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The Journal of Immunology, 2007, 179, 236 -246
Copyright © 2007 by The American Association of Immunologists, Inc.
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Role of Phosphatidylinositol-3 Kinase in Transcriptional Regulation of TLR-Induced IL-12 and IL-10 by Fc{gamma} Receptor Ligation in Murine Macrophages1

Swamy Kumar Polumuri2, Vladimir Y. Toshchakov2 and Stefanie N. Vogel3

Department of Microbiology and Immunology, School of Medicine, University of Maryland, Baltimore, MD 21201


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Ligation of Fc{gamma}R 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 Fc{gamma}R ligation on TLR-induced IL-12. Fc{gamma}R 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 Fc{gamma}R ligation on TLR-induced IL-12 and IL-10. LPS-induced binding of NF-{kappa}B to the IL-12 p40 promoter NF-{kappa}B-binding site was not affected by Fc{gamma}R ligation at 1 h; however, by 4 h, NF-{kappa}B binding was markedly inhibited, confirmed in situ by chromatin immunoprecipitation analysis. This effect was wortmannin sensitive. Although TLR-induced I{kappa}B{alpha} degradation was not affected by Fc{gamma}R ligation, I{kappa}B{alpha} accumulated in the nuclei of cells treated with LPS and Fc{gamma}R 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 Fc{gamma}R ligation, and this was also reversed by wortmannin. Thus, Fc{gamma}R 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Stimulation of cells with the prototype TLR4 agonist, LPS, triggers production of various cytokines including IL-12, which plays a crucial role in the Th1 response (1). This resultant inflammatory response is important for the elimination of many infectious microorganisms. However, excessive and prolonged activation of innate immunity is detrimental to the host and, in some cases, even lethal, owing to severe tissue damage and circulatory failure, which leads to septic shock. To avoid such adverse outcomes, numerous anti-inflammatory, counterregulatory strategies are invoked by the host: limited activation of necessary DNA-binding proteins, e.g., NF-{kappa}B and AP-1 (2, 3), up-regulation of negative regulators (e.g., IL-1R-associated kinase M (4) and suppressor of cytokine signaling-1 (5)), and the production of anti-inflammatory cytokines (e.g., IL-10 (6, 7)) have all been shown to regulate TLR4-mediated signaling negatively. Additionally, membrane-bound proteins harboring the "Toll-IL-1 resistance" domain, such as single Ig IL-1R-related molecule and ST2, have also been found to be involved in negative regulation of TLR signaling (8, 9).

Transgenic mice that express constitutively active AKT (also referred to as protein kinase B (PKB)4), a downstream target of PI3K, were protected from a lethal dose of LPS when compared with wild-type littermates (10). These data suggest that the PI3K-AKT pathway may be involved as a mechanism for protecting against an otherwise lethal LPS challenge. PI3K is a heterodimeric enzyme that consists of regulatory (p85) and catalytic (p110) subunits. Once PI3K is activated, its catalytic activity results in local production of phosphatidylinositol-3, 4,5-triphosphate (PIP3). PIP3 can act as second messenger that recruits and activates intracellular signaling molecules such as the Ser/Thr protein kinase AKT/PKB (11, 12). Evidence for the involvement of PI3K in TLR signaling was provided by Arbibe et al. (13) who demonstrated that mutations within multiple p85 docking sites of TLR2 resulted in a loss of the ability of p85 to associate with TLR2 and loss of NF-{kappa}B transcriptional activity. Martin et al. (14) demonstrated that Porphyromonas gingivalis LPS, a TLR2 agonist, activates the PI3K-AKT pathway. Blocking this pathway with the PI3K inhibitors resulted in decreased IL-10 and increased IL-12 production (14). In monocytes, blocking the PI3K-AKT pathway with PI3K inhibitors further enhanced LPS-induced TNF-{alpha} and tissue factor production, as well as activation of MAPKs, AP-1, Egr-1, NF-{kappa}B, and decreased the phosphorylation of GSK3{alpha}/GSK3beta (15). Interestingly, PI3K-deficient mice exhibit defective Th2-associated immunity, i.e., IL-12 levels and Th1 responses are strongly elevated (16, 17). Thus, PI3K activity may be central to the development of cell-mediated immunity by affecting IL-12 synthesis directly or indirectly by exerting effects on counterregulatory circuits.

Earlier reports suggested that activation of bone marrow-derived macrophages by LPS in the presence of IgG-opsonized erythrocytes or soluble immune complexes increased production of IL-10 and decreased production of IL-12 (18, 19, 20). Similarly, several other receptors on the macrophage surface behave like Fc{gamma}R when engaged during TLR-mediated signaling, resulting in down-regulation of LPS-induced IL-12 production. These include the PGD2 receptor (21), A2A receptors (22), calcitonin gene-related peptide receptor (23), H2 receptor (24), cannabinoid CB2 receptor (25), G protein-coupled receptors (26), complement receptors (27), scavenger receptors (18), CD47 receptor (28), beta2 adrenergic receptors (29), and others. Likewise, IL-10 is a potent inhibitor of IL-12 production in LPS-stimulated cells (6, 7) and exposure of macrophages to vitamin D3 (30) or to macrophage stimulatory protein (31) can also inhibit IL-12 production. Finally, infection of macrophages or dendritic cells with measles virus (32), herpes virus (33), Toxoplasma gondii (34) and Leishmania major (35) can result in decreased LPS-induced IL-12 secretion. The molecular mechanism(s) for this inhibition appear to be quite complex, yet inhibition appears to occur at the level of IL-12 transcription, resulting in decreased steady-state mRNA production and protein synthesis.

In the present studies, we hypothesized that down-regulation of IL-12 in response to treatment with LPS and immune complexes is mediated through the PI3K-AKT signaling pathway. Our data indicate that Fc{gamma}R ligation modulates the PI3K/AKT pathway induced by TLR engagement and affects IL-12 production at multiple sites along the signaling pathway. Accordingly, increased PI3K activation resulted in down-regulation of TLR4- and TLR2-induced IL-12 production and reciprocal up-regulation of IL-10. Treatment of macrophages with PI3K inhibitors, LY294002 or wortmannin, reversed the effect immune complexes had on TLR4- and TLR2-induced IL-12 and IL-10. The presence of opsonized erythrocytes resulted in the down-regulation of LPS-induced binding of NF-{kappa}B at a late time point (4 h). This correlated with increased nuclear translocation of I{kappa}B{alpha} and inhibition of Rel family members binding to an NF-{kappa}B site found in the IL-12 p40 promoter, the latter being confirmed in chromatin immunoprecipitation (ChIP) assays. Both effects were reversed by PI3K inhibitors. Induction of steady-state mRNA levels of LPS-induced transcription factors, including Rel family members and IFN regulatory factor-8 (IRF-8)/IFN consensus sequence-binding protein (ICSBP), as well as IRF-8-inducible gene expression, was also down-regulated upon Fc{gamma}R ligation, and may serve as an additional mechanism that suppresses tissue damage due to overproduction of IL-12. Collectively, these data support the hypothesis that Fc{gamma}R 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.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents

Protein-free Escherichia coli K235 LPS (<0.008% protein) were prepared as described previously (36). The synthetic lipoprotein S-[2,3-Bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-Cys-(S)-Ser-Lys4-OH, trihydrochloride (P3C) was purchased from EMC Microcollections. PI3K inhibitors, wortmannin and LY294002, were obtained from Cell Signaling and Sigma-Aldrich, respectively. Ab for pAKT (Ser473), pGSK3{alpha}/pGSK3beta, pp38, pERK1/2, pJNK1/2, and pSTAT1 were from Cell Signaling, and anti-I{kappa}B{alpha} and anti-beta-actin from Santa Cruz Biotechnology. Rabbit polyclonal antisera directed to mouse p50, p65, c-Rel, p-52, and RelB were a gift from Dr. N. Rice (Frederick Cancer Research and Development Center, National Cancer Institute, Frederick, MD).

