Suppressed production of pro-inflammatory cytokines by LPS-activated macrophages after treatment with Toxoplasma gondii lysate

During Toxoplasma gondii infection, macrophages, dendritic cells, and neutrophils are important sources of pro-inflammatory cytokines from the host. To counteract the pro-inflammatory activities, T. gondii is known to have several mechanisms inducing down-regulation of the host immunity. In the present study, we analyzed the production of proand anti-inflammatory cytokines from a human myelomonocytic cell line, THP-1 cells, in response to treatment with T. gondii lysate or lipopolysaccharide (LPS). Treatment of THP-1 cells with LPS induced production of IL-12, TNF-alpha, IL-8, and IL-10. Co-treatment of THP-1 cells with T. gondii lysate inhibited the LPS-induced IL-12, IL-8 and TNF-alpha expression, but increased the level of IL-10 synergistically. IL-12 and IL-10 production was down-regulated by anti-human toll-like receptor (TLR)-2 and TLR4 antibodies. T. gondii lysate triggered nuclear factor (NF)-kappaB-dependent IL-8 expression in HEK293 cells transfected with TLR2. It is suggested that immunosuppression induced by T. gondii lysate treatment might occur via TLR2-mediated NF-kappaB activation.     

Bacterial lipopolysaccharide activates nuclear factor-kappaB through interleukin-1 signaling mediators in cultured human dermal endothelial cells and mononuclear phagocytes

Bacterial lipopolysaccharide (LPS)-mediated immune responses, including activation of monocytes, macrophages, and endothelial cells, play an important role in the pathogenesis of Gram-negative bacteria-induced sepsis syndrome. Activation of NF-kappaB is thought to be required for cytokine release from LPS-responsive cells, a critical step for endotoxic effects. Here we investigated the role and involvement of interleukin-1 (IL-1) and tumor necrosis factor (TNF-alpha) signal transducer molecules in LPS signaling in human dermal microvessel endothelial cells (HDMEC) and THP-1 monocytic cells. LPS stimulation of HDMEC and THP-1 cells initiated an IL-1 receptor-like NF-kappaB signaling cascade. In transient cotransfection experiments, dominant negative mutants of the IL-1 signaling pathway, including MyD88, IRAK, IRAK2, and TRAF6 inhibited both IL-1- and LPS-induced NF-kappaB-luciferase activity. LPS-induced NF-kappaB activation was not inhibited by a dominant negative mutant of TRAF2 that is involved in TNF signaling. LPS-induced activation of NF-kappaB-responsive reporter gene was not inhibited by IL-1 receptor antagonist. TLR2 and TLR4 were expressed on the cell surface of HDMEC and THP-1 cells. These findings suggest that a signal transduction molecule in the LPS receptor complex may belong to the IL-1 receptor/toll-like receptor (TLR) super family, and the LPS signaling cascade uses an analogous molecular framework for signaling as IL-1 in mononuclear phagocytes and endothelial cells.     

Novel and detrimental effects of lipopolysaccharide on in vitro generation of immature dendritic cells: involvement of mitogen-activated protein kinase p38

Dendritic cells (DCs) are recognized as major players in the regulation of immune responses to a variety of Ags, including bacterial agents. LPS, a Gram-negative bacterial cell wall component, has been shown to fully activate DCs both in vitro and in vivo. LPS-induced DC maturation involves activation of p38, extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinases, and NF-kappaB. Blocking p38 inhibits LPS-induced maturation of DCs. In this study we investigated the role of LPS in the in vitro generation of immature DCs. We report here that in contrast to the observed beneficial effects on DCs, the presence of LPS in monocyte culture retarded the generation of immature DCs. LPS not only impaired the morphology and reduced the yields of the cultured cells, but also inhibited the up-regulation of surface expression of CD1a, costimulatory and adhesion molecules. Furthermore, LPS up-regulated the secretion of IL-1beta, IL-6, IL-8, IL-10, and TNF-alpha; reduced Ag presentation capacity; and inhibited phosphorylation of ERK, but activated p38, leading to a reduced NF-kappaB activity in treated cells. Neutralizing Ab against IL-10, but not other cytokines, partially blocked the effects of LPS. Inhibiting p38 (by inhibitor SB203580) restored the morphology, phenotype, and Ag presentation capacity of LPS-treated cells. SB203580 also inhibited LPS-induced production of IL-1beta, IL-10, and TNF-alpha; enhanced IL-12 production; and recovered the activity of ERK and NF-kappaB. Thus, our study reveals that LPS has dual effects on DCs that are biologically important: activating existing DCs to initiate an immune response, and inhibiting the generation of new DCs to limit such a response.     

