. TNF-alpha expression was preceded by p38 MAPK phosphorylation, and selective inhibition of p38 MAPK abrogated LPS-induced TNF-alpha expression. Deficiency in eNOS decreased basal and LPS-stimulated TNF-alpha expression in cardiomyocytes. NOS inhibitor l-NAME attenuated LPS-induced p38 MAPK phosphorylation and TNF-alpha production in wild-type cardiomyocytes, whereas NO donor 2,2'-(hydroxynitrosohydrazono)bis-ethanamine (DETA-NO) (2 microm) or overexpression of eNOS by adenoviral gene transfer restored the response of eNOS(-/-) cardiomyocytes to LPS. These effects of NO were mediated through cAMP-dependent pathway based on the following facts. First, deficiency in eNOS decreased basal levels of intracellular cAMP, and DETA-NO elevated intracellular cAMP levels in eNOS(-/-) cardiomyocytes. Second, a cAMP analogue 8-Br-cAMP mimicked the effect of NO in eNOS(-/-) cardiomyocytes. Third, either inhibition of cAMP or cAMP-dependent protein kinase attenuated LPS-stimulated p38 MAPK phosphorylation and TNF-alpha production in wild-type cardiomyocytes. In conclusion, eNOS enhances LPS-stimulated TNF-alpha expression in cardiomyocytes. Activation of p38 MAPK is essential in LPS-stimulated TNF-alpha expression. Moreover, the effects of NO on LPS-stimulated TNF-alpha expression are mediated through cAMP/cAMP-dependent protein kinase-dependent p38 MAPK pathway in neonatal cardiomyocytes.     

MPS1: a small, evolutionarily conserved zinc finger protein from the protozoan Toxoplasma gondii

Production of tissue factor (TF) in response to lipopolysaccharide (LPS) was examined in human umbilical vein endothelial cells (HUVECs) transfected with human CD14 DNA. The expression of CD14 on HUVECs dramatically enhanced the production of TF at a low concentration of LPS in the absence of fetal calf serum (FCS). On the other hand, mock-transfected HUVECs did not respond to even a high concentration of LPS. TF production in CD14-expressing HUVECs was significantly inhibited by anti-CD14 monoclonal antibody. Addition of FCS to the culture of CD14-expressing HUVECs markedly augmented the LPS-induced TF production, whereas only a marginal effect was observed in mock-transfected HUVECs. The findings suggested that the integration of membrane CD14 rendered HUVECs highly sensitive to LPS in the production of TF irrespective of the presence of FCS.     

Inhibitory effects of biseokeaniamide A against lipopolysaccharide-induced signal transduction

Lipopolysaccharides (LPS) are associated with various inflammatory diseases; therefore, the inhibition of LPS-induced nitric oxide (NO) production may have extensive therapeutic applications. We searched for inhibitors of NO production in the LPS-stimulated murine macrophage-like cell line RAW264.7 from MeOH extracts of marine organisms. The MeOH extract of the marine cyanobacterium Okeania sp., collected in Okinawa, Japan, showed inhibitory activity. Biseokeaniamide A was isolated from the MeOH extract by chromatographic separation. Biseokeaniamide A inhibited NO production without cytotoxicity. It reduced inducible nitric oxide synthase levels and suppressed the expression of IL-1β in LPS-stimulated RAW264.7 cells. Biseokeaniamide A did not inhibit IκBα degradation but inhibited IκBα expression. Thus, biseokeaniamide A, a naturally occurring lipopeptide, was identified as a selective inhibitor of LPS signal transduction.     

IL-4 pretreatment selectively enhances cytokine and chemokine production in lipopolysaccharide-stimulated mouse peritoneal macrophages

Although well recognized for its anti-inflammatory effect on gene expression in stimulated monocytes and macrophages, IL-4 is a pleiotropic cytokine that has also been shown to enhance TNF-alpha and IL-12 production in response to stimulation with LPS. In the present study we expand these prior studies in three areas. First, the potentiating effect of IL-4 pretreatment is both stimulus and gene selective. Pretreatment of mouse macrophages with IL-4 for a minimum of 6 h produces a 2- to 4-fold enhancement of LPS-induced expression of several cytokines and chemokines, including TNF-alpha, IL-1alpha, macrophage-inflammatory protein-2, and KC, but inhibits the production of IL-12p40. In addition, the production of TNF-alpha by macrophages stimulated with IFN-gamma and IL-2 is inhibited by IL-4 pretreatment, while responses to both LPS and dsRNA are enhanced. Second, the ability of IL-4 to potentiate LPS-stimulated cytokine production appears to require new IL-4-stimulated gene expression, because it is time dependent, requires the activation of STAT6, and is blocked by the reversible protein synthesis inhibitor cycloheximide during the IL-4 pretreatment period. Finally, IL-4-mediated potentiation of TNF-alpha production involves specific enhancement of mRNA translation. Although TNF-alpha protein is increased in IL-4-pretreated cells, the level of mRNA remains unchanged. Furthermore, LPS-stimulated TNF-alpha mRNA is selectively enriched in actively translating large polyribosomes in IL-4-pretreated cells compared with cells stimulated with LPS alone.     