Cell culture

Primary murine macrophages were obtained by peritoneal lavage from 6- to 8-wk-old C57BL/6J mice (The Jackson Laboratory) 4 days after i.p. injection with sterile thioglycolate as described previously (37). Macrophages were cultured in RPMI 1640 supplemented with 2% FCS, 2 mM glutamine, penicillin, and streptomycin as described previously (37).

Opsonized sheep erythrocytes

IgG-opsonized erythrocytes were prepared by incubating sheep RBC (Lampire Biological Laboratories) with anti-sheep RBC IgG (Cordis Laboratories) at nonagglutinating titers for 60 min in a 37°C water bath with mixing once every 20 min. Opsonized erythrocytes were washed twice with saline and resuspended in complete medium. Erythrocytes were added to macrophages at a ratio of 10:1 as described previously (18).

Cell stimulation

Macrophages were cultured at 4 x 106 cells/well in 6-well plates. After overnight incubation, cells were pretreated with medium only, or medium containing 2 µM LY294002 or 500 nM wortmannin for 60 min before stimulation with medium only, LPS (100 ng/ml), or P3C (1 µg/ml), without or with Fc{gamma}R (opsonized erythrocytes) ligation for the times indicated. Macrophages were washed with PBS and then lysed in buffer (1% Triton X-100, 5 mM EDTA, 50 mM NaCl, 50 mM NaF, 10 mM Tris with protease inhibitor mixture (Roche) and 1 mM sodium vanadate), and boiled for 5 min with Laemmli lysis buffer for SDS-PAGE and subsequent Western blot analysis.

Measurement of steady-state mRNA by quantitative real-time PCR

Total RNA was isolated using RNA Stat60 isolation reagent from Tel-Test "B," as specified by the manufacturer’s instructions and quantified by spectrophotometric analysis. cDNA was prepared from 1 µg of total RNA using AMV Reverse Transcriptase (Promega) and poly-oligo (dT) priming. The resulting cDNA was quantified by real-time PCR using SYBR Green PCR Master Mix (Applied Biosystems) and ABI Prism 7900HT cycler as described (38, 39). Primers for detection of IL-12 p40, IL-12 p35, IL-10, Rel family members, ICSBP/IRF-8, and GAPDH mRNAs were designed using the Primer Express 2.0 program (Applied Biosystems).

Western blot analysis

Twenty micrograms of total protein in Laemmli buffer was boiled for 5 min, resolved by 10% SDS-PAGE in Tris/glycine/SDS buffer (25 mM Tris, 250 mM glycine, 0.1% SDS) from Bio-Rad, and then electrotransferred onto Immobilon-P transfer membranes (Millipore) at 100 V for 1.5 h (4°C). After blocking for 1 h in TBST (20 mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20) containing 5% nonfat milk, membranes were washed three times in TBST and probed for 20 h at 4°C with the respective Abs, according to the manufacturer’s instructions. Following washing in TBST, membranes were incubated with secondary HRP-conjugated, anti-rabbit IgG from Cell Signaling (1/2000 dilution) for 1 h at room temperature, washed three times in TBST, and bands were detected using ECL plus reagents (Amersham Pharmacia Biotech).

Preparation of nuclear extracts and EMSA

Nuclear extracts were prepared using a nuclear extraction kit (Active Motif) according to the manufacturer’s instructions. The NF-{kappa}B consensus oligonucleotide, 5'-agttgaggggactttcccaggc-3', from the murine Ig{kappa}B L chain gene enhancer (40) and NF-{kappa}B-specific oligonucleotide, 5'-acttcttaaaattcccccagaatg-3', from the murine IL-12 p40 promoter (41), were synthesized by the Biopolymer and Genomics Core Laboratory (University of Maryland, Baltimore, MD). DNA probes were 32P end-labeled with T4 polynucleotide kinase (Invitrogen Life Technologies), as recommended by the manufacturer. EMSA was conducted as described previously (2). The polyacrylamide gels were dried at 80°C for 2 h and exposed to a phosphor screen overnight. The phosphor screen images were visualized using a scanner Storm 680 (Molecular Dynamics) and the signal intensities were quantified with the ImageQuant program (Molecular Dynamics; Amersham). Counts from NF-{kappa}B consensus sequences and specific NF-{kappa}B-binding sequences derived from the murine IL-12 p40 promoter were normalized to counts obtained from NF-Y binding used as a loading control.

