The PI3K family is thought to participate in TLR signaling, and it has been reported to be a negative regulator of TLR-mediated production of IL-12, a key inducer of Th1 responses. However, the role of individual PI3K subtypes in IL-12 production remains obscure. We defined the distinct regulation of LPS-mediated IL-12 production by p110α and p110β catalytic subunits of PI3K in human APCs. We observed that knockdown of PI3K p110β by small interfering RNA (siRNA) suppressed both LPS-induced IL-12 protein production and mRNA expression in monocyte-derived macrophages and dendritic cells (DCs). Knockdown of PI3K p110α by siRNA reduced LPS-induced IL-12 protein production in both cell types. Conversely, knockdown of PI3K p110α suppressed LPS-induced IL-12 mRNA expression in monocyte-derived macrophages but minimally affected monocyte-derived DCs. PI3K p110β siRNA inhibited JNK activation, but not p38 MAPK or ERK activation, stimulated by LPS, while PI3K p110α siRNA did not affect LPS-induced JNK, p38 MAPK, or ERK activation in both cell types. Transfection of siRNA against JNK1, JNK2, and both decreased LPS-induced IL-12 production. Furthermore, PI3K p110β siRNA attenuated LPS-induced JNK1 phosphorylation, while not affecting JNK2 phosphorylation. Our findings indicate that PI3K p110β positively controls LPS-induced IL-12 production through the JNK1-dependent pathway in human macrophages and DCs.

IL-12 is a heterodimeric 70-kDa (p70) cytokine composed of two disulfide-linked glycosylated chains of 40 (p40) and 35 kDa (p35) encoded by two distinct genes (1). This cytokine is mainly produced by APCs, such as dendritic cells (DCs)3 and monocytes-macrophages, in response to bacterial products such as LPS and intracellular pathogens (1, 2), or upon interaction with activated T cells (1, 3). IL-12 is a potent inducer of IFN-γ production by NK cells, NKT cells, and CD8+ T cells. Additionally, IL-12 is critical for the differentiation of naive CD4+ T cells into the Th1 cells required to eliminate intracellular pathogens and tumor cells (4). In this way, APCs bridge the innate and adaptive immune responses through IL-12 production. The expression of IL-12 appears to be regulated primarily at the level of transcription, and the promoter of the gene encoding p40 has been studied in great detail (5). Additionally, some researchers have demonstrated the early signaling events underlying the regulation of IL-12 expression. The stimulation of TLR4 by LPS induces the nuclear transport of NF-κB and MAPKs through MyD88, protein kinase IL-1R-associated kinase, and TNF receptor-associated factor 6 (6). The MAPK cascades constitute an important group of serine/threonine signaling kinases that modulate the phosphorylation, and therefore the activation status, of transcription factors, and link transmembrane signaling with gene induction events in the nucleus. JNK, p38 MAPK, and ERK constitute three major subfamilies of MAPK, and we have already reported the JNK- and p38 MAPK-mediated signaling to play a negative and a positive role, respectively, in LPS-induced IL-12 production from monocytes (7, 8). Three JNK genes have been identified (JNK1, JNK2, and JNK3), and their alternative splicing results in at least 10 isoforms of JNK (9). JNK1 and JNK2 are ubiquitously expressed, whereas JNK3 is expressed mainly in the brain, testis, and heart. Mice deficient in JNK1 and JNK2 have distinct defects in their T cell function (10, 11). However, the role of either JNK1 or JNK2 in IL-12 production from human APCs has not yet been previously investigated.

PI3Ks are a subfamily of lipid kinases that catalyze the addition of a phosphate molecule specifically to the 3-position of the inositol ring of phosphoinositides. PI3Ks have been divided into three classes: class I, class II, and class III. Class I PI3Ks are further divided into class IA and class IB. Class IA PI3Ks are heterodimers composed of a regulatory subunit (p85) and a catalytic subunit (p110) that are encoded by different genes, termed p110α, p110β, and p110δ (12). Fukao et al. (13) reported that gene targeting of the regulatory subunit of PI3K p85α results in augmentation of LPS-induced IL-12 production, suggesting that class IA PI3K negatively regulates LPS-induced IL-12 production in murine DCs. However, little is known in regard to how each of the class IA PI3K subtypes controls IL-12 production in APCs, because disruption of either p110α or p110β has been reported to cause death in the early stage of embryonic development (14).