Activation of AMPK attenuates neutrophil proinflammatory activity and decreases the severity of acute lung injury

AMP-activated protein kinase (AMPK) is activated by increases in the intracellular AMP-to-ATP ratio and plays a central role in cellular responses to metabolic stress. Although activation of AMPK has been shown to have anti-inflammatory effects, there is little information concerning the role that AMPK may play in modulating neutrophil function and neutrophil-dependent inflammatory events, such as acute lung injury. To examine these issues, we determined the effects of pharmacological activators of AMPK, 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) and barberine, on Toll-like receptor 4 (TLR4)-induced neutrophil activation. AICAR and barberine dose-dependently activated AMPK in murine bone marrow neutrophils. Exposure of LPS-stimulated neutrophils to AICAR or barberine inhibited release of TNF-alpha and IL-6, as well as degradation of IkappaBalpha and nuclear translocation of NF-kappaB, compared with findings in neutrophil cultures that contained LPS without AICAR or barberine. Administration of AICAR to mice resulted in activation of AMPK in the lungs and was associated with decreased severity of LPS-induced lung injury, as determined by diminished neutrophil accumulation in the lungs, reduced interstitial pulmonary edema, and diminished levels of TNF-alpha and IL-6 in bronchoalveolar lavage fluid. These results suggest that AMPK activation reduces TLR4-induced neutrophil activation and diminishes the severity of neutrophil-driven proinflammatory processes, including acute lung injury.     

A heteroglycan from the cyanobacterium Nostoc commune modulates LPS-induced inflammatory cytokine secretion by THP-1 monocytes through phosphorylation of ERK1/2 and Akt

Cyanobacteria (blue-green algae) have been consumed as food and used in folk medicine since ancient times to alleviate a variety of diseases. Cyanobacteria of the genus Nostoc have been shown to produce complex exopolysaccharides with antioxidant and antiviral activity. Furthermore, Nostoc sp. are common in cyanolichen symbiosis and lichen polysaccharides are known to have immunomodulating effects. Nc-5-s is a heteroglycan isolated from free-living colonies of Nostoc commune and its structure has been characterized in detail. The aim of this study was to determine the effects of Nc-5-s on the inflammatory response of lipopolysaccharide (LPS)-stimulated human THP-1 monocytes and how the effects are mediated. THP-1 monocytes primed with interferon-γ and stimulated with LPS in the presence of Nc-5-s secreted less of the pro-inflammatory cytokine interleukin (IL)-6 and more of the anti-inflammatory cytokine IL-10 than THP-1 monocytes stimulated without Nc-5-s. In contrast, Nc-5-s increased LPS-induced secretion of the pro-inflammatory cytokines tumor necrosis factor (TNF)-α and IL-8. Nc-5-s decreased LPS-induced phosphorylation of the extracellular regulated kinase (ERK)1/2 and Akt kinase, but did not affect phosphorylation of the p38 kinase, activation of the nuclear factor kappa B pathway, nor DNA binding of c-fos. These results show that Nc-5-s has anti-inflammatory effects on IL-6 and IL-10 secretion by THP-1 monocytes, but its effects are pro-inflammatory when it comes to TNF-α and IL-8. Furthermore, they show that the effects of Nc-5-s may be mediated through the ERK1/2 pathway and/or the Akt/phosphoinositide 3-kinase pathway and their downstream effectors. The ability of Nc-5-s to decrease IL-6 secretion, increase IL-10 secretion and moderate ERK1/2 activation indicates a potential for its development as an anti-inflammatory agent.     