ChIP assay

After stimulation, peritoneal macrophages were fixed by adding 1% formaldehyde (Sigma-Aldrich) in RPMI 1640 medium and incubating at room temperature for 15 min. Cells were washed with ice-cold PBS and quenched by adding glycine (0.125 M) in PBS at the room temperature for 5 min. Plates were washed with ice-cold PBS and cells were collected by scraping. After centrifugation, cells were lysed with buffer (50 mM Tris (pH 8.0), 2 mM EDTA, 0.1% Nonidet P-40, 10% glycerol) supplemented with protease inhibitor mixture (Roche) for 15 min on ice to remove the remove the cytoplasmic proteins. Nuclei were precipitated by centrifugation (1200 x g, 5 min) and resuspended in 1 ml of SDS lysis solution (50 mM Tris (pH 8.0), 10 mM EDTA, 1% SDS) supplemented with protease inhibitor mixture (Roche). Chromatin was sheared by sonication (10 x 20 s pulses, with 30-s intervals on ice, at one-third the strength of the sonicator capacity (Sonic Dismembrator, model 60; Fisher) and centrifuged to pellet the debris. The sheared chromatin was an average length of 200–800 bp. Chromatin was diluted 1/4 in dilution buffer (16.7 mM Tris (pH 8.0), 1.2 mM EDTA, 0.01% SDS, 1.1% Triton X-100, and 167 mM NaCl) and immunoprecipitated with 2 µg of Abs against p65 (sc-372X), c-Rel (sc-71X), and isotype control normal IgG (sc-2027) from Santa Cruz Biotechnology overnight at 4°C with rotation. Magnetic protein A beads (Dynal; Invitrogen Life Technologies) were washed and pre-equilibrated with dilution buffer in the presence of 100 µg/ml herring sperm DNA (Invitrogen Life Technologies) and 25 µl of 50% slurry was added to each immunoprecipitated sample and incubated for 30 min at 4°C with rotation. Complexes attached to beads were captured using a magnetic stand (Dynal), rinsed with 1 ml of wash buffer 1 (20 mM Tris (pH 8.0), 2 mM EDTA, 1% Triton X-100, 0.1% SDS, 150 mM NaCl) once, followed by 1 ml of wash buffer 2 (20 mM Tris (pH 8.0), 2 mM EDTA, 1% Triton X-100, 0.1% SDS, 500 mM NaCl) and further washed with 1 ml of lithium chloride wash buffer (10 mM Tris (pH 8.0), 1 mM EDTA, 1% Igepal CA-630, 1% deoxycholic acid, sodium salt, 0.25 M LiCl). The complex was further washed twice with TE buffer. The elution buffer was freshly prepared (1% SDS, 0.1 M NaHCO3) and precipitated complexes were eluted twice with 250 µl for 15 min at room temperature. Protein-DNA cross-linked complexes were reverted by adding 20 µl of 5 M NaCl per tube and heating at 65°C for 4 h to overnight. Each sample was treated with 40 µg of proteinase K for 1 h at 55°C, then DNA was extracted by phenol-chloroform and ethanol precipitation. One-fifteenth of the immunoprecipitated DNA was used as the template for each PCR for 35 cycles. The primers used were: IL-12 p40 (sense 5'-TTCCGACGTCTATATTCCCTCT-3' and antisense 5'-AGCTGCCTGCTCTGATGTGC-3'), IL-12 p35 (sense 5'-GACCTGTCCCGGGACAAGAGT-3' and antisense 5'-CGCTGACCTTGGGAGACACAT-3'), inducible NO synthase (iNOS) (sense 5'-GTGAGTCCCAGTTTTGAAGTGACTAC-3' and antisense 5'-GAGCTATTTTGCATAACTGTTCCCA-3') I{kappa}B{alpha} (sense 5'-CCAGTGGCTCATCGCAGG-3' and antisense 5'-CTGGCGAGGTCTGACTGTTGT-3') and TNF-{alpha} (sense 5'-CAGTTCTCAGGGTCCTATACAACACA-3' and antisense 5'-GGTAGTGGCCCTACACCTCTGTC-3').

Measurement of cytokine production

Murine IL-12 p40, IL-12 p70, and IL-10 levels were measured in supernatants by ELISA (R&D Systems) obtained after overnight stimulation of peritoneal macrophages with LPS or P3C, without or with Fc{gamma}R ligation.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
The effect of Fc{gamma}R ligation on TLR signaling in peritoneal macrophages

Previous studies have shown that coligation of Fc{gamma}R on bone marrow-derived macrophages results in a reprogramming of gene expression induced by LPS to an anti-inflammatory profile in which IL-12 production is repressed while induction of IL-10 is enhanced (18, 19, 20). To confirm and extend these findings, we used TLR4 and TLR2 agonists, LPS and P3C, respectively, to stimulate primary peritoneal exudate macrophages, without or with Fc{gamma}R ligation of macrophages with IgG-opsonized erythrocytes. Fig. 1, A and B, show that Fc{gamma}R engagement on macrophages resulted in a significant inhibition of IL-12 p40 and IL-12 p35 steady-state mRNA levels in both LPS- and P3C-stimulated macrophages, whereas LPS- and P3C-induced IL-10 mRNA levels were significantly increased. Protein levels of IL-12 p40, bioactive IL-12 p70, and IL-10 were measured in macrophage culture supernatants by ELISA and confirmed these findings (Fig. 1C), although the inhibition of P3C-induced IL-12 p40 did not reach the level of statistical significance despite an inhibitory trend. Thus, Fc{gamma}R ligation of primary murine macrophages, in the absence of TLR stimulation, does not induce IL-12 and IL-10. However, when macrophages are concurrently stimulated by Fc{gamma}R ligation and TLR4 or TLR2 agonists, both mRNA and protein levels of IL-12 and IL-10 are reciprocally modulated when compared with levels in macrophages stimulated with TLR agonists only.


Figure 1
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  		FIGURE 1. Effect of Fc{gamma}R ligation on LPS- and P3C-induced IL-12 and IL-10 production in murine peritoneal macrophages. A, IL-12 p40, IL-12 p35, and IL-10 mRNA were measured by real-time PCR at the indicated time intervals following stimulation with LPS (100 ng/ml), without or with Fc{gamma}R ligation. B, IL-12 p40, IL-12 p35, and IL-10 mRNA were measured by real-time PCR at the indicated time intervals following stimulation with P3C (1 µg/ml), without or with Fc{gamma}R ligation (E). C, IL-12p40, IL-12 p70, and IL-10 protein were measured in macrophage supernatants by ELISA after overnight stimulation with LPS or P3C, without or with Fc{gamma}R (E) ligation. M, medium (unstimulated control). Data shown are the average ± SEM of three separate experiments (*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001).

 
The effect of Fc{gamma}R ligation on the TLR-induced PI3K-AKT pathway in peritoneal macrophages

Fukao et al. (17) demonstrated that TLR stimulation of dendritic cells derived from wild-type mice treated with PI3K inhibitors, or from PI3K–/– mice, exhibited increased IL-12 production, suggesting that PI3K negatively regulates IL-12 production. Martin et al. (14) showed that P. gingivalis LPS, a TLR2 agonist, activates the PI3K pathway and that pharmacologic inhibition of this pathway also resulted in increased levels of IL-12 in human monocytes. To explore a possible role for PI3K in the regulation of TLR-induced signaling by Fc{gamma}R ligation, we examined the effect of concurrent engagement of TLR and Fc{gamma}R on phosphorylation of AKT, a downstream target of PI3K. Fig. 2A shows that unstimulated macrophages exhibited little phosphorylation of AKT. LPS- and P3C-induced phosphorylation of AKT was barely detectable at 30 min, while strong phosphorylation could be detected at 60 and 240 min. In contrast to gene expression, Fc{gamma}R ligation alone also activated AKT as early as 15 min. The effect of LPS or P3C stimulation with Fc{gamma}R ligation was seen as early as 15 min and was clearly increased by 30 min when compared with either treatment alone. Similar responses to stimulation by LPS or P3C and Fc{gamma}R ligation were observed in different macrophage types including the RAW 264.7 murine macrophage cell line (data not shown), as well as bone marrow-derived macrophages from BALB/c mice (Fig. 2B). LPS-unresponsive C3H/HeJ macrophages failed to activate AKT in response to LPS, while P3C induced pAKT that was similarly augmented by concurrent Fc{gamma}R ligation (data not shown). Finally, similar patterns of AKT phosphorylation were also observed if soluble OVA-IgG complexes were used to ligate the Fc{gamma}R, rather than erythrocyte IgG (Fig. 2B). These findings support the hypothesis that activation of the PI3K-AKT pathway may be involved in negative regulation of TLR-induced IL-12 production by Fc{gamma}R ligation in peritoneal macrophages.