In the present study, we investigated the roles of class I PI3K p110 isoforms, p110α and p110β, in LPS-induced IL-12 production in human monocyte-derived macrophages (MD-macrophages) and DCs (MD-DCs). The results indicated that PI3K 110β positively regulates IL-12 synthesis induced by LPS. Moreover, we examined the molecular mechanism that underlies the positive regulation of IL-12 production by PI3K p110β and found the activation of PI3K p110β by LPS to positively control the JNK1 activity, but not JNK2, thus leading to the regulation of IL-12 production.

PMA and LPS (from Escherichia coli, serotype 0111:B4) were obtained from Sigma-Aldrich. Wortmannin to specifically inhibit PI3K activity was obtained from Calbiochem and dissolved in DMSO (Sigma-Aldrich).

MD-macrophages and MD-DCs were generated as previously described (15, 16). PBMCs were separated by density gradient centrifugation from leukocyte concentrates obtained from healthy adult donors who signed informed consent to allow the use of their blood for research purposes, and monocytes were purified from PBMCs by positive selection with anti-CD14 magnetic beads (Miltenyi Biotec). For differentiation into macrophages, monocytes were cultured in RPMI 1640 supplemented with 10% FBS (Equitech-Bio) and 50 U/ml penicillin G sodium, 50 μg/ml streptomycin sulfate (Invitrogen) (complete medium) supplemented with 100 ng/ml human rM-CSF (R&D Systems) for 7 days. For differentiation into DCs, monocytes were cultured in complete medium supplemented with 10 ng/ml human rGM-CSF and 10 ng/ml human rIL-4 (R&D Systems) for 6 days. Every 2 days, 10 ng/ml rGM-CSF and 10 ng/ml rIL-4 were added until 6 days of culture, when nonadherent cells corresponding to the DC-enriched fraction were harvested and washed. The phenotypic distinctions between DC and macrophage as expressions of CD1a (DC positive, macrophage negative) and CD14 (the reverse) were confirmed using flow cytometry. For cytokine production, MD-macrophages and DCs were stimulated with 1 μg/ml and 100 ng/ml LPS, respectively, for 24 h, and were sedimented by centrifugation. The supernatants were stored at −20°C before cytokine quantitation.

We assessed the effect of inducing RNA interference on PI3K p110α, p110β, and JNKs using Silencer siRNA (Ambion): PI3K p110α (nos. 3052 and 144251), p110β (nos. 144255 and 144254), JNK1 (nos. 1320 and 143167), and JNK2 (nos. 1637 and 142317). Control siRNA is an siRNA sequence that will not cause the specific degradation of any cellular message (no. 1; Ambion). MD-macrophages and MD-DCs were transfected with 100 and 200 nM final concentrations, respectively, of siRNA using TransIT-TKO transfection reagent (Mirus Bio) according to the manufacturer’s instructions. After siRNA transfection, we determined PI3K p110α, p110β, JNK1, and JNK2 contents in whole cell lysates by Western blotting using anti-p110α (Cell Signaling Technology), p110β (S-19; Santa Cruz Biotechnology), JNK1 (FL; Santa Cruz Biotechnology), and JNK2 (Cell Signaling Technology), respectively, to confirm the silencing of these protein.

Western blotting proceeded as previously described (17). We used the primary Abs selectivity to recognize phosphorylated forms of Akt (Thr308), JNK (Thr183/Tyr185), p38 MAPK (Thr180/Tyr182), and ERK (Thr202/Tyr204) (all from Cell Signaling Technology).

Cytokine concentrations in the supernatants were determined using specific ELISA kits. Levels of IL-12p40 and IL-12p70 protein were measured using ELISA kits purchased from R&D Systems (DuoSet ELISA Development Kit) according to the manufacturer’s instructions.

We measured the cytokine mRNA expression after MD-macrophages and immature MD-DCs with 1 μg/ml LPS for 8 h and with 100 ng/ml LPS for 8 h, respectively. Total RNA was extracted and the RNAs were treated by DNase using an RNeasy Mini kit and RNase-Free DNase set (Qiagen) and conversed to cDNA using a high-capacity cDNA archive kit (PE Applied Biosystems). The transcription levels of IL-12p40, IL-12p35, IL-10, and IL-23p19 were detected using real-time PCR (7500 real-time PCR system; PE Applied Biosystems) with the TaqMan Universal PCR Master mix (PE Applied Biosystems). The primers and the probes of target genes were TaqMan Assay-on-Demand gene expression assays (PE Applied Biosystems): IL-12p40 (Hs00233688_1), IL-12p35 (Hs01073447_m1), IL-10 (Hs00174086_m1), and IL-23p19 (Hs00372324_m1), and, as a control, G3PDH (Hs99999905_m1). The threshold cycle (CT) was recorded for each sample to reflect the RNA expression level, and ΔCT (average CT of target genes − average CT of G3PDH) was used to reflect the relative expression levels of target genes. To determine effects of PI3K p110α, p110β, or JNKs siRNA on cytokine expression, ΔΔCT was calculated as follows: ΔΔCT = ΔCT PI3K p110α, p110β, or JNKs siRNA-treated cells − ΔCT control siRNA-treated cells. The value of 2−ΔΔCT was calculated to demonstrate the fold changes in the expression of the target genes in PI3K p110α, p110β, or JNKs siRNA-treated cells in comparison to that seen in the control siRNA-treated cells.

