Lipoteichoic Acids from Lactobacillus Strains Elicit Strong Tumor Necrosis Factor Alpha-Inducing Activities in Macrophages through Toll-Like Receptor 2

Lactobacilli are nonpathogenic gram-positive inhabitants of microflora. At least some Lactobacillus strains have been postulated to have health beneficial effects, such as the stimulation of the immune system. Here we examined the stimulatory effects of lactobacilli on mouse immune cells. All six heat-killed Lactobacillus strains examined induced the secretion of tumor necrosis factor alpha (TNF-α) from mouse splenic mononuclear cells, albeit to various degrees. When fractionated subcellular fractions of Lactobacillus casei were tested for NF-κB activation and TNF-α production in RAW264.7, a mouse macrophage cell line, the activity was found to be as follows: protoplast > cell wall polysaccharide-peptidoglycan complex. Both crude extracts and purified lipoteichoic acids (LTAs) from two Lactobacillus strains, L. casei and L. fermentum, significantly induced TNF-α secretion from RAW264.7 cells and splenocytes of C57BL/6, C3H/HeN, and C3H/HeJ mice but not from splenocytes of C57BL/6 TLR2^−/− mice. Lactobacillus LTA induced activation of c-Jun N-terminal kinase activation in RAW264.7 cells. Furthermore, in HEK293T cells transected with a combination of CD14 and Toll-like receptor 2 (TLR2), NF-κB was activated in response to Lactobacillus LTA. Taken together, these data suggest that LTAs from lactobacilli elicit proinflammatory activities through TLR2.     

Hepatic stellate cells primed with cytokines upregulate inflammation in response to peptidoglycan or lipoteichoic acid

Gram-positive bacterial products such as peptidoglycan (PGN) and lipoteichoic acid (LTA) are potent stimulators of innate inflammatory responses. We previously reported that lipopolysaccharide (LPS), a major biologically active agent of gram-negative bacteria, induces a proinflammatory response via the Toll-like receptor (TLR) 4 in hepatic stellate cells (HSCs). Here we investigated the mechanism of proinflammatory action by PGN and LTA in activated human HSCs. Following treatment with either TNF-alpha or IL-1beta, expression of TLR2 and CD14 was determined by real-time PCR and Western blotting. NF-kappaB activation was assessed by NF-kappaB-driven luciferase assay and electrophoretic mobility shift assay. Interleukin-8 (IL-8) from culture supernatant was measured by ELISA. Activated human HSCs express TLR2 and CD14, which are receptors for PGN and LTA signaling. TNF-alpha and IL-1beta significantly upregulated the expression of TLR2 mRNA and protein in HSCs. PGN and LTA induced NF-kappaB activation and stimulated production of IL-8 in HSCs. Pretreatment with TNF-alpha or IL-1beta augmented NF-kappaB activation and IL-8 production in response to PGN or LTA. Both PGN- and LTA-induced NF-kappaB activation and IL-8 secretion were completely inhibited by anti-TLR2 blocking antibody (T2.5). These findings suggest that TNF-alpha or IL-1beta primed HSCs enhance the production of IL-8 in response to PGN and LTA through augmentation of the TLR2 system.     

The xanthine oxidase–NFAT5 pathway regulates macrophage activation and TLR‐induced inflammatory arthritis

NFAT5 (nuclear factor of activated T cells), a well‐known osmoprotective factor, can be activated by isotonic stimuli such as Toll‐like receptor (TLR) triggering. However, it is unclear how NFAT5 discriminates between isotonic and hypertonic stimuli to produce different functional and molecular outcomes. Here, we identified a novel XO–ROS–p38 MAPK–NFAT5 pathway (XO is xanthine oxidase, ROS is reactive oxygen species) that is activated in RAW 264.7 macrophages upon isotonic TLR stimulation. Unlike what is seen under hypertonic conditions, XO‐derived ROS were selectively required for the TLR‐induced NFAT5 activation and NFAT5 binding to the IL‐6 promoter in RAW 264.7 macrophages under isotonic conditions. In mouse peritoneal macrophages and human macrophages, TLR ligation also induced NFAT5 activation, which was dependent on XO and p38 kinase. The involvement of XO in NFAT5 activation by TLR was confirmed in RAW 264.7 macrophages implanted in BALB/c mice. Moreover, allopurinol, an XO inhibitor, suppressed arthritis severity and decreased the expression of NFAT5 and IL‐6 in splenic macrophages in C57BL/6 mice. Collectively, these data support a novel function of the XO–NFAT5 axis in macrophage activation and TLR‐induced arthritis, and suggest that XO inhibitor(s) could serve as a therapeutic agent for chronic inflammatory arthritis.     