Figure 2
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  		FIGURE 2. Effect of Fc{gamma}R ligation on TLR-induced PI3K-dependent phosphorylation of AKT in murine macrophages. A, Murine peritoneal macrophages were stimulated with LPS (100 ng/ml) or P3C (1 µg/ml), without or with Fc{gamma}R ligation (E). Whole cell lysates were prepared at the indicated time points (minutes) and were analyzed by Western analysis with anti-phospho-AKT (Ser473) and anti-beta-actin Abs, the latter of which was used as loading control. B, Bone marrow-derived macrophages were stimulated with LPS (10 ng/ml) or with Fc{gamma}R ligation either opsonized erythrocytes (E) or OVA-IgG soluble complexes (OVA). Whole cell lysates were prepared at the indicated time points (minutes (M)) and were analyzed by Western analysis with anti-phospho-AKT (Ser473) and total ERK1/2 Abs, the latter of which was used as loading control. M, medium (unstimulated control); IgG (HC), IgG H chain. Data shown are representative of three separate experiments.

 
PI3K inhibitors reversed the effect of Fc{gamma}R ligation on TLR signaling

To determine whether the ability of LPS and P3C to activate the PI3K-AKT pathway plays a functional role in the regulation of cytokine production, we sought to determine whether PI3K-AKT inhibition affects the induction of IL-12 p40, IL-12 p35, and IL-10 at the level of steady-state mRNA. Two different PI3K inhibitors, wortmannin (42) and LY294002 (43), were used to study the involvement of PI3K in macrophages stimulated with TLR agonists, without or with Fc{gamma}R ligation. Many previous studies have used 20 µM LY294002, but we found this concentration to be toxic to murine peritoneal macrophages (data not shown). Therefore, preliminary experiments were conducted in which we varied the concentration of wortmannin from 50 to 500 nM and LY294002 from 0.5 to 5 µM. Inhibition of IL-12 p40 and IL-12 p35 mRNA and increase in IL-10 mRNA in macrophages treated with LPS and Fc{gamma}R ligation were reversed by both inhibitors in a dose-dependent manner (data not shown) and in all subsequent experiments, 500 nM wortmannin and 2 µM LY294002 were used to avoid cellular toxicity.

As shown in Fig. 3A, the already high levels of LPS-induced IL-12 p40 mRNA were not significantly affected by wortmannin or LY294002. In contrast, both inhibitors reversed the effect of Fc{gamma}R ligation on LPS-induced IL-12 p40 significantly. The significant decrease in LPS-induced IL-12 p35 mRNA with Fc{gamma}R ligation was reversed in the presence of wortmannin, with a similar trend seen with LY294002. Fig. 3B shows that the effect of PI3K inhibitors on P3C-induced IL-12 gene expression is more complex. In macrophages treated with P3C, IL-12 p40 mRNA levels were significantly increased in the presence of wortmannin only, while both wortmannin and LY294002 significantly increased steady-state levels of IL-12 p35 mRNA. Neither inhibitor significantly reversed the inhibitory effect of Fc{gamma}R ligation on P3C-induced IL-12 p40 mRNA levels, despite an upward trend. In contrast, IL-12 p35 mRNA levels induced in the presence of concurrent Fc{gamma}R ligation and P3C were significantly increased when either wortmannin or LY294002 was included in the culture (Fig. 3B). Both LPS- and P3C-induced increases in IL-10 mRNA levels in macrophage with Fc{gamma}R ligation were significantly decreased in the presence of PI3K inhibitors (Fig. 3). Thus, inhibition of PI3K inhibitors fully or partially reversed the effects of Fc{gamma}R ligation on TLR4- and TLR2-mediated IL-12 and IL-10 mRNA expression.


Figure 3
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  		FIGURE 3. PI3K inhibitors reverse the effect Fc{gamma}R ligation on LPS- and P3C-induced IL-12 and IL-10 mRNA expression. IL-12 p40, IL-12 p35, and IL-10 mRNA were measured by real-time PCR at the indicated time intervals in peritoneal macrophages stimulated with (A) LPS (100 ng/ml) or (B) P3C (1 µg/ml), without or with Fc{gamma}R (E) ligation, in absence or presence of PI3K inhibitors, wortmannin (W; 500 nM) or LY294002 (LY; 2 µM). Data shown are the average ± SEM of three separate experiments (*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001).

 
We next used the two PI3K inhibitors to evaluate their effect on phosphorylation of AKT and other targets. Whole cell lysates from LPS- or P3C-stimulated macrophages, without or with Fc{gamma}R ligation, in the absence or presence of PI3K inhibitors, were screened for AKT phosphorylation by Western analysis. As shown in Fig. 4, 0.5 and 2 µM of wortmannin and LY294002, respectively, blocked phosphorylation of AKT completely. Importantly, LPS- or P3C-induced phosphorylation of GSK3{alpha}/GSK3beta, p38, or ERK1/2 MAPKs, or STAT1 on serine 727 (a downstream target of p38) were not inhibited under these same conditions. Taken collectively, these data strongly suggest that the effect of Fc{gamma}R ligation on LPS- or P3C-induced expression of IL-12 p40, IL-12 p35, and IL-10 could be correlated with augmentation of PI3K activity and subsequent phosphorylation of AKT, but not with modulation of GSK3{alpha}/GSK3beta, p38, or ERK1/2 MAPKs.


Figure 4
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  		FIGURE 4. The PI3K inhibitors, wortmannin (W) and LY294002 (L), block AKT phosphorylation induced by LPS or P3C, without or with Fc{gamma}R ligation. Macrophages were stimulated for 30 min with LPS (100 ng/ml) or P3C (1 µg/ml), without or with Fc{gamma}R (E) ligation, in absence or presence of PI3K inhibitors. Whole cell extracts were analyzed by Western blot analysis using phosphospecific Abs to AKT (Ser473), GSK3{alpha}/GSK3beta, ERK1/2, p38, and STAT1 (Ser727). beta-actin was used as a loading control. M, medium (unstimulated control). Data shown are representative of three separate experiments.

 
The effect of Fc{gamma}R ligation on LPS-induced NF-{kappa}B binding

Five Rel family members have been identified in mammalian cells: p50, p65 (Rel A), c-Rel, p52, and RelB. These proteins can form various homodimeric and heterodimeric complexes that are maintained in an inactive state in the cytoplasm by their association with I{kappa}B proteins (44). Upon cellular activation, I{kappa}B{alpha} is phosphorylated, ubiquitinated, and then degraded, leading to the release of NF-{kappa}B, which subsequently translocates to the nucleus and enables inflammatory gene transcription (45, 46). Therefore, activation of the NF-{kappa}B pathway was first measured by the degradation of I{kappa}B{alpha} as a surrogate marker. Whole cell lysates from LPS- and P3C-stimulated macrophages, without or with Fc{gamma}R ligation, were screened for I{kappa}B{alpha} degradation by Western blotting. The results in Fig. 5 show that no degradation of I{kappa}B{alpha} was observed in medium-treated cells or cells subjected to Fc{gamma}R ligation only. In contrast, I{kappa}B{alpha} degradation was detected as early as 15 min and I{kappa}B{alpha} was completely degraded by 30 min after stimulation with TLR agonists. Normal levels of I{kappa}B{alpha} were restored as early as 60 min after LPS or P3C stimulation. Concurrent Fc{gamma}R ligation did not alter this process. Thus, I{kappa}B{alpha} degradation is not affected by Fc{gamma}R ligation without or with TLR4- or TLR2-mediated signaling.