JNK1 was immunoprecipitated from equal amounts of cytoplasmic protein using agarose-linked rabbit polyclonal anti-JNK1 Ab (C-17; Santa Cruz Biotechnology) in 1× lysis buffer for 8 h at 4°C with rocking. The immunoprecipitates were washed three times with 1× lysis buffer and electrotransferred. Phosphorylation of JNK1 was measured by Western blotting using mouse polyclonal anti-phospho-JNK Ab (G-7; Santa Cruz Biotechnology). Phosphorylation of JNK2 was detected in the supernatants of the immunoprecipitates of JNK1 by Western blotting using anti-phospho-JNK Ab (G-7).

All values are expressed as means ± SEM of the indicated numbers of experiments. An ANOVA was used to analyze the statistical significance of differences. When statistical significance was reached, a post hoc analysis using the Bonferroni/Dunn test was performed. A p value <0.05/m (where m is the number of comparisons) was considered to be statistically significant according to the Bonferroni/Dunn method.

To evaluate roles of PI3K p110 isoforms in LPS-induced IL-12 production and signal transduction, we performed experiments using siRNA. Transfection of MD-macrophages for 48 h with 100 nM PI3K p110α (no. 3052) and p110β (no. 144255) siRNA attenuated the PI3K p110α (by 60%; Fig. 1,A) and p110β (by 54%; Fig. 1,B) content, respectively. Under these conditions, effects of PI3K p110 siRNAs on LPS signal transduction were assessed by detecting activation of Akt, which is situated downstream of PI3K. Fig. 1,E shows the stimulation of LPS-caused phosphorylation of Akt, as well as the reduction in PI3K p110α and p110β content by specific siRNA to decrease Akt phosphorylation at 30 min after adding LPS (by 53% and 52%, respectively). Similarly, the transfection of MD-DCs for 72 h with 200 nM PI3K p110β siRNA attenuated the PI3K p110β content (by 51%; Fig. 1,D) and LPS-induced Akt phosphorylation (by 44%; Fig. 1,F). On the other hand, PI3K p110α siRNA reduced the PI3K p110α content of MD-DCs (by 53%; Fig. 1,C), but it did not affect LPS-induced Akt phosphorylation of MD-DCs (Fig. 1 F). The expression patterns of class IA PI3K family members were similar in MD-macrophages and MD-DCs (supplemental Fig. 1).4 These results suggest that PI3K p110β is involved in LPS signal transduction in human macrophages and DCs.

FIGURE 1.

The effect of siRNA specific for PI3K p110 isoforms on LPS-induced Akt activation in human macrophages and DCs. A–D, Western blots of MD-macrophages (A and B) or MD-DCs (C and D) with control siRNA, p110α siRNA (no. 3052) or p110β siRNA (no. 144255) transfection for 48 (MD-macrophages) or 72 h (MD-DCs). Nitrocellulose filters were probed with anti-p110α Ab (A and C) or anti-p110β Ab (B and D) and with anti-p42 MAPK Ab to confirm equal protein loading. E and F, MD-macrophages (E) or MD-DCs (F) were transfected with siRNA before stimulation with LPS for 30 min. Thr308 phosphorylation of Akt was analyzed by Western blot. Total Akt analysis acts as a protein loading control.

FIGURE 1.

The effect of siRNA specific for PI3K p110 isoforms on LPS-induced Akt activation in human macrophages and DCs. A–D, Western blots of MD-macrophages (A and B) or MD-DCs (C and D) with control siRNA, p110α siRNA (no. 3052) or p110β siRNA (no. 144255) transfection for 48 (MD-macrophages) or 72 h (MD-DCs). Nitrocellulose filters were probed with anti-p110α Ab (A and C) or anti-p110β Ab (B and D) and with anti-p42 MAPK Ab to confirm equal protein loading. E and F, MD-macrophages (E) or MD-DCs (F) were transfected with siRNA before stimulation with LPS for 30 min. Thr308 phosphorylation of Akt was analyzed by Western blot. Total Akt analysis acts as a protein loading control.