Saccharomyces boulardii produces a soluble anti-inflammatory factor that inhibits NF-kappaB-mediated IL-8 gene expression

Saccharomyces boulardii (Sb) is a non-pathogenic yeast that ameliorates intestinal injury and inflammation caused by a wide variety of enteric pathogens. We hypothesized that Sb may exert its probiotic effects by modulation of host cell signaling and pro-inflammatory gene expression. Human HT-29 colonocytes and THP-1 monocytes were stimulated with IL-1beta, TNFalpha or LPS in the presence or absence of Sb culture supernatant (SbS). IL-8 protein and mRNA levels were measured by ELISA and RT-PCR, respectively. The effect of SbS on IkappaB alpha degradation was studied by Western blotting and on NF-kappaB-DNA binding by EMSA. NF-kappaB-regulated gene expression was evaluated by transient transfection of THP-1 cells with a NF-kappaB-responsive luciferase reporter gene. SbS inhibited IL-8 protein production in IL-1beta or TNFalpha stimulated HT-29 cells (by 75% and 85%, respectively; P<0.001) and prevented IL-1beta-induced up-regulation of IL-8 mRNA. SbS also inhibited IL-8 production, prevented IkappaB alpha degradation, and reduced both NF-kappaB-DNA binding and NF-kappaB reporter gene up-regulation in IL-1beta or LPS-stimulated THP-1 cells. Purification and characterization studies indicate that the S. boulardii anti-inflammatory factor (SAIF) is small (<1 kDa), heat stable, and water soluble. The probiotic yeast Saccharomyces boulardii exerts an anti-inflammatory effect by producing a low molecular weight soluble factor that blocks NF-kappaB activation and NF-kappaB-mediated IL-8 gene expression in intestinal epithelial cells and monocytes. SAIF may mediate, at least in part, the beneficial effects of Saccharomyces boulardii in infectious and non-infectious human intestinal disease.     

Implications of proteasome inhibition: an enhanced macrophage phenotype

The objective of this study was to elucidate the role of the cellular proteasome on endotoxin-mediated activation of the macrophage. To study this role, THP-1 cells were stimulated with lipopolysaccharide (LPS) with selective cells being pretreated with the proteasome inhibitor, lactacystin or MG-132. LPS stimulation led to the phosphorylation and degradation of IRAK, followed by activation of JNK/SAPK, ERK 1/2, and p38. Subsequently, LPS induced the degradation of IkappaB, and the nuclear activation of NF-kappaB and AP-1. Activation of these pathways was associated with the production of IL-6, IL-8, IL-10, and TNF-alpha. Proteasome inhibition with either lactacystin or MG-132 attenuated LPS-induced IRAK degradation, and enhanced activation of JNK/SAPK, ERK 1/2, and p38. Proteasome inhibition, also, led to increased LPS-induced AP-1 activation, and attenuated LPS-induced IkappaB degradation resulting in abolished NF-kappaB activation. Proteasome inhibition led to significant modulation of LPS-induced cytokine production; increased IL-10, no change in IL-6, and decreased IL-8, and TNF-alpha. Thus, this study demonstrates that cellular proteasome is critical to regulation of LPS-induced signaling within the macrophage, and inhibition of the proteasome results in a conversion to an anti-inflammatory phenotype.     

15-deoxy-Delta12,14-prostaglandin J2 inhibits IFN-inducible protein 10/CXC chemokine ligand 10 expression in human microglia: mechanisms and implications

Regulation of cytokine and chemokine expression in microglia may have implications for CNS inflammatory disorders. In this study we examined the role of the cyclopentenone PG 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) in microglial inflammatory activation in primary cultures of human fetal microglia. 15d-PGJ(2) potently inhibited the expression of microglial cytokines (IL-1, TNF-alpha, and IL-6). We found that 15d-PGJ(2) had differential effects on the expression of two alpha-chemokines; whereas the Glu-Lys-Arg (ELR)(-) chemokine IFN-inducible protein-10/CXCL10 was inhibited, the ELR(+) chemokine IL-8/CXCL8 was not inhibited. These findings were shown in primary human microglia and the human monocytic cells line THP-1 cells, using diverse cell stimuli such as bacterial endotoxin, proinflammatory cytokines (IL-1 and TNF-alpha), IFN-beta, and HIV-1. Furthermore, IL-8/CXCL8 expression was induced by 15d-PGJ(2) alone or in combination with TNF-alpha or HIV-1. Combined results from EMSA, Western blot analysis, and immunocytochemistry showed that 15d-PGJ(2) inhibited NF-kappaB, Stat1, and p38 MAPK activation in microglia. Adenoviral transduction of super-repressor IkappaBalpha, dominant negative MKK6, and dominant negative Ras demonstrated that NF-kappaB and p38 MAPK were involved in LPS-induced IFN-inducible protein 10/CXCL10 production. Interestingly, although LPS-induced IL-8/CXCL8 was dependent on NF-kappaB, the baseline or 15d-PGJ(2)-mediated IL-8/CXCL8 production was NF-kappaB independent. Our results demonstrate that 15d-PGJ(2) has opposing effects on the expression of two alpha-chemokines. These data may have implications for CNS inflammatory diseases.     