Inhibition of lipopolysaccharide-induced I-kappaB degradation and tumor necrosis factor-alpha expression by acriflavine, an antimicrobial agent

Acriflavine neutral (ACF) has been used for treatment of microbial infections for humans and fishes. Effects of ACF on the nuclear factor-kappaB (NF-kappaB) activation and tumor necrosis factor-alpha (TNF-alpha) production by lipopolysaccharide (LPS), an endotoxin, were examined in rat and RAW264.7 cells. Gel retardation analysis revealed that LPS (1 microg/kg) activated NF-kappaB in the liver, whereas pretreatment of rats with ACF (10 mg/kg) completely prevented the NF-kappaB activation. Selectivity of the NF-kappaB DNA binding was confirmed by immunodepletion with anti-p65 and anti-p50 antibodies. Translocation of NF-kappaB to the nucleus is preceded by phosphorylation and proteolytic degradation of inhibitor-kappaBalpha (I-kappaBalpha) subunit. Whereas the level of I-kappaBalpha protein was rapidly decreased after treatment of rats with LPS (1 microg/kg), ACF treatment prior to LPS attenuated the decrease in I-kappaBalpha protein level. LPS-induced increase in the production of TNF-alpha, the principal inflammatory mediator, was prevented by ACF pretreatment by 80%. Stimulation of RAW264.7 cells with 1 microg/ml of LPS caused an increase in DNA binding activity of NF-kappaB, which was 80% inhibited by 1 microg/ml of ACF. LPS reduced I-kappaBalpha level in RAW264.7 cells by 77%. ACF attenuated LPS-induced decrease in I-kappaBalpha protein in a concentration-dependent manner. Production of TNF-alpha by LPS from RAW264.7 cells was decreased by 84% in the presence of ACF. Data showed that ACF inhibited LPS-induced NF-kappaB activation through inhibition of I-kappaBalpha degradation and TNF-alpha production in both rat and RAW264.7 cells. Inhibition of NF-kappaB activation and TNF-alpha production may be associated with the anti-inflammatory activity of ACF.     

Susceptibility to Coccidioides species in C57BL/6 mice is associated with expression of a truncated splice variant of Dectin-1 (Clec7a)

Coccidioides posadasii spherules stimulate macrophages to make cytokines via TLR-2 and Dectin-1. We used formalin-killed spherules and 1,3-beta-glucan purified from spherules to stimulate elicited peritoneal macrophages and myeloid dendritic cells (mDCs) from susceptible (C57BL/6) and resistant (DBA/2) mouse strains. DBA/2 macrophages produced more TNF-alpha and IL-6 than macrophages from C57BL/6 mice, and the amount of TNF-alpha made was dependent on both TLR2 and Dectin-1. DCs from C57BL/6 mice made more IL-10 and less IL-23p19 and IL-12p70 than did DBA/2 DC. These responses were inhibited by a monoclonal antibody to Dectin-1. DBA/2 mice expressed full-length Dectin-1, whereas C57BL/6 mice spliced out exon 3, which encodes most of the stalk. RAW cells transduced to express the full-length Dectin-1 responded better to FKS than cells expressing truncated Dectin-1. We compared the isoform of Dectin-1 expressed by 34 C57BL/6 X DBA/2 recombinant inbred (BXD RI) lines with their susceptibility to Coccidioides immitis. In 25 of 34 RI lines susceptibility or resistance corresponded to short or full-length isoforms, respectively. These results suggest that alternative splicing of the Dectin-1 gene contributes to susceptibility of C57BL/6 mice to coccidioidomycosis, and affects the cytokine responses of macrophages and mDCs to spherules.     