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We next investigated effects of PI3K p110 siRNAs on LPS-induced IL-12 production. Knockdown of PI3K 110β by specific siRNA significantly (p < 0.05) reduced LPS-induced IL-12p40 production in comparison to the cells transfected with control siRNA in MD-macrophages (Fig. 2,A) and MD-DCs. (Fig. 3,A). LPS-induced IL-12 p70 production from MD-DCs also suppressed by PI3K 110β siRNA (Fig. 3,B). Although IL-12p70 protein was detected in control siRNA-transfected MD-macrophages stimulated by LPS (18.66 ± 3.93 pg/ml, n = 4), PI3K 110β siRNA decreased LPS-induced IL-12p70 production to undetectable levels (<7.8 pg/ml). Furthermore, we analyzed the mRNA expression by real-time PCR. LPS increased IL-12p40, p35, IL-10, and IL-23p19 mRNA expression >10,000-fold (data not shown). Consistent with the protein data, PI3K 110β siRNA significantly (p < 0.05) inhibited both IL-12p40 and IL-12p35 mRNA expression induced by LPS in MD-macrophages (Fig. 2,B) and MD-DCs (Fig. 3,C). Similarly, other constructs of PI3K p110β siRNA (no. 144254) reduced not only the PI3K p110β content (supplemental Fig. 2B) but also LPS-induced IL-12 mRNA expression from MD-macrophages (supplemental Fig. 2C) and MD-DCs (supplemental Fig. 2D). Although IL-10 is involved in a major regulatory mechanism that limits IL-12 production, PI3K 110β siRNA decreased LPS-induced IL-10 mRNA expression in MD-DCs (Fig. 3,D) or did not affected it in MD-macrophages (Fig. 2 C). These findings suggest that PI3K 110β plays a positive role in LPS-induced IL-12 production in human macrophages and DCs.

FIGURE 2.

PI3K p110β siRNA inhibited LPS-induced IL-12 production in MD-macrophages. MD-macrophages were transfected with siRNA for 48 h and then were stimulated with LPS. A, After 24 h, IL-12p40 protein in the culture supernatants was evaluated by ELISA. B and C, After 8 h, cells were collected and cDNA was generated from the RNA preparations. IL-12p40 (B, open bars), p35 (B, filled bars), and IL-10 (C) mRNA expressions were determined by real-time PCR quantification, and results are expressed as percentages of the control value (control siRNA-transfected cells). Values represent means ± SEM (n = 4–5). ∗, p < 0.05 in comparison to control siRNA-transfected cells.

FIGURE 2.

PI3K p110β siRNA inhibited LPS-induced IL-12 production in MD-macrophages. MD-macrophages were transfected with siRNA for 48 h and then were stimulated with LPS. A, After 24 h, IL-12p40 protein in the culture supernatants was evaluated by ELISA. B and C, After 8 h, cells were collected and cDNA was generated from the RNA preparations. IL-12p40 (B, open bars), p35 (B, filled bars), and IL-10 (C) mRNA expressions were determined by real-time PCR quantification, and results are expressed as percentages of the control value (control siRNA-transfected cells). Values represent means ± SEM (n = 4–5). ∗, p < 0.05 in comparison to control siRNA-transfected cells.

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FIGURE 3.

PI3K p110β siRNA inhibited LPS-induced IL-12 production in MD-DCs. MD-DCs were transfected with siRNA for 72 h and then were stimulated with LPS. A and B, After 24 h, IL-12p40 (A) and IL-12p70 (B) proteins in the culture supernatants were evaluated by ELISA. C–E, After 8 h, IL-12p40 (C, open bars), IL-12p35 (C, filled bars), IL-10 (D), and IL-23p19 (E) mRNA expressions were determined by real-time PCR quantification, and results are expressed as percentages of the control value (control siRNA-transfected cells). Values represent means ± SEM (n = 4–5). ∗, p < 0.05 in comparison to control siRNA-transfected cells.

FIGURE 3.

PI3K p110β siRNA inhibited LPS-induced IL-12 production in MD-DCs. MD-DCs were transfected with siRNA for 72 h and then were stimulated with LPS. A and B, After 24 h, IL-12p40 (A) and IL-12p70 (B) proteins in the culture supernatants were evaluated by ELISA. C–E, After 8 h, IL-12p40 (C, open bars), IL-12p35 (C, filled bars), IL-10 (D), and IL-23p19 (E) mRNA expressions were determined by real-time PCR quantification, and results are expressed as percentages of the control value (control siRNA-transfected cells). Values represent means ± SEM (n = 4–5). ∗, p < 0.05 in comparison to control siRNA-transfected cells.