Magnesium isoglycyrrhizinate suppresses LPS-induced inflammation and oxidative stress through inhibiting NF-κB and MAPK pathways in RAW264.7 cells

Magnesium Isoglycyrrhizinate (MgIG), a novel molecular compound extracted from licorice root, has exhibited greater anti-inflammatory activity and hepatic protection than glycyrrhizin and β-glycyrrhizic acid. In this study, we investigated the anti-inflammatory effect and the potential mechanism of MgIG on Lipopolysaccharide (LPS)-treated RAW264.7 cells. MgIG down-regulated LPS-induced pro-inflammatory mediators and enzymes in LPS-treated RAW264.7 cells, including TNF-α, IL-6, IL-1β, IL-8, NO and iNOS. The generation of reactive oxygen species (ROS) in LPS-treated RAW264.7 cells was also reduced. MgIG attenuated NF-κB translocation by inhibiting IKK phosphorylation and IκB-α degradation. Simultaneously, MgIG also inhibited LPS-induced activation of MAPKs, including p38, JNK and ERK1/2. Taken together, these results suggest that MgIG suppresses inflammation by blocking NF-κB and MAPK signaling pathways, and down-regulates ROS generation and inflammatory mediators.     

Distinct role of p38 and c-Jun N-terminal kinases in IL-10-dependent and IL-10-independent regulation of the costimulatory molecule B7.2 in lipopolysaccharide-stimulated human monocytic cells

The costimulatory molecule B7.2 (CD86) plays a vital role in immune activation and development of Th responses. The molecular mechanisms by which B7.2 expression is regulated are not understood. We investigated the role of mitogen-activated protein kinases (MAPK) in the regulation of B7.2 expression in LPS-stimulated human monocytic cells. LPS stimulation of human monocytes resulted in the down-regulation of B7.2 expression that could be abrogated by anti-IL-10 Abs. Furthermore, SB202190, a specific inhibitor of p38 MAPK, inhibited LPS-induced IL-10 production and reversed B7.2 down-regulation, suggesting that LPS-induced B7.2 down-regulation may be mediated, at least in part, via regulation of IL-10 production by p38 MAPK. In contrast to human promonocytic THP-1 cells that are refractory to the inhibitory effects of IL-10, LPS stimulation enhanced B7.2 expression. This IL-10-independent B7.2 induction was not influenced by specific inhibitors of either p38 or p42/44 MAPK. To ascertain the role of the c-Jun N-terminal kinase (JNK) MAPK, dexamethasone, an inhibitor of JNK activation, was used, which inhibited LPS-induced B7.2 expression. Transfection of THP-1 cells with a plasmid expressing a dominant-negative stress-activated protein/extracellular signal-regulated kinase kinase 1 significantly reduced LPS-induced B7.2 expression, thus confirming the involvement of JNK. To study the signaling events downstream of JNK activation, we show that dexamethasone did not inhibit LPS-induced NF-kappaB activation in THP-1 cells, suggesting that JNK may not be involved in NF-kappaB activation leading to B7.2 expression. Taken together, our results reveal the distinct involvement of p38 in IL-10-dependent, and JNK in IL-10-independent regulation of B7.2 expression in LPS-stimulated monocytic cells.     

Autocrine Regulation of Macrophage Activation via Exocytosis of ATP and Activation of P2Y11 Receptor