Immunomodulatory effects of dead Lactobacillus on murine splenocytes and macrophages

To elucidate the immunomodulation effects of dead lactobacilli, whole cells and gastrointestinal enzymatic hydrolysates of supernatants and precipitates from Lactobacillus paracasei subsp. paracasei NTU 101 and L. plantarum NTU 102 on RAW264.7 macrophages and splenocytes were investigated. Increased NO, COX-2 expression, IL-10 and IL-12 were observed in high-dose precipitates and whole cells of both strains after 24-h stimulation. All of the hydrolysates and whole cells from both strains induced lower pro-inflammatory cytokines (IL-1β and IL-6) than LPS. The supernatants activated cell division to the S phase or promoted advance to the G2/M phase. Regardless of the Lactobacillus strains, higher levels of TNF-α, IL-6, IL-10 and IL-12 in splenocytes were induced by the precipitates. Supernatant of NTU 101 increased the amounts of IFN-γ than precipitate in splenocytes. It shows that hydrolysates of NTU 101 induce the proliferations of macrophage and splenocyte and the release of IL-10 and IL-12 cytokines to modulate the innate and adaptive immune systems and inflammatory response.     

Probiotic Lactobacillus-induced improvement in murine chronic inflammatory bowel disease is associated with the down-regulation of pro-inflammatory cytokines in lamina propria mononuclear cells

IL-6/STAT-3 signals play key roles in inflammatory bowel disease (IBD). It is known that Lactobacillus casei strain Shirota (LcS) improves inflammatory disorders. This study aimed to elucidate the effect of LcS on murine chronic IBD and to clarify the mechanism. We focused the inhibitory effect of LcS on the production of IL-6 in lipopolysaccharide (LPS)-stimulated large intestinal lamina propria mononuclear cells (LI-LPMC) isolated from mice with chronic colitis and in RAW264.7 cells in vitro. We also determined in vivo the effect of LcS on murine chronic IBD models induced with dextran sodium sulphate and SAMP1/Yit mice. Finally, we examined the cellular determinants of LcS for the down-regulation of IL-6 secretion by LI-LPMC, RAW264.7 cells and peripheral blood mononuclear cells (PBMC) derived from patients with ulcerative colitis (UC). LcS, but not other strains of Lactobacillus, inhibited the production of IL-6 in LPS-stimulated LI-LPMC and RAW264.7 cells, down-regulating the nuclear translocation of NF-kappaB. The LcS-diet-improved murine chronic colitis is associated with the reduction of IL-6 synthesis by LI-LPMC. LcS also improved chronic ileitis in SAMP1/Yit mice. The release of IL-6 in vitro in LPS-stimulated LI-LPMC, RAW 264.7 cells and UC-PBMC was inhibited by a polysaccharide-peptidoglycan complex (PSPG) derived from LcS. This probiotic-induced improvement in murine chronic inflammatory bowel disease is associated with the down-regulation of pro-inflammatory cytokines such as IL-6 and IFN-gamma production in LPMC. Therefore, LcS may be a useful probiotic for the treatment of human inflammatory bowel disease.     

Activation of toll-like receptor-mediated NF-kappa beta by zymosan-derived water-soluble fraction: possible contribution of endotoxin-like substances

Zymosan is a well-known reagent for the examination of inflammatory response and is prepared from yeast, Saccharomyces cerevisiae. In the activation process, Toll-like receptor (TLR) 2 and TLR6 act as functional receptors for NF-kappaB activation. Although zymosan is primarily composed of beta-glucans, little is known about the active component of zymosan-mediated biological activities. The active moiety of zymosan was fractionated by its solubility in water, and its biological activity on macrophages and TLRs-transfectants examined. The macrophage cell line, RAW264.7, was treated with zymosan-derived preparations, and tumor necrosis factor alpha (TNF-alpha) produced in the culture supernatant was measured by ELISA. Increased TNF-alpha production was observed by stimulation with water-soluble (ZWS) or water-insoluble fraction (ZWIS). ZWS showed higher activity in TNF-alpha production. NF-kappaB activation via TLR2, TLR1/ TLR2, TLR2/TLR6, and TLR4/MD-2/CD14 also was enhanced by stimulation with ZWS and ZWIS. In particular, ZWS showed higher activity via TLR1/TLR2, TLR2/TLR6, and TLR4/MD-2/CD14 than other preparations. ZWS activity was decreased by treatment with polymyxin B, but not with lysozyme and zymolyase. Furthermore, ZWS contained significant more endotoxin than any other preparations. Therefore, we suggest that the active moiety of ZWS for the NF-kappaB activation has an endotoxin-like substance, that is abundantly observed in Gram-negative bacteria. These results imply that the inflammatory activity of zymosan is induced not only by beta-glucans, but also by other endotoxin-like water-soluble substances.     