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Knockdown of PI3K 110α by specific siRNA significantly (p < 0.05) inhibited IL-12p40 (Figs. 2,A and 3,A) and IL-12p70 (Fig. 3,B) protein production induced by LPS in MD-macrophages and DCs. Although PI3K 110α siRNA significantly (p < 0.05) suppressed IL-12p40 and IL-12p35 mRNA expression stimulated by LPS in MD-macrophages (Fig. 2,B), it did not affect either IL-12p35 mRNA expression or minimally affected IL-12p40 mRNA expression in MD-DCs (Fig. 3,C). This siRNA significantly (p < 0.05) reduced IL-23p19 mRNA expression by LPS in MD-DCs (Fig. 3 E). Additionally, these results correlated with the data using other constructs of PI3K p110α siRNA (no. 144251; supplemental Fig. 2). These observations propose that PI3K 110α positively regulates LPS-induced IL-12 production in MD-macrophages and DCs at the transcriptional and posttranscriptional level, respectively.

To analyze the mechanism by which PI3K 110β is involved in LPS-induced IL-12 production, we tested the influence of PI3K 110β siRNA on LPS-induced MAPK superfamily. We measured levels of phosphorylated JNK and p38 MAPK at 30 min after adding LPS, because LPS-induced activation of JNK and p38 MAPK reaches a maximum at 30 min in THP-1 cells (8). Fig. 4 demonstrates that PI3K p110β siRNA reduced LPS-induced JNK phosphorylation, whereas it did not affect LPS-induced p38 MAPK or ERK phosphorylation in MD-macrophages (Fig. 4,B) and MD-DCs (Fig. 4,D). A densitometric analysis showed that PI3K p110β siRNA caused an ∼50% reduction in the phosphorylation of both p46 and p54 JNK induced by LPS. On the other hand, PI3K p110α siRNA did not change LPS-induced JNK, p38 MAPK, or ERK phosphorylation in both cell types (Fig. 4, A and C). These data indicate that PI3K p110β positively regulates JNK activity without controlling p38 MAPK or ERK activity in LPS-mediated signal transduction of human macrophages and DCs.

FIGURE 4.

PI3K p110β siRNA inhibited LPS-induced JNK activation. MD-macrophages (A and B) or MD-DCs (C and D) were transfected with siRNA before stimulation with LPS. After 30 min, phosphorylation of JNK, p38 MAPK, and ERK was determined by Western blotting. Bottom panels, Western blots using Abs to PI3K p110α (A and C) and p110β (B and D), thus indicating a reduction in the contents of PI3K p110α and p110β, respectively, by specific siRNA. One representative finding of two or three experiments is shown.

FIGURE 4.

PI3K p110β siRNA inhibited LPS-induced JNK activation. MD-macrophages (A and B) or MD-DCs (C and D) were transfected with siRNA before stimulation with LPS. After 30 min, phosphorylation of JNK, p38 MAPK, and ERK was determined by Western blotting. Bottom panels, Western blots using Abs to PI3K p110α (A and C) and p110β (B and D), thus indicating a reduction in the contents of PI3K p110α and p110β, respectively, by specific siRNA. One representative finding of two or three experiments is shown.

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We previously reported that JNK negatively controls IL-12 production in PMA-treated THP-1 cells and human monocytes using SP600125, a selective inhibitor of JNK (8). However, this consideration is difficult to explain in that the positive regulation of IL-12 production by PI3K p110β is through JNK because the activation of JNK is positively controlled by PI3K p110β (Fig. 4). Therefore, we investigated the role of the JNK pathway in LPS-induced IL-12 production from MD-macrophages and DCs using JNK1 and JNK2 siRNA. JNK has apparent molecular masses of 46 (p46 JNK) and 54 (p54 JNK) kDa that are largely, but not exclusively, composed of the JNK1 and JNK2 isoforms, respectively (18). After transfection of MD-DCs with siRNA for 36 h, JNK1 or JNK2 contents were determined by Western blotting of whole cell lysates using anti-JNK1 Ab (FL) that also recognizes JNK2. Fig. 5,A demonstrates that JNK1 siRNA (no. 1320) resulted in 53% and 52% reduction of the endogeneous level of p46 JNK and p54 JNK, respectively. JNK2 siRNA (no. 1637) resulted in a 52% reduction of the endogeneous level of p54 JNK, which was different from p54 JNK attenuated by JNK1 siRNA, whereas JNK2 siRNA did not change cellular amounts of p46 JNK (Fig. 5 B). These results suggest that JNK1 is composed of p46 and p54 JNK, while JNK2 is composed of only p54 JNK in MD-DCs.