It is important to understand the mechanisms that regulate macrophage activation to establish novel therapies for inflammatory diseases, such as sepsis; a systemic inflammatory response syndrome generally caused by bacterial lipopolysaccharide (LPS). In this study, we investigated the involvement of extracellular ATP-mediated autocrine signaling in LPS-induced macrophage activation. We show here that ATP release via exocytosis, followed by activation of P2Y11 receptor, is a major pathway of the macrophage activation, leading to release of cytokines. Treatment of human monocyte THP-1 cells with LPS induced rapid ATP release from cells, and this release was blocked by knockdown of SLC17A9 (vesicular nucleotide transporter, VNUT), which is responsible for exocytosis of ATP. ATP-enriched vesicles were found in cytosol of THP-1 cells. These data suggest the involvement of vesicular exocytosis in the release of ATP. Knockdown of SLC17A9, the P2Y11 antagonist NF157 or knockdown of P2Y11 receptor significantly suppressed both M1-type polarization and IL-6 production in THP-1 cells, indicating an important role of activation of P2Y11 receptor by released ATP in macrophage activation. Next, the effect of NF157 on LPS-induced immune activation was examined in vivo. Administration of LPS to mice caused increase of serum IL-1ß, IL-6, IL-12 and TNF-alpha levels at 3–24 h after the administration. Pre-treatment of LPS-treated mice with NF157 suppressed both elevation of proinflammatory cytokines in serum and M1 polarization of peritoneal/spleen macrophages. Moreover, post-treatment with NF157 at 30 min after administration of LPS also suppressed the elevation of serum cytokines levels. We conclude that vesicular exocytosis of ATP and autocrine, positive feedback through P2Y11 receptors is required for the effective activation of macrophages. Consequently, P2Y11 receptor antagonists may be drug candidates for treatment of inflammatory diseases such as sepsis.     

The tyrosine kinase Pyk2 mediates lipopolysaccharide-induced IL-8 expression in human endothelial cells

Secretion of proinflammatory cytokines by LPS activated endothelial cells contributes substantially to the pathogenesis of sepsis. However, the mechanism involved in this process is not well understood. In the present study, we determined the role of a nonreceptor proline-rich tyrosine kinase, Pyk2, in LPS-induced IL-8 (CXCL8) production in endothelial cells. First, we observed a marked activation of Pyk2 in response to LPS. Furthermore, inhibition of Pyk2 activity in these cells by transduction with the catalytically inactive Pyk2 mutant, transfection with Pyk2-specific small interfering RNA, or treatment with Tyrphostin A9 significantly blocked LPS-induced IL-8 production. The supernatants of LPS-stimulated cells exhibiting attenuated Pyk2 activity blocked transendothelial neutrophil migration in comparison to the supernatants of LPS-treated controls, thus confirming the inhibition of functional IL-8 production. Investigations into the molecular mechanism of this pathway revealed that LPS activates Pyk2 leading to IL-8 production through the TLR4. In addition, we identified the p38 MAPK pathway to be a critical step downstream of Pyk2 during LPS-induced IL-8 production. Taken together, these results demonstrate a novel role for Pyk2 in LPS-induced IL-8 production in endothelial cells.     

Interaction of lipopolysaccharide with human small intestinal lamina propria fibroblasts favors neutrophil migration and peripheral blood mononuclear cell adhesion by the production of proinflammatory mediators and adhesion molecules

Fibroblasts are important effector cells having a potential role in augmenting the inflammatory responses in various diseases. In infantile diarrhea caused by enteropathogenic Escherichia coli (EPEC), the mechanism of inflammatory reactions at the mucosal site remains unknown. Although the potential involvement of fibroblasts in the pathogenesis of cryptococcus-induced diarrhea in pigs has been suggested, the precise role of lamina propria fibroblasts in the cellular pathogenesis of intestinal infection and inflammation caused by EPEC requires elucidation. Earlier we reported the lipopolysaccharide (LPS)-induced cell proliferation, and collagen synthesis and downregulation of nitric oxide in lamina propria fibroblasts. In this report, we present the profile of cytokines and adhesion molecules in the cultured and characterized human small intestinal lamina propria fibroblasts in relation to neutrophil migration and adhesion in response to lipopolysaccharide (LPS) extracted from EPEC 055:B5. Upon interaction with LPS (1-10 micrograms/ml), lamina propria fibroblasts produced a high level of proinflammatory mediators, interleukin (IL)-1alpha, IL-1beta, IL-6, IL-8, tumor necrosis factor (TNF)-alpha and cell adhesion molecules (CAM) such as intercellular cell adhesion molecule (ICAM), A-CAM, N-CAM and vitronectin in a time-dependent manner. LPS induced cell-associated IL-1alpha and IL-1beta, and IL-6, IL-8 and TNF-alpha as soluble form in the supernatant. Apart from ICAM, vitronectin, A-CAM, and N-CAM proteins were strongly induced in lamina propria fibroblasts by LPS. Adhesion of PBMC to LPS-treated lamina propria fibroblasts was ICAM-dependent. LPS-induced ICAM expression in lamina propria fibroblasts was modulated by whole blood, PBMC and neutrophils. Conditioned medium of LPS-treated lamina propria fibroblasts remarkably enhanced the neutrophil migration. The migration of neutrophils was inhibited by anti-IL-8 antibody. Co-culture of fibroblasts with neutrophils using polycarbonate membrane filters exhibited time-dependent migration of neutrophils. These findings indicate that the coordinate production of proinflammatory cytokines and adhesion molecules in lamina propria fibroblasts which do not classically belong to the immune system can influence the local inflammatory reactions at the intestinal mucosal site during bacterial infections and can influence the immune cell population residing in the lamina propria.     