Figure 5
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  		FIGURE 5. Kinetics of I{kappa}B{alpha} degradation in LPS- and P3C-stimulated macrophages, without or with Fc{gamma}R ligation. Macrophages were stimulated with LPS (100 ng/ml) and P3C (1 µg/ml), without or with IgG-opsonized erythrocytes (E). Whole cell lysates were prepared at the indicated time points and were analyzed by Western blot analysis using total anti-I{kappa}B{alpha} Ab. beta-actin was used as loading control. M, medium (unstimulated control). Data shown are representative of three separate experiments.

 
We next analyzed nuclear extracts from LPS-activated macrophages by EMSA for binding to NF-{kappa}B sites, which is known to be important in the regulation of IL-12 p40 transcription (41, 47). We sought to determine whether specific patterns of NF-{kappa}B binding might correlate with the dysregulated expression of IL-12 p40 seen in the LPS-induced macrophage after Fc{gamma}R ligation. Two NF-{kappa}B-binding sequences were used: one is a consensus sequence, which is universally used for measuring NF-{kappa}B binding by EMSA, and the other is the specific NF-{kappa}B site found in the murine IL-12 p40 promoter. For each probe, binding was compared with binding of NF-Y as a loading control. Fig. 6A reveals differences in NF-{kappa}B binding in nuclear extracts after LPS activation for 1 and 4 h. At the early time point (1 h), there was no effect of Fc{gamma}R ligation on LPS-induced NF-{kappa}B binding to either probe; however, we observed a significant inhibition in NF-{kappa}B binding to both probes in 4 h samples in the presence of Fc{gamma}R ligation. This inhibition in NF-{kappa}B binding was reversed significantly in both the NF-{kappa}B consensus sequence and the NF-{kappa}B-specific IL-12 p40 sequence by wortmannin. No significant differences were observed in binding to the NF-Y probe. The data in Fig. 6A were quantified and were found statistically different at 4 h (Fig. 6B). A supershift assay revealed that binding to the NF-{kappa}B site in the IL-12 p40 promoter is predominantly composed of p50 or p52/p65 heterodimers, with faint detection of c-Rel also. No NF-{kappa}B binding was seen in unstimulated cells or in the presence of unlabeled probe (Fig. 6C). Therefore, NF-{kappa}B binding was very specific and PI3K dependent and the data suggest that inhibition of late NF-{kappa}B binding may, in part, underlie inhibition of LPS-induced IL-12 in macrophages with Fc{gamma}R ligation.


Figure 6
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  		FIGURE 6. EMSA for NF-{kappa}B and translocation of I{kappa}B{alpha} to the nuclei upon LPS stimulation in murine peritoneal macrophages. A, Macrophages were stimulated with LPS (100 ng/ml) without or with Fc{gamma}R ligation (E), in the absence or presence of wortmannin (W, 500 nM). Nuclear extracts were analyzed by EMSA using both an NF-{kappa}B consensus sequence and NF-{kappa}B site specific for the IL-12 p40 promoter region. EMSA for NF-Y was used as the loading control. B, Quantification of the results obtained in A was obtained from the counts derived from the phosphor screen. Data for binding to NF-{kappa}B-specific sequences were normalized with NF-Y data for individual samples. The results represent the mean ± SEM of three separate experiments. C, Supershift assay was done for the specific NF-{kappa}B-binding site of IL-12 p40 promoter region. The Rel family antisera (p50, p52, p65, c-Rel, and RelB) were preincubated with nuclear extracts before mixing with the labeled probe. D, Translocation of I{kappa}B{alpha} protein upon LPS stimulation in peritoneal macrophages. Macrophages were stimulated with LPS (100 ng/ml) or LPS plus IgG-opsonized erythrocytes (E) at the indicated time intervals in the absence or presence of PI3K inhibitor, wortmannin (W, 500 nM). Nuclear extracts were analyzed by Western blot analysis for I{kappa}B{alpha} protein and beta-actin as the loading control. Data shown are representative of three separate experiments. E, Translocation of Rel family members into the nucleus upon LPS stimulation in peritoneal macrophages. Macrophages were stimulated with LPS (100 ng/ml), without or with IgG-opsonized erythrocytes (E) for 4 h, in the absence or presence of PI3K inhibitor, wortmannin (W, 500 nM). Nuclear extracts were analyzed by EMSA and Western blot for Rel family members (p65, p50, and c-rel), I{kappa}B{alpha} and beta-actin, as the loading control. Data shown are representative of three separate experiments. F, ChIP assay. Macrophages were stimulated with LPS (100 ng/ml) without or with IgG-opsonized erythrocytes (E) for 1 and 4 h. ChIP was conducted using p65 and c-Rel Abs. The promoters for Nfkbia, Tnf, Il12b, Il12a, and Nos2 were analyzed for the recruitment of NF-{kappa}B/Rel A or c-Rel by ChIP as described in Materials and Methods.

 
We measured three NF-{kappa}B components (p65, p50, and c-Rel) directly in samples of nuclear proteins after LPS stimulation of macrophages in the presence of immune complexes without or with PI3K inhibitor wortmannin. LPS-induced translocation of p65, p50, and c-Rel was not affected by Fc{gamma}R ligation after 4 h of treatment. Activation of NF-{kappa}B is dependent on phosphorylation and degradation of its inhibitor, I{kappa}B{alpha}. I{kappa}B{alpha} mRNA expression is, itself, NF-{kappa}B dependent, and this protein is synthesized de novo within 1 h of LPS stimulation (see Fig. 5). However, I{kappa}B{alpha} has also been found in nuclei, where it has been shown to be responsible for postinduction inhibition of NF-{kappa}B-dependent transcription (48). Therefore, we measured the inhibitor protein, I{kappa}B{alpha}, in the nucleus by Western analysis. Fig. 6, D and E, illustrates an accumulation of I{kappa}B{alpha} protein in the nuclei of cells stimulated by LPS concurrently with ligation of Fc{gamma}R at 4 h. The PI3K inhibitor, wortmannin, reversed the effect of Fc{gamma}R ligation on translocation of I{kappa}B{alpha} protein into the nucleus at 4 h after treatment with LPS. Because the inhibitory effects of Fc{gamma}R ligation on LPS-induced NF-{kappa}B translocation and NF-{kappa}B binding were not observed until 4 h, we hypothesized that transcription of Rel family members may also be affected.