Stimulus (polyphenol, IFN-gamma, LPS)-dependent nitric oxide production and antileishmanial effects in RAW 264.7 macrophages

The effects of interferon (IFN-γ), lipopolysaccharide (LPS), and some polyphenols as individual stimuli, as well as in various combinations on NO production in non-infected and infected macrophage-like RAW 264.7 cells were investigated, with emphasis on the NO/parasite kill relationship. In non-infected and in Leishmania parasitized cells, gallic acid significantly inhibited the IFN-γ and LPS-induced NO detected in the supernatant. This effect was less prominent in IFN-γ- than in LPS-stimulated cells. Interestingly, and in contrast to non-infected cells, gallic acid inhibited NO production only when added within 3 h after IFN-γ + LPS. Addition of gallic acid following prolonged incubation with IFN-γ + LPS periods (24 h) no longer inhibited, sometimes even enhanced NO release. Notably, an excellent NO/parasite kill relationship was evident from all the experiments. This study was extended to a series of polyphenols (3-O-shikimic acid, its 3,5-digalloylated analogue, catechin, EGCG, and a procyanidin hexamer) with proven immunostimulatory activities. Although these compounds themselves were found to be weak NO-inducers, the viability of intracellular Leishmania parasites was considerably reduced. Furthermore, their dose-dependent effects on macrophage NO release was determined in the presence of IFN-γ and/or LPS. Again, non-infected and infected cells differed significantly in the NO response, while inhibition of IFN-γ and/or LPS-induced NO production by the tested polyphenols strongly depended on the given time of exposure and the sequence of immunological stimuli. A strong inverse correlation between NO levels and intracellular survival rates of Leishmania parasites supported the assumption that the observed inhibition of NO was not simply due to interference with the Griess assay used for detection.     

C-Mannosylated peptides derived from the thrombospondin type 1 repeat enhance lipopolysaccharide-induced signaling in macrophage-like RAW264.7 cells

C-Mannosylation is a unique type of glycosylation occurring at the first Trp (W) in the WXXW motif, which is found in the thrombospondin type 1 repeat (TSR) of proteins. However, the biological function of C-mannosylation is not fully understood. In this study, we investigated the effect of C-mannosylated TSR-derived peptides on lipopolysaccharide (LPS)-induced signaling in macrophage-like RAW264.7 cells. The cells were stimulated with LPS in the presence or absence of chemically synthesized peptides with or without C-mannose (e.g., (C-Man)-Trp-Ser-Pro-Trp [C-Man-WSPW], C-Man-W, WSPW, etc.), then the effects of the peptides on cellular viability and signaling were examined. We found a cytotoxic effect in the cells treated with LPS and C-Man-WSPW, but not in the cells solely treated with LPS or C-Man-WSPW. We also found that production of tumor necrosis factor-alpha (TNF-alpha) was upregulated more in response to LPS plus C-Man-WSPW, than in response to LPS plus WSPW or LPS alone. Among the LPS-induced signaling pathways that induce production of TNF-alpha, the activation of c-Jun N-terminal kinase (JNK) was greatly enhanced by LPS and C-Man-WSPW, and the production of TNF-alpha was suppressed by an inhibitor for JNK. Together, these results demonstrate a novel function of the C-mannosylated TSR-derived peptide motif, to promote LPS-induced JNK signaling, and this leads to an enhancement of cytotoxicity via the upregulation of TNF-alpha production.     

IL-9 inhibits oxidative burst and TNF-alpha release in lipopolysaccharide-stimulated human monocytes through TGF-beta