FIGURE 5.

JNKs siRNA transfection of MD-DCs and MD-macrophages decreased LPS-induced IL-12 production. A and B, Western blots of MD-DCs with control siRNA or JNK1 siRNA (no. 1320; A) or JNK2 siRNA (no. 1637; B) transfection for 36 h. Nitrocellulose filters were probed with anti-JNK1 Ab (FL) that also recognizes JNK2. NF-κB p65 analysis acts as protein loading control. C–G, MD-DCs (C–E) or MD-macrophages (F and G) were transfected with siRNA and then were stimulated with LPS. C, After 24 h, IL-12p40 protein in culture supernatants was evaluated by ELISA. D–G, After 8 h, IL-12p40 (D and F, open bars), p35 (D and F, filled bars), and IL-10 (E and G) mRNA expressions were determined by real-time PCR, and results are expressed as percentages of the control value. Values represent means ± SEM (n = 3–4). ∗, p < 0.05 in comparison to control siRNA-transfected cells.

FIGURE 5.

JNKs siRNA transfection of MD-DCs and MD-macrophages decreased LPS-induced IL-12 production. A and B, Western blots of MD-DCs with control siRNA or JNK1 siRNA (no. 1320; A) or JNK2 siRNA (no. 1637; B) transfection for 36 h. Nitrocellulose filters were probed with anti-JNK1 Ab (FL) that also recognizes JNK2. NF-κB p65 analysis acts as protein loading control. C–G, MD-DCs (C–E) or MD-macrophages (F and G) were transfected with siRNA and then were stimulated with LPS. C, After 24 h, IL-12p40 protein in culture supernatants was evaluated by ELISA. D–G, After 8 h, IL-12p40 (D and F, open bars), p35 (D and F, filled bars), and IL-10 (E and G) mRNA expressions were determined by real-time PCR, and results are expressed as percentages of the control value. Values represent means ± SEM (n = 3–4). ∗, p < 0.05 in comparison to control siRNA-transfected cells.

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We next examined the susceptibility to LPS-induced IL-12 production under these conditions. Fig. 5,C shows that reduction in JNK1 or JNK2 content by siRNA significantly (p < 0.05) suppressed LPS-induced IL-12p40 production from MD-DCs. Additionally, simultaneous transfection with JNK1 and JNK2 siRNA significantly (p < 0.05) reduced LPS-induced IL-12p40 production (Fig. 5,C). At the mRNA level, JNK1 or JNK2 siRNA significantly (p < 0.05) suppressed both IL-12p40 and IL-12p35 mRNA expression by LPS in MD-DCs (Fig. 5,D) and MD-macrophages (Fig. 5,F). Similarly, the reduction in JNK1 (supplemental Fig. 3A) and JNK2 (supplemental Fig. 3B) content by other constructs of siRNA against JNK1 (no. 143167) and JNK2 (no. 142317), respectively, decreased LPS-induced IL-12 expression from MD-DCs (supplemental Fig. 3C). LPS-induced IL-10 expression was also decreased by JNK1 or JNK2 siRNA (Fig. 5, E and G). These data indicate that JNK1 and JNK2 positively control LPS-induced IL-12 production in both MD-macrophages and MD-DCs.

Next, to clarify the relationship between PI3K p110β and JNK isoforms in LPS signal transduction, we evaluated the influence of PI3K p110β siRNA on LPS-induced JNK1 and JNK2 phosphorylation. We separately detected phosphorylated JNK1 by immunoprecipitation using specific Abs against JNK1 (C-17). The immunoprecipitates with anti-JNK1 Ab included both phosphorylated p46 and p54 JNK, which were enhanced by LPS and reduced by JNK1 siRNA, but not by JNK2 siRNA (data not shown), thus indicating that these phosphorylated forms of JNK are phosphorylated JNK1. Transfection of MD-DCs (Fig. 6,A, top) or MD-macrophages (Fig. 6,B, top) with PI3K 110β siRNA decreased the levels of JNK1 phosphorylation enhanced by LPS. Conversely, the supernatants of the immunoprecipitates of JNK1 included only phosphorylated p54 JNK, which were enhanced by LPS and reduced by JNK2 siRNA (data not shown), thus indicating that these phosphorylated forms of JNK are phosphorylated JNK2. PI3K 110β siRNA did not change LPS-induced JNK2 phosphorylation (Fig. 6, middle panels). These findings indicate that the positive regulatory effect of PI3K 110β on LPS-induced JNK activation is dependent on JNK1 phosphorylation, but is independent of JNK2 phosphorylation, in human macrophages and DCs.