Saccani et al. (49) demonstrated that LPS-stimulated macrophages showed NF-{kappa}B recruitment to targeted genes appears to occur in two distinct phases, with one subset of genes (e.g., Nfkbia, mip2, and Sod2) that are already acetylated before stimulation, i.e., constitutively and immediately accessible to NF-{kappa}B binding. Another set of genes (e.g., Il12b, Ccl5, and Il6) was regulated late and required hyperacetylation of the promoter to make it accessible to NF-{kappa}B. We sought to measure recruitment of NF-{kappa}B to the promoters of Nfkbia, Tnf, Il12b, Il12a, and Nos2 by ChIP assay. As shown in Fig. 6F, recruitment of NF-{kappa}B p65 to late, LPS-induced gene promoters Il12b, Il12a, and Nos2 was significantly inhibited by Fc{gamma}R ligation at 4 h, consistent with the patterns observed in the EMSA shown in Fig. 6A. Recruitment of NF-{kappa}B c-Rel to the IL-12 p35 promoter, but not the IL-12 p40 promoter was also significantly inhibited in LPS-stimulated cells with 4 h of Fc{gamma}R ligation. Recruitment of NF-{kappa}B p65 or c-Rel to promoters of LPS-induced early genes (Nfkbia and Tnf) was not inhibited by Fc{gamma}R ligation. Moreover, both I{kappa}B{alpha} and TNF-{alpha} promoter recruitment of NF-{kappa}B c-Rel was very robust in 1-h samples but markedly reduced by 4 h.

To investigate this possibility, steady-state mRNA levels of Rel family members (p65, p50, p52, c-Rel, and RelB) were measured by real-time PCR. Fig. 7 shows that LPS-induced expression of p65 and p50, and to a lesser extent, c-Rel mRNA were inhibited by concurrent Fc{gamma}R ligation; however, there was no effect of Fc{gamma}R ligation on LPS-induced p52 (Fig. 7) or RelB mRNA (data not shown).


Figure 7
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  		FIGURE 7. Steady-state mRNA levels of Rel family members upon LPS stimulation without or with Fc{gamma}R ligation. Relative gene expression was measured by real-time PCR at the indicated time intervals after stimulation of LPS (100 ng/ml), without or with Fc{gamma}R ligation (E) in peritoneal macrophages. The results represent the mean ± SEM, of three separate experiments (*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001).

 
The effect of Fc{gamma}R engagement on LPS induced IRF-8/ICSBP mRNA

IRF-8, also called ICSBP, is a transcription factor that is essential for the induction of IL-12 (50, 51). Previous studies have shown that Fc{gamma}R engagement down-regulates LPS-induced IRF-8/ICSBP protein (52). Therefore, we sought to determine whether IRF-8/ICSBP transcription was also dysregulated by Fc{gamma}R engagement in LPS- and P3C-stimulated macrophages. Fig. 8A shows that the LPS-induced IRF-8/ICSBP mRNA was significantly decreased by Fc{gamma}R ligation. The decreased levels of IRF-8/ICSBP mRNA were reversed by the PI3K inhibitor, wortmannin. However, there was no induction of IRF-8/ICSBP observed in P3C-stimulated peritoneal macrophages (data not shown). These data suggest that the inhibitory effect of Fc{gamma}R on LPS-induced IL-12 may be, in part, due to inhibition of IRF-8/ICSBP transcription.


Figure 8
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  		FIGURE 8. A, The mRNA levels of transcription factor IRF-8/ICSBP. IRF-8/ICSBP mRNA levels were measured by real-time PCR at indicated time intervals (H) after LPS stimulation in peritoneal macrophages in the absence or presence of the PI3K inhibitor, wortmannin (W; 500 nM), without or with Fc{gamma}R ligation (E). The results represent the mean ± SEM of three separate experiments. B, The mRNA levels of LPS-induced iNOS. Relative gene expression was measured by real-time PCR at the indicated time intervals after stimulation of LPS (100 ng/ml) without or with Fc{gamma}R ligation (E) in peritoneal macrophages. The results represent the mean ± SEM of three separate experiments (*, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001).

 
The effect of Fc{gamma}R engagement on LPS-induced iNOS mRNA

LPS-induced iNOS production has been shown to be dependent on PI3K in macrophages (53) and c6 glioma cells (54). LPS-induced iNOS expression is also dependent on the NF-{kappa}B transcription factor (55) and very recent studies described that IFN-{gamma}-induced iNOS gene expression was dependent on IRF-8/ICSBP and IRF-1 in murine macrophages (56). Therefore, we sought to determine whether LPS-induced iNOS mRNA was inhibited by Fc{gamma}R ligation. Fig. 8B shows that the LPS-induced iNOS was significantly decreased by Fc{gamma}R ligation. Therefore, the down-regulation of IRF-8/ICSBP by LPS plus Fc{gamma}R ligation results in down-regulation of iNOS mRNA expression as well.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
This report supports and extends previous studies that have shown that ligation of Fc{gamma}R on bone marrow-derived murine macrophages results in a reprogramming of gene expression induced by LPS to an anti-inflammatory profile such that IL-12 production is repressed while induction of IL-10 is enhanced (18, 19, 20). That this creates an anti-inflammatory milieu in vivo is supported by the fact that administration of IgG-opsonized erythrocytes protects mice from lethal endotoxicity (20). Our data extend these findings significantly: in peritoneal exudate macrophages concurrently stimulated by Fc{gamma}R ligation and either TLR4 or TLR2/1 agonists, e.g., LPS or P3C, respectively, both mRNA and protein levels of IL-12 and IL-10 are reciprocally modulated when compared with macrophages stimulated with TLR agonists only (Fig. 1). Although this dichotomous and rather selective regulation of IL-12 and IL-10 has been recognized for a number of years, the precise mechanisms that underlie this skewing of the cytokine response to an anti-inflammatory, "Th2" profile have not been fully delineated.

A role for PI3K in the regulation of TLR-induced signaling by Fc{gamma}R ligation was evidenced in our study by the observed synergistic activation of AKT, a downstream target of PI3K, upon TLR signaling with concurrent Fc{gamma}R ligation in macrophages (Figs. 2 and 4) that correlated with negative regulation of IL-12 production (Figs. 1 and 3). Previous reports have provided evidence that Fc{gamma}R-mediated phagocytosis was dependent upon PI3K activation: phagocytosis was inhibited in the presence of wortmannin in the human monocytic cell line U937 and in guinea pig neutrophils (57) and Fc{gamma}R engagement was reported to be coupled functionally to activation of PI3K via the protein tyrosine kinase, Syk (58, 59). Recent studies on the role of PI3K in innate immunity suggested that negative regulation of IL-12 by activation of the PI3K-AKT pathway perhaps serves as a possible "safety" mechanism to control the magnitude of cellular responses to pathogens (15, 16, 17). Fukao et al. (17) demonstrated that TLR stimulation of dendritic cells derived from wild-type mice treated with PI3K inhibitors, or from PI3K–/– mice, exhibited increased IL-12 production, supporting the hypothesis that PI3K negatively regulates IL-12 production. Martin et al. (14) later showed that P. gingivalis LPS, a TLR2 agonist, activates the PI3K-AKT pathway and that pharmacologic inhibition of this pathway resulted in increased levels of IL-12 in human monocytes. To determine the role of the PI3K-AKT pathway in the cross-talk between Fc{gamma}R ligation and TLR signaling by LPS or P3C, we studied the effect of PI3K-AKT inhibitors on induction of IL-12 p40, IL-12 p35, and IL-10 in primary murine macrophages (Fig. 3). Both inhibitors reversed the inhibition of IL-12 p40 and IL-12 p35 and the increase in IL-10 seen in macrophages concurrently stimulated with LPS and Fc{gamma}R. This was correlated with the observation that phosphorylation of AKT was completely blocked by PI3K inhibitors (Fig. 4). Our data support earlier studies suggesting that PI3K may provide a compensatory mechanism that suppresses proinflammatory cytokine production in response to sepsis or septic shock (60). Thus, enhanced activation of the PI3K-AKT pathway upon concurrent Fc{gamma}R and TLR engagement may dampen the host inflammatory response by diminishing IL-12 production while stimulating IL-10.