IL-9 is a Th2 cytokine that exerts pleiotropic activities on T cells, B cells, mast cells, hematopoietic progenitors, and lung epithelial cells, but no effect of this cytokine has been reported so far on mononuclear phagocytes. Human blood monocytes preincubated with IL-9 for 24 h before LPS or PMA stimulation exhibited a decreased oxidative burst, even in the presence of IFN-gamma. The inhibitory effect of IL-9 was specifically abolished by anti-hIL-9R mAb, and the presence of IL-9 receptors was demonstrated on human blood monocytes by FACS. IL-9 also down-regulated TNF-alpha and IL-10 release by LPS-stimulated monocytes. In addition, IL-9 strongly up-regulated the production of TGF-beta1 by LPS-stimulated monocytes. The suppressive effect of IL-9 on the respiratory burst and TNF-alpha production in LPS-stimulated monocytes was significantly inhibited by anti-TGF-beta1, but not by anti-IL-10Rbeta mAb. Furthermore, IL-9 inhibited LPS-induced activation of extracellular signal-regulated kinase 1/2 mitogen-activated protein kinases in monocytes through a TGF-beta-mediated induction of protein phosphatase activity. In contrast, IL-4, which exerts a similar inhibitory effect on the oxidative burst and TNF-alpha release by monocytes, acts primarily through a down-regulation of LPS receptors. Thus, IL-9 deactivates LPS-stimulated blood mononuclear phagocytes, and the mechanism of inhibition involves the potentiation of TGF-beta1 production and extracellular signal-regulated kinase inhibition. These findings highlight a new target cell for IL-9 and may account for the beneficial activity of IL-9 in animal models of exaggerated inflammatory response.     

Surfactant protein A inhibits alveolar macrophage cytokine production by CD14-independent pathway

The lung collectin surfactant protein A (SP-A) has both anti-inflammatory and prophagocytic activities. We and others previously showed that SP-A inhibits the macrophage production of tumor necrosis factor (TNF)-alpha stimulated by the gram-negative bacterial component LPS. We propose that SP-A decreases the production of proinflammatory cytokines by alveolar macrophages via a CD14-independent mechanism. SP-A inhibited LPS-simulated TNF-alpha production in rat and mouse macrophages in the presence and absence of serum (72% and 42% inhibition, respectively). In addition, SP-A inhibited LPS-induced mRNA levels for TNF-alpha, IL-1 alpha, and IL-1 beta as well as NF-kappa B DNA binding activity. SP-A also diminished ultrapure LPS-stimulated TNF-alpha produced by wild-type and CD14-null mouse alveolar macrophages by 58% and 88%, respectively. Additionally, SP-A inhibited TNF-alpha stimulated by PMA in both wild-type and TLR4-mutant macrophages. These data suggest that SP-A inhibits inflammatory cytokine production in a CD14-independent manner and also by mechanisms independent of the LPS signaling pathway.     

Minocycline inhibits apoptotic cell death via attenuation of TNF-alpha expression following iNOS/NO induction by lipopolysaccharide in neuron/glia co-cultures

We attempted to ascertain the neuroprotective effects and mechanisms of minocycline in inflammatory-mediated neurotoxicity using primary neuron/glia co-cultures treated with lipopolysaccharide (LPS). Neuronal cell death was induced by treatment with LPS for 48 h, and the cell damage was assessed using lactate dehydrogenase (LDH) assays and by counting microtubule-associated protein-2 (MAP-2) positive cells. Through terminal transferase deoxyuridine triphosphate-biotin nick end labeling (TUNEL)-staining and by measuring caspase-3 activity, we found that LPS-induced neuronal cell death was mediated by apoptosis. We determined that pre-treatment with minocycline significantly inhibited LPS-induced neuronal cell death. In addition, LPS induced inducible nitric oxide synthase (iNOS) expression significantly, resulting in nitric oxide (NO) production within glial cells, but not in neurons. Both nitric oxide synthase (NOS) inhibitors (N(G)-monomethyl-L-arginine monoacetate (L-NMMA) and S-methylisothiourea sulfate (SMT)) and minocycline inhibited iNOS expression and NO release, and increased neuronal survival in neuron/glia co-cultures. Pre-treatment with minocycline significantly inhibited the rapid and extensive production of tumor necrosis factor-alpha (TNF-alpha) mediated by LPS in glial cells. We also determined that the signaling cascade of LPS-mediated iNOS induction and NO production was mediated by TNF-alpha by using neutralizing antibodies to TNF-alpha. Consequently, our results show that the neuroprotective effect of minocycline is associated with inhibition of iNOS induction and NO production in glial cells, which is mediated by the LPS-induced production of TNF-alpha.     

Reduced lipopolysaccharide (LPS)-induced nitric oxide production in peritoneal macrophages and inhibited LPS-induced lethal shock in mice by a sugar cane (Saccharum officinarum L.) extract

A sugar cane extract (SCE) has been found to have an immunostimulating effect in several animals. Lipopolysaccharide (LPS) is known to induce endotoxin shock via the production of inflammatory modulators such as tumor necrosis factor (TNF)-alpha and nitric oxide (NO). We examined in the present study the effects of SCE on the TNF-alpha and NO production in LPS-stimulated mice peritoneal cells and the endotoxin shock in mice. The supplementation of SCE to peritoneal macrophages cultured with LPS resulted in a significant decrease in NO production. All the mice injected intraperitoneally with LPS and D-galactosamine (LPS+GalN) died within 24 h. However, a peritoneal injection, but no intravenous or oral administration, of SCE (500-1,000 mg/kg) at 3 to 48 h before the LPS+GalN-challenge resulted in a significantly improved survival rate. These results suggest that SCE had a protective effect on LPS-induced endotoxin shock via one of possible mechanisms involving the suppression of NO production in the mouse peritoneal cavity.     