Differential Toll-like receptor recognition and induction of cytokine profile by Bifidobacterium breve and Lactobacillus strains of probiotics

The use of probiotics as a food supplement has gained tremendous interest in the last few years as beneficial effects were reported in gut homeostasis and nutrient absorption but also in immunocompromised patients, supporting protection from colonization or infection with pathogenic bacteria or fungi. As a treatment approach for inflammatory bowel diseases, a suitable probiotic strain would ideally be one with a low immunogenic potential. Insight into the immunogenicities and types of T-cell responses induced by potentially probiotic strains allows a more rational selection of a particular strain. In the present study, the bacterial strains Bifidobacterium breve (NumRes 204), Lactobacillus rhamnosus (NumRes1), and Lactobacillus casei (DN-114 001) were compared concerning their capacity to induce inflammatory responses in terms of cytokine production by human and mouse primary immune cells. It was demonstrated that the B. breve strain induced lower levels of the proinflammatory cytokine gamma interferon (IFN-γ) than the tested L. rhamnosus and L. casei strains. Both B. breve and lactobacilli induced cytokines in a Toll-like receptor 9 (TLR9)-dependent manner, while the lower inflammatory profile of B. breve was due to inhibitory effects of TLR2. No role for TLR4, NOD2, and C-type lectin receptors was apparent. In conclusion, TLR signaling is involved in the differentiation of inflammatory responses between probiotic strains used as food supplements.     

The role of MyD88 and TLR4 in the LPS-mimetic activity of Taxol

Taxol can mimic bacterial lipopolysaccharide (LPS) by activating mouse macrophages in a cell cycle-independent, LPS antagonist-inhibitable manner. Macrophages from C3H/HeJ mice, which have a spontaneous mutation in Toll-like receptor 4 (TLR4), are hyporesponsive to both LPS and Taxol, suggesting that LPS and Taxol may share a signaling pathway involving TLR4. To determine whether TLR4 and its interacting adaptor molecule MyD88 are necessary for Taxol's LPS mimetic actions, we examined Taxol responses of primary macrophages from genetically defective mice lacking either TLR4 (C57BL/10ScNCr) or MyD88 (MyD88 knockout). When stimulated with Taxol, macrophages from wild-type mice responded robustly by secreting both TNF and NO, while macrophages from either TLR4-deficient C57BL/10ScNCr mice or MyD88 knockout mice produced only minimal amounts of TNF and NO. Taxol-induced NF-kappa B-driven luciferase activity was reduced after transfection of RAW 264.7 macrophages with a dominant negative version of mouse MyD88. Taxol-induced microtubule-associated protein kinase (MAPK) activation and NF-kappa B nuclear translocation were absent from TLR4-null macrophages, but were preserved in MyD88 knockout macrophages with a slight delay in kinetics. Neither Taxol-induced NF-kappa B activation, nor I kappa B degradation was affected by the presence of phosphatidylinositol 3-kinase inhibitors. These results suggest that Taxol and LPS not only share a TLR4/MyD88-dependent pathway in generating inflammatory mediators, but also share a TLR4-dependent/MyD88-independent pathway leading to activation of MAPK and NF-kappa B.     

Lipoteichoic acid induces nuclear factor-kappaB activation and nitric oxide synthase expression via phosphatidylinositol 3-kinase, Akt, and p38 MAPK in RAW 264.7 macrophages

We previously demonstrated that lipoteichoic acid (LTA) might activate phosphatidylcholine-phospholipase C (PC-PLC) and phosphatidylinositol-phospholipase C (PI-PLC) to induce protein kinase C activation, which in turn initiates nuclear factor-kappaB (NF-kappaB) activation and finally induces inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) release in RAW 264.7 macrophages. In this study, we further investigated the roles of tyrosine kinase, phosphatidylinositiol 3-kinase (PI3K)/Akt, and p38 mitogen-activated protein kinase (MAPK) in LTA-induced iNOS expression and NO release in RAW 264.7 macrophages. Tyrosine kinase inhibitors (genistein and tyrphostin AG126), PI3K inhibitors (wortmannin and LY 294002), and a p38 MAPK inhibitor (SB 203580) attenuated LTA-induced iNOS expression and NO release in concentration-dependent manners. Treatment of RAW 264.7 macrophages with LTA caused time-dependent activations of Akt and p38 MAPK. The LTA-induced Akt activation was inhibited by wortmannin, LY 294002, genistein, and tyrphostin AG126. The LTA-induced p38 MAPK activation was inhibited by genistein, tyrphostin AG126, wortmannin, LY 294002, and SB 203580. The LTA-induced formation of an NF-kappaB-specific DNA-protein complex in the nucleus was inhibited by wortmannin, LY 294002, genistein, tyrphostin AG126, and SB 203580. Treatment of macrophages with LTA caused an increase in kappaB-luciferase activity, and this effect was inhibited by tyrphostin AG126, wortmannin, LY 294002, the Akt dominant negative mutant (AktDN), and SB 203580. Based on those findings, we suggest that LTA might activate the PI3K/Akt pathway through tyrosine kinase to induce p38 MAPK activation, which in turn initiates NF-kappaB activation, and ultimately induces iNOS expression and NO release in RAW 264.7 macrophages.     