Recently, Martin et al. (61) correlated phosphorylation of GSK3beta to the cytokine profiles produced upon engagement of various TLRs and suggested that GSK3 is key to the differential regulation of IL-12 and IL-10. In accordance with this report, we observed that LPS- and P3C-induced GSK3{alpha}/GSK3beta phosphorylation and this was augmented upon coordinate engagement of TLRs and Fc{gamma}R (Fig. 4). However, in contrast to previous findings (15, 61, 62, 63), phosphorylation of GSK3{alpha}/GSK3beta was not inhibited by wortmannin or LY294002, suggesting that PI3K is not solely responsible for the activation of GSK{alpha}/GSKbeta in our system. This may be attributable to the concentration of inhibitors used in these previous studies, which we found to be toxic to the macrophage systems used herein. Alternatively, several earlier studies have suggested that, in addition to PI3K-AKT (PKB), GSK3{alpha}/GSK3beta could also be phosphorylated and deactivated by protein kinase A at serine 21 of GSK3{alpha} and serine 9 of GSK3beta (64, 65). It also has been known for more than 20 years that Fc{gamma}R ligation increases levels of intracellular cAMP in murine macrophages (66, 67). This increase in intracellular cAMP, in turn, may activate protein kinase A and account for the PI3K-independent phosphorylation of GSK3beta observed in our experiments. Thus, our findings that PI3K inhibitors affect the IL-12/IL-10 expression profile, while not affecting GSK3 phosphorylation, illustrate a case where GSK3 activity could not be correlated with the reciprocal regulation of IL-12/IL-10 induced by Fc{gamma}R and TLR coengagement and suggests that additional pathways apart from inhibition of GSK3{alpha}/GSK3beta contribute to the dichotomous regulation of IL-12 and IL-10.

Lucas et al. (68) attributed the LPS-induced increase in levels of IL-10 production with Fc{gamma}R ligation to augmented ERK1/2 activation in bone marrow-derived macrophages. However, in our system there was no detectable increase in ERK1/2 activation in response to stimulation of macrophages with IgG-opsonized erythrocytes and LPS or P3C above that seen with TLR stimulation alone (Fig. 4). We also did not observe any modulation of TLR agonist-induced activation of JNK, p38, or STAT1 (serine 727) by PI3K inhibitors or Fc{gamma}R ligation, under conditions where phosphorylation of AKT was greatly inhibited (Fig. 4). This suggests that the difference between our study and that of Lucas et al. (68) may be attributable to cross-talk between ERK1/2-mediated signaling and the PI3K-AKT pathway because only the latter was inhibited in our study. The regulation of IL-12 mRNA gene expression has been described extensively in a recent review (69) and is dependent upon the coordinated expression of IL-12 IL-12b and IL-12a genes, which are encoded on different chromosomes. Many studies have confirmed that the primary regulatory step for the expression of both IL-12 IL-12b and IL-12a genes is at the level of transcription: multiple transcription factors are known to be involved in regulation of these genes, e.g., NF-{kappa}B (47), C/EBPbeta (41), PU.1 (70), IRF-1 (37, 71), IRF-2 (37), IRF-8/ICSBP (50, 51), NFAT (72), AP-1 (73), and activator and repressor factors such as Kruppel-like factor (74). TLR-mediated NF-{kappa}B binding and translocation is dependent upon the degradation of an inhibitory protein, I{kappa}B{alpha}. I{kappa}B{alpha} sequesters Rel family proteins in the cytoplasm, preventing the activation and translocation of NF-{kappa}B to the nucleus. Stimulation of macrophages with LPS induces rapid phosphorylation of I{kappa}B{alpha} that leads to its degradation (Fig. 5), thus enabling Rel proteins to translocate to nucleus where they bind to different promoters to initiate transcription (45, 46). Our finding that Fc{gamma}R ligation failed to interfere with the normal degradation of I{kappa}B{alpha} in cells stimulated with LPS or P3C indicates that this mechanism cannot account for diminished IL-12 transcription.

In contrast, analysis of NF-{kappa}B binding by EMSA revealed that while there was no difference in binding of nuclear proteins to the NF-{kappa}B-specific oligomers 1 h after LPS stimulation with Fc{gamma}R ligation, there was a highly significant diminution in NF-{kappa}B binding at 4 h (Fig. 6). It is likely that the generation of such a complex repertoire of activated transcription factors (e.g., IRF-8/ICSBP, NF-{kappa}B, IRF-1, AP-1, and others) and their coordinated assembly within both the IL-12 p40 and IL-12 p35 promoters contributes, in part, to the relatively late induction of IL-12 (Fig. 1) when compared with early LPS-inducible genes (e.g., Tnf, etc.). Inhibition of LPS-induced NF-{kappa}B binding to IL-12 p40 promoter 4 h after treatment of cells with IgG-opsonized erythrocytes may also contribute to the relatively selective down-regulation of LPS-induced IL-12 production upon Fc{gamma}R ligation. The reversal of inhibition of NF-{kappa}B binding by PI3K inhibitors 4 h after Fc{gamma}R ligation of TLR-stimulated macrophages suggests that the inhibition of NF-{kappa}B binding to IL-12 p40 promoter and transcription of IL-12 p40 are PI3K dependent. Inhibition of PI3K enhanced LPS-induced NF-{kappa}B binding in THP-1 cells (15) and PI3K negatively regulates LPS-induced NF-{kappa}B binding and IL-12 production (15, 17). In a more recent study (75), Zhu et al. (72) demonstrated that microtubule-associated serine/threonine kinase 205 kDa (MAST 205) is required for LPS-induced IL-12 production. These authors also showed that macrophage Fc{gamma}R ligation resulted in rapid ubiquitination and proteasomal degradation of MAST 205 and that dominant-negative MAST 205 mutants inhibited LPS-induced IL-12 synthesis and NF-{kappa}B binding in macrophages that were ligated through the Fc{gamma}R for 2 h, followed by LPS treatment for 1 h (75), for a total of 3 h Fc{gamma}R engagement. LPS-induced TNF-{alpha} (18) and I{kappa}B{alpha} protein expression (Fig. 5) were not inhibited by Fc{gamma}R ligation, even though both TNF-{alpha} and I{kappa}B{alpha} expression are dependent on NF-{kappa}B (76, 77). Our data support the hypothesis that transcription of these two "immediate early" genes is dependent on "early" NF-{kappa}B binding to their promoters, which we have shown is not affected by Fc{gamma}R ligation, rather than later NF-{kappa}B binding to their promoters. Taken collectively, our data support a kinetic basis for the prior observation that there was no effect of Fc{gamma}R ligation on LPS-induced TNF-{alpha} (18) and I{kappa}B{alpha} protein (Fig. 5), while IL-12 mRNA and protein were inhibited. Our EMSA findings were strengthened by ChIP analyses, in which late LPS-inducible, NF-{kappa}B-dependent genes were affected by Fc{gamma}R ligation, with the recruitment of NF-{kappa}B/Rel A or c-Rel to the promoters of the genes that encode IL-12 p40, IL-12 p35, and iNOS being significantly inhibited at 4 h. In contrast, the early genes like Nfkbia and Tnf did not show any difference in the recruitment of NF-{kappa}B. The recruitment of NF-{kappa}B to these "early" promoters was confirmed in earlier reports (49).