FIGURE 6.

PI3K p110β siRNA inhibited LPS-induced JNK1 phosphorylation but not JNK2 phosphorylation. Transfection of MD-DCs (A) or MD-macrophages (B) with siRNAs before stimulation with LPS for 30 min. Aliquots of lysates were immunoprecipitated with anti-JNK1 Ab (C-17). The immunoprecipitates (upper panel) or supernatants (middle panel) were collected and subjected to Western blotting with anti-phospho-JNK Ab. Bottom panels, Western blots using anti-Rac Ab to confirm equal protein loading. One representative finding of two experiments is shown.

FIGURE 6.

PI3K p110β siRNA inhibited LPS-induced JNK1 phosphorylation but not JNK2 phosphorylation. Transfection of MD-DCs (A) or MD-macrophages (B) with siRNAs before stimulation with LPS for 30 min. Aliquots of lysates were immunoprecipitated with anti-JNK1 Ab (C-17). The immunoprecipitates (upper panel) or supernatants (middle panel) were collected and subjected to Western blotting with anti-phospho-JNK Ab. Bottom panels, Western blots using anti-Rac Ab to confirm equal protein loading. One representative finding of two experiments is shown.

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Previous reports have shown wortmannin, a global PI3K inhibitor, to augment LPS-induced IL-12 production and p38 MAPK activation in mouse DCs (13). Finally, we examined the effect of wortmannin on LPS-induced IL-12 production and MAPK activation in MD-DCs. Fig. 7,A demonstrates that wortmannin significantly (p < 0.05) enhanced LPS-induced both IL-12p40 and IL-12p35 mRNA expression. These results were consistent with those in mouse DCs, but they contrasted with those using PI3K p110β siRNA in human macrophages and DCs. Additionally, wortmannin increased JNK and p38 phosphorylation stimulated by LPS, while it did not affect ERK phosphorylation in MD-DCs (Fig. 7 B).

FIGURE 7.

Wortmannin enhanced LPS-induced IL-12 production and MAPK activation. MD-DCs were cultured with 0.1% DMSO (control vehicle) or wortmannin for 30 min before stimulation with LPS. A, After 8 h, IL-12p40 (open bars) and IL-12p35 (filled bars) mRNA expressions were determined by real-time PCR quantification. Values represent means ± SEM (n = 3). ∗, p < 0.05 in comparison to LPS-stimulated cells. B, After 30 min, phosphorylation of JNK, p38 MAPK, and ERK were determined by Western blot. One representative finding of two experiments is shown.

FIGURE 7.

Wortmannin enhanced LPS-induced IL-12 production and MAPK activation. MD-DCs were cultured with 0.1% DMSO (control vehicle) or wortmannin for 30 min before stimulation with LPS. A, After 8 h, IL-12p40 (open bars) and IL-12p35 (filled bars) mRNA expressions were determined by real-time PCR quantification. Values represent means ± SEM (n = 3). ∗, p < 0.05 in comparison to LPS-stimulated cells. B, After 30 min, phosphorylation of JNK, p38 MAPK, and ERK were determined by Western blot. One representative finding of two experiments is shown.

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Since targeted disruption of either PI3K p110α or p110β has been reported to be embryonically lethal (15), it may not be possible to assign a function to these PI3K isoforms using conventional knockout mice approaches. We tried in the present study to perform knockdown of PI3K p110α or p110β in human APCs using the technique of RNA interference, and thus demonstrated a reduction of the PI3K p110α and p110β contents of human APCs by PI3K p110α and p110β siRNA, respectively (Fig. 1). Recently, studies using mouse macrophage-like RAW 264.7 cells, which are deficient in PI3K p110β, suggested that PI3K p110β negatively regulates IL-12 production in response to LPS (19). Conversely, we herein demonstrated that a disruption of PI3K p110β by specific siRNA caused a significant reduction of LPS-induced IL-12 protein production and mRNA expression from MD-macrophages and MD-DCs (Figs. 2 and 3), thus indicating that PI3K p110β positively regulates IL-12 production in response to LPS in human macrophages and DCs. IL-10 is involved in a major negative regulatory mechanism of IL-12 production (20). We consider that IL-10 is not involved in the PI3K p110β-mediated positive regulation of IL-12 expression induced by LPS because LPS-induced IL-10 expression is also positively controlled in MD-DCs (Fig. 3) or is not controlled in MD-macrophages (Fig. 2) by PI3K p110β.