Peripheral blood neutrophil activation patterns are associated with pulmonary inflammatory responses to lipopolysaccharide in humans

Increased nuclear accumulation of NF-kappaB in LPS-stimulated peripheral blood neutrophils has been shown to be associated with more severe clinical course in patients with infection associated acute lung injury. Such observations suggest that differences in neutrophil response may contribute to the pulmonary inflammation induced by bacterial infection. To examine this question, we sequentially measured LPS-induced DNA binding of NF-kappaB in neutrophils collected from healthy humans on at least three occasions, each separated by at least 2 wk, and then determined pulmonary inflammatory responses after instillation of LPS into the lungs. Consistent patterns of peripheral blood neutrophil responses, as determined by LPS-induced NF-kappaB DNA binding, were present in volunteers, with a >80-fold difference between individuals in the mean area under the curve for NF-kappaB activation. The number of neutrophils recovered from bronchoalveolar lavage after exposure to pulmonary LPS was significantly correlated with NF-kappaB activation in peripheral blood neutrophils obtained over the pre-LPS exposure period (r = 0.65, p = 0.009). DNA binding of NF-kappaB in pulmonary neutrophils also was associated with the mean NF-kappaB area under the curve for LPS-stimulated peripheral blood neutrophils (r = 0.63, p = 0.01). Bronchoalveolar lavage levels of IL-6 and TNFRII were significantly correlated with peripheral blood neutrophil activation patterns (r = 0.75, p = 0.001 for IL-6; and r = 0.48, p = 0.049 for TNFRII. These results demonstrate that stable patterns in the response of peripheral blood neutrophils to LPS exist in the human population and correlate with inflammatory response following direct exposure to LPS in the lung.     

Role of phosphatidylinositol-3 kinase in transcriptional regulation of TLR-induced IL-12 and IL-10 by Fc gamma receptor ligation in murine macrophages

Ligation of FcgammaR concurrent with LPS stimulation of murine macrophages results in decreased IL-12 and increased IL-10 production. Because PI3K deficiency has been associated with increased IL-12, we hypothesized that PI3K was central to the anti-inflammatory effect of FcgammaR ligation on TLR-induced IL-12. FcgammaR ligation of macrophages increased pAKT, a correlate of PI3K activity, above levels induced by TLR4 or TLR2 agonists. This increase was blocked by PI3K inhibitors, wortmannin or LY294002, as was the effect of FcgammaR ligation on TLR-induced IL-12 and IL-10. LPS-induced binding of NF-kappaB to the IL-12 p40 promoter NF-kappaB-binding site was not affected by FcgammaR ligation at 1 h; however, by 4 h, NF-kappaB binding was markedly inhibited, confirmed in situ by chromatin immunoprecipitation analysis. This effect was wortmannin sensitive. Although TLR-induced IkappaBalpha degradation was not affected by FcgammaR ligation, IkappaBalpha accumulated in the nuclei of cells treated with LPS and FcgammaR ligation for 4 h, and was blocked by PI3K inhibitors. LPS-induced IFN regulatory factor-8/IFN consensus sequence-binding protein mRNA, and an IFN regulatory factor-8-dependent gene, Nos2, were inhibited by concurrent FcgammaR ligation, and this was also reversed by wortmannin. Thus, FcgammaR ligation modulates LPS-induced IL-12 via multiple PI3K-sensitive pathways that affect production, accumulation, and binding of key DNA-binding proteins required for IL-12 induction.