Activation of NF-{kappa}B is dependent upon phosphorylation and degradation of its inhibitor, I{kappa}B{alpha}. I{kappa}B{alpha} mRNA expression is NF-{kappa}B dependent, is induced within 1 h of LPS stimulation, and has been reported to be responsible for postinduction inhibition of NF-{kappa}B-dependent transcription (48). Newly synthesized I{kappa}B{alpha} is targeted to the nucleus by a novel nuclear import sequence within the second ankyrin repeat (78). Nuclear translocation of I{kappa}B{alpha} results from a specific, energy-dependent transport process (79). I{kappa}B{alpha} negatively regulates transcription by inhibiting binding of NF-{kappa}B to the DNA and ultimately exports NF-{kappa}B from the nucleus back into cytoplasm. The export function of I{kappa}B{alpha} is due to a nuclear export sequence located in its C-terminal domain (80). In our studies, I{kappa}B{alpha} protein accumulated in the nucleus to a much greater extent in samples treated with LPS in combination with Fc{gamma}R ligation at 4 h (Fig. 6D) where it could bind to the Rel family members in the nucleus and form inactive complexes, thereby competitively inhibiting NF-{kappa}B binding to the promoter IL-12 p40. Increased levels of I{kappa}B{alpha} protein in the nucleus were completely blocked by the presence of wortmannin in macrophages treated with LPS and Fc{gamma}R ligation. These data strongly suggest that the accumulation of I{kappa}B{alpha} protein in the nucleus is also mediated by the activation of the PI3K-AKT pathway. Thus, the regulation of I{kappa}B{alpha} distribution, first in the cytoplasm and then in the nucleus, coupled with the wortmannin-sensitive accumulation in the nucleus, suggests the possibility that I{kappa}B{alpha} protein ultimately down-regulates the NF-{kappa}B binding to the Il12b promoter in cells treated with LPS along with Fc{gamma}R ligation.

Two members of the IRF family of transcription factors, IRF-1 and IRF-8/ICSBP, play a significant role in the LPS-induced IL-12 in vivo and in vitro. LPS-induced IL-12 p40 and IL-12 p35 mRNA are dependent on de novo synthesis of transcription factors IRF-8/ICSBP and IRF-1. Both IRF-1- and IRF-8/ICSBP-deficient mice and macrophages are highly defective in the production of IL-12 (37, 50, 51): IRF-8/ICSBP was found to be essential for the transcription of both IL-12 p40 and IL-12 p35 mRNA (51) and IRF-1 for IL-12 p35 (71). BXH-2 mice, that carry a point mutation within the DNA-binding domain of IRF-8/ICSBP, fail to produce IL-12, even though expression of mutated ICSBP was normal (81). Earlier studies indicated that LPS-induced IRF-8/ICSBP and IRF-1 proteins in the nucleus were down-regulated by Fc{gamma}R engagement (52). Our data extend this finding by demonstrating a significant repression of IRF-8/ICSBP mRNA expression upon Fc{gamma}R ligation and TLR signaling. Moreover, LPS-induced inhibition of IRF-8/ICSBP mRNA expression by Fc{gamma}R ligation was also reversed in the presence of PI3K inhibitor wortmannin. Thus, this study is the first to demonstrate that PI3K negatively regulates the LPS-induced IRF-8/ICSBP steady-state mRNA. IRF-8/ICSBP was induced only in cells stimulated with TLR4 agonist, LPS, whereas stimulation of macrophages with the TLR2 agonist P3C failed to induce the IRF-8/ICSBP. A similar observation was also seen in macrophages stimulated with the TLR9 agonist, CpG DNA, which did not depend upon IRF-8/ICSBP for induction of IL-12 (82). Collectively, the decrease in production of critical transacting factors, IRF-8/ICSBP and IRF-1, in cells that are concurrently stimulated by TLR4 and Fc{gamma}R ligation provides yet an additional mechanism by which IL-12 production is inhibited. LPS-induced iNOS mRNA expression has been shown to be regulated by PI3K (53, 54) as well as NF-{kappa}B (55) and IRF-8 (56) dependent. That LPS-induced iNOS mRNA was inhibited by Fc{gamma}R ligation further confirms our hypothesis.

IL-12 is a key cytokine in the control of many pathogens and the inflammation that accompanies infection. IL-12 also induces IFN-{gamma} production, favoring the differentiation of Th (Th1) cells and creating a link between innate resistance and adaptive immunity. Our findings show that in macrophages coordinately engaged through TLR and Fc{gamma}R ligation, PI3K plays a significant regulatory role in the balance of IL-12 and IL-10. We have identified multiple novel mechanisms by which this complex system is regulated including altered kinetics of NF-{kappa}B binding, redistribution of I{kappa}B{alpha} between cytoplasmic and nuclear compartments, and diminished transcription of a key transactivating factor, IRF-8/ICSBP.


   Acknowledgments
 
We acknowledge Zachary Roberts, Leah Cole, and Karen Thomas for their help and advice during these studies. We are grateful for technical advice on the ChIP assay from Drs. Stephen Smale, Ruslan Medzhitov, Xia Zhang, and David Mosser and their laboratories.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grant AI18797 (to S.N.V.). Back

2 S.K.P. and V.Y.T. contributed equally to the work in this article. Back

3 Address correspondence and reprint requests to Dr. Stefanie N. Vogel, Department of Microbiology and Immunology, School of Medicine, University of Maryland, Howard Hall Room 324, 660 West Redwood Street, Baltimore, MD 21201. E-mail address: svogel{at}som.umaryland.edu Back

4 Abbreviations used in this paper: PKB, protein kinase B; ChIP, chromatin immunoprecipitation; IRF-8, IFN regulatory factor-8; ICSBP, IFN consensus sequence-binding protein; P3C, S-[2,3-Bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-Cys-(S)-Ser-Lys4-OH trihydrochloride; iNOS, inducible NO synthase; GSK3, glycogen synthase kinase-3; MAST 205, microtubule-associated serine/threonine kinase 205 kDa. Back

Received for publication March 26, 2006. Accepted for publication April 26, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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