Involvement of protein kinase C in the inhibition of lipopolysaccharide-induced nitric oxide production by thapsigargin in RAW 264.7 macrophages

This study explored the effects of inhibition of endoplasmic reticulum (ER) Ca²⁺-ATPase on lipopolysaccharide (LPS)-induced protein kinase C (PKC) activation, nuclear factor-κB (NF-κB) translocation, inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production in RAW 264.7 macrophages. Thapsigargin (TG) irreversibly inhibits ER Ca²⁺-ATPase and LPS-induced NO production is reduced even after washout. TG also attenuated LPS-stimulated iNOS expression by using immunoblot analysis. However, another distinct fully reversible ER Ca²⁺-ATPase inhibitor, 2,5-di-tert-butylhydroquinone (DBHQ), ionophore A23187 and ionomycin could exert a similar effect to TG in increasing intracellular calcium concentration; however, these agents could not mimic TG in reducing iNOS expression and NO production. LPS increased PKC- and -β activation, and TG pretreatment attenuated LPS-stimulated PKC activation. Not did pretreatment with DBHQ, A23187 and ionomycin reduce LPS-stimulated PKC activation. Furthermore, NF-κB-specific DNA–protein-binding activity in the nuclear extracts was enhanced by treatment with LPS, and TG pretreatment attenuated LPS-stimulated NF-κB activation. None of DBHQ, A23187 and ionomycin pretreatment reduced LPS-stimulated NF-κB activation. These data suggest that persistent inhibition of ER Ca²⁺-ATPase by TG would influence calcium release from ER Ca²⁺ pools that was stimulated by the LPS activated signal processes, and might be the main mechanism for attenuating PKC and NF-κB activation that induces iNOS expression and NO production.     

The inhibitory action of sodium arsenite on lipopolysaccharide-induced nitric oxide production in RAW 267.4 macrophage cells: a role of Raf-1 in lipopolysaccharide signaling

The effect of sodium arsenite (SA) on LPS-induced NO production in RAW 267.4 murine macrophage cells was studied. SA pretreatment of LPS-stimulated RAW cells resulted in a striking reduction in NO production. No significant difference in LPS binding was observed between RAW cells pretreated with SA and control untreated RAW cells, suggesting that SA might impair the intracellular signal pathway for NO production. SA inhibited LPS-induced NF-kappaB activation by preventing loss of IkappaB-alpha and -beta. Furthermore, SA blocked phosphorylation of extracellular signal-regulated kinase 1/2 (Erk1/2), but not phosphorylation of p38 and c-Jun N-terminal kinase. SA treatment resulted in the disappearance of Raf-1, suggesting that it might cause the inhibition of the Erk1/2 mitogen-activated protein (MAP) kinase pathway. The SA-mediated loss of Raf-1 also abolished LPS-induced NF-kappaB activation as well as the Erk1/2 pathway. The dominant negative mutant of MAP kinase kinase 1 inhibited both NO production and NF-kappaB activation in LPS-stimulated RAW cells. Taken together, these results indicate that the inhibitory action of SA on NO production in LPS-stimulated macrophages might be due to abrogation of inducible NO synthase induction, and it might be closely related to inactivation of the NF-kappaB and Erk1/2 MAP kinase pathways through loss of Raf-1.     

IgG-coated erythrocytes augment LPS-stimulated TNF-alpha secretion, TNF-alpha mRNA levels, and TNF-alpha mRNA stability in macrophages

Previous studies have shown that IgG-coated erythrocytes (EIgG) augment the LPS-stimulated increase in serum TNF-alpha levels in animals and the LPS-stimulated secretion of TNF-alpha by isolated macrophages. The present study evaluated the mechanism for the effect of EIgG on LPS-stimulated TNF-alpha secretion in the murine macrophage cell line, RAW 264.7. Incubation of the macrophages with EIgG or IgG-coated glass beads caused a dose-dependent augmentation of LPS-stimulated TNF-alpha secretion. The addition of EIgG increased the rate of LPS-stimulated TNF-alpha protein secretion between 2 and 4 hr after LPS. Accordingly, EIgG increased the levels of TNF-alpha mRNA at 2 and 3 hr after LPS. The increase in the LPS-stimulated TNF-alpha mRNA levels caused by EIgG was associated with an increase in TNF-alpha mRNA stability. Thus, the augmentation of LPS-stimulated TNF-alpha secretion by EIgG was associated with an increase in TNF-alpha mRNA levels which at least partly resulted from an increase in the stability of TNF-alpha mRNA.