Hyperthermia differentially regulates TLR4 and TLR2-mediated innate immune response

Fever influences multiple parameters of the immune response. However, the mechanisms by which fever manipulates immune response remain undefined. Here we present the evidences that fever range hyperthermia differentially regulates immune response to lipopolysaccharide (LPS) and lipoteichoic acids (LTA) through modulating Toll-like receptor (TLR) signaling. Pretreatment with 39.5 degrees C temperature enhanced LPS, but not LTA, induced NF-kappaB activation and TNF-alpha, IL-6 production in human macrophages. Consistently, expression of TLR4, but not TLR2, was up-regulated by 39.5 degrees C treatment. The increase in LPS-induced cytokine production was inhibited by TLR4-blocking antibody, indicating the enhancement of LPS-induced cytokine production by 39.5 degrees C pretreatment was TLR4-dependent. Pretreatment of mice with 39.5 degrees C temperature also enhanced LPS, but not LTA, induced TNF-alpha and IL-6 production in vivo. These results support the concept that fever range hyperthermia might activate innate immune response by promoting TLR4 expression and signaling, providing a possible mechanistic explanation for the function of fever in regulating innate immune responses.     

Interaction of lipoteichoic acid and CpG-DNA during activation of innate immune cells

The innate immune system recognizes pathogen-associated molecular patterns (PAMP) to cope with evolving infections. Toll-like receptors (TLRs) play a pivotal role in recognition of PAMPs. In the course of infection not a single but rather a full panel of different microbial components interacts with distinct TLRs simultaneously. Only limited information is available on effects of combinations of TLR agonists. Here, we have analyzed the effects of lipoteichoic acid (LTA), CpG-DNA and combinations thereof on innate immune cells in vitro. Although proinflammatory cytokines like TNF-alpha were induced by these agonists in quite similar amounts, CpG DNA was superior in its potency to induce IL-12p40 reflecting important differences in the biological valence of LTA and CpG-DNA. When given in combination, LTA and CpG-DNA were additive in induction of TNF-alpha, IL-6 and nitric oxide in RAW 264 macrophages, peritoneal macrophages and dendritic cells. Additive effects were also observed in regard to TNF-alpha mRNA. In contrast, LTA suppressed IL12p40 secretion induced by CpG-DNA in RAW cells and peritoneal macrophages but not in dendritic cells. Intracellular signal cascades (NFkappaB and p38 MAP kinase) showed additive effects after simultaneous triggering. mRNA expression ofTLRs showed only minor regulation after CpG or LTA application and thus does not account for the additive/suppressive effects observed. These results indicate that the consequences of interaction of innate immune cells with microbial pattern depend on the responding cell type and might be differential for certain effector mechanisms. Thus, the pathogen-characteristic panel of TLR ligands will induce pathogen-specific innate responses decisive for the inflammatory reactions.     

TLR2 and TLR4 agonists synergistically up-regulate SR-A in RAW264.7 through p38

It is known that macrophage scavenger receptor A (SR-A) can protect mice from endotoxemia. In addition, Escherichia coli O111:B4 LPS from Sigma (sLPS), which contains both TLR4 and TLR2 agonists, was previously reported to be able to induce SR-A expression on murine macrophage cell line RAW264.7. However, the relative role of both TLR4 and TLR2 agonists from Sigma (sLPS) in the up-regulation of SR-A on RAW264.7 is still undefined. Here, we found that sLPS could only slightly up-regulate SR-A on RAW264.7 following removing its TLR4 and TLR2 agonists, respectively. In contrast, the combination of TLR4 agonist uLPS (re-extracted sLPS) and TLR2 agonist Pam3CSK4 dramatically induced SR-A expression, and synergistically promoted RAW264.7 to bind and internalize FITC-LPS specifically through SR-A. The combination had no such effect either on TLR2 or TLR4 expression, and incubation with IL-6, IL-10, IL-12 or TNF-alpha alone could not induce SR-A expression on RAW264.7. In addition, treatment with a NF-kappaB inhibitor pyrrolidine dithiocarbamate (PDTC) could only weakly suppress the up-regulation of SR-A by the combination. However, the combination synergistically promoted MAPK p38 phosphorylation, and p38 specific inhibitor SB203580 completely suppressed its inducible effect on SR-A expression. Hence, we demonstrated that up-regulation of SR-A by sLPS was resulted from the cooperation of its TLR4 and TLR2 agonists through p38, and we also presented a novel synergy effect of TLR2 and TLR4 agonists.     

Commercial preparations of lipoteichoic acid contain endotoxin that contributes to activation of mouse macrophages in vitro

Lipoteichoic acids (LTA), cell wall components of gram-positive bacteria, have been reported to induce various inflammatory mediators and to play a key role in gram-positive-microbe-mediated septic shock. In a large number of these studies, investigators used commercially available LTA purified from a variety of gram-positive bacteria, including Staphylococcus aureus, Bacillus subtilis, and Streptococcus sanguis. We report here that, although these commercially available LTA could be readily shown to stimulate production of nitric oxide (NO) in RAW 264.7 mouse macrophages, the activity was dramatically inhibited by polymyxin B, a relatively specific inhibitor of endotoxin biological activity. One-step purification of the commercially available S. aureus LTA using hydrophobic interaction chromatography resulted in two well-separated peak fractions, one highly enriched for LTA and a second highly enriched for endotoxin. The LTA-enriched fractions did not induce production of NO in RAW 264.7 macrophages, although they caused a dose-dependent induction of NO in the presence of low concentrations of gamma interferon (IFN-gamma) (which by itself induced little NO), regardless of the presence of polymyxin B. In contrast, the endotoxin-enriched fractions by themselves inhibited in high levels of NO in RAW 264.7 macrophages but activity was almost completely inhibited in the presence of polymyxin B. Consistent with these findings, our data also indicate that commercial LTA preparations from S. aureus, B. subtilis, and S. sanguis were not able to induce NO from lipopolysaccharide-hyporesponsive C3H/HeJ mouse peritoneal macrophages, but in the presence of IFN-gamma, these LTA preparations were able to induce relatively high levels of NO from C3H/HeJ macrophages. These results indicate that commercially available LTA can contain contaminating and potentially significant levels of endotoxin that can be expected to contribute to the putative macrophage-stimulating effects of LTA as assessed by NO production. The fact that the purified LTA, by itself, was not able to induce significant levels of NO secretion in RAW 264.7 macrophages supports the conclusion that caution in attributing high-level biological activity to this microbial cell wall constituent should be exercised.     

Nitric oxide production by high molecular weight water-soluble chitosan via nuclear factor-kappaB activation

High molecular weight water-soluble chitosan (WSC), having an average molecular weight of 300000 Da and a degree of deacethylation over 90%, can be produced using a simple multi-step membrane separation process. In this study, the effect of WSC on the production of nitric oxide (NO) in RAW 264.7 macrophages was evaluated. Water-insoluble chitosan alone has been previously shown to exhibit in vitro stimulatory effect on macrophages NO production. However, WSC had no effect on NO production by itself. When WSC was used in combination with recombinant interferon-gamma (rIFN-gamma), there was a marked cooperative induction of NO synthesis in a dose-dependent manner. The optimal effect of WSC on NO synthesis was shown 24 h after treatment with rIFN-gamma. The increased production of NO from rIFN-gamma plus WSC-stimulated RAW 264.7 macrophages was decreased by the treatment with N(G)-monomethyl-L-arginine (N(G)MMA). The increase in NO synthesis was reflected, as an increased amounts of inducible NO synthase protein. In addition, synergy between rIFN-gamma and WSC was mainly dependent on WSC-induced tumor necrosis factor-alpha (TNF-alpha) and nuclear factor-kappaB (NF-kappaB) activation. The present results indicate that the capacity of WSC to increase NO production from rIFN-gamma-primed RAW 264.7 macrophages is the result of WSC-induced TNF-alpha secretion via the signal transduction pathway of NF-kappaB activation.