Additonally, we showed that PI3K 110α siRNA significantly inhibited LPS-induced IL-12 protein production and mRNA expression from MD-macrophages (Fig. 2). These results are consistent with the recent reports by Lee et al. (21). They demonstrated that a deficiency in PI3K p110α in THP-1 monocytic cells suppressed IL-12p40 protein production and mRNA expression induced by LPS (21). On the other hand, PI3K p110α siRNA significantly suppressed IL-12 protein production, while it had little effect on IL-12 mRNA expression in MD-DCs (Fig. 3). The level of reduction of PI3K p110α content by siRNA was moderate in MD-DCs (by ∼50%; Fig. 1,C). Therefore, the effect of a more robust inhibition of PI3K p110α on IL-12 mRNA expression remains unknown. However, IL-23p19 expression in MD-DCs was decreased by the addition of PI3K p110α siRNA (Fig. 3), thus suggesting that the reduction in PI3K p110α content by this siRNA is sufficient to affect LPS-induced cytokine expression. We consider that PI3K p110α plays a positive role in LPS-induced IL-12 production through the transcriptional and posttranscriptional regulation in MD-macrophages and MD-DCs, respectively.

Previous studies in mice null for PI3K p85α revealed the augmentative effect on LPS-induced IL-12 production in murine DCs (13). The p85α knockout mice also exhibited increases in p38 MAPK activity, suggesting that class IA PI3K negatively controls LPS-induced IL-12 production through p38 MAPK activation. In the present study, we found that wortmannin, a global PI3K inhibitor, enhanced both IL-12p40 and IL-12p35 expression from MD-DCs induced by LPS (Fig. 7). Additionally, wortmannin augmented LPS-induced p38 MAPK activity (Fig. 7). These results correlate with the data from DCs in p85α knockout mice (13). We therefore consider that wortmannin affects activities of multiple PI3K subtypes, such as classes IA, IB, II, and III, resulting in the negative regulation of IL-12 production. Conversely, knockdown of PI3K p110β by specific siRNA did not affect LPS-induced p38 MAPK activity, but instead inhibited LPS-induced IL-12 production and JNK activity (Figs. 2–4), which contrasted with our findings on wortmannin-treated cells (Fig. 7). We think that the technique of RNA interference therefore helps to elucidate the precise roles (positive or negative) of individual subtypes of PI3K, and the present data confirm that PI3K p110β plays a positive role in IL-12 production induced by LPS in human macrophages and DCs. Additionally, we consider that the differential regulation mechanisms by PI3K p110β between human APCs and RAW 264.7 cells in IL-12 production are due to differences in species because the same results of PI3K p110β siRNA on LPS-induced IL-12 production and JNK activation were obtained with MD-macrophages and MD-DCs (Figs. 2–4) and PI3K p110β siRNA did not affect LPS-induced JNK activation in RAW 264.7 cells (19).

We have previously demonstrated the blockade of JNK activation by SP600125, a global inhibitor of JNK, to increase LPS-induced IL-12 production in PMA-treated THP-1 cells (8). In contrast, the present study discovered a JNK1- and a JNK2-mediated positive feedback mechanism of LPS-induced IL-12 production in MD-macrophages and DCs, because JNK1 and JNK2 siRNA inhibited LPS-induced IL-12p40 production and both p40 and p35 expression (Fig. 5). These observations are consistent with a previous report that SP600125 inhibited the production of IL-12 by LPS in MD-DCs (22). Interestingly, knockdown of PI3K p110β induced a decrease in phosphorylation of JNK in LPS-stimulated MD-macrophages and DCs (Fig. 3). Moreover, PI3K p110β siRNA decreased LPS-induced phosphorylation of JNK1 but did not affect JNK2 phosphorylation (Fig. 6), thus confirming that the positive regulation of IL-12 production by PI3K p110β is mediated by JNK1 in human macrophages and DCs. Conversely, wortmannin augmented LPS-induced not only IL-12 production but also JNK phosphorylation in MD-DCs (Fig. 7). These observations suggest that the JNK pathway contributes to the regulation of IL-12 production by the PI3K family as well as by the PI3K p110β subtype in human macrophages and DCs.

In summary, we report that PI3K p110β plays a positive role in LPS-induced IL-12 production through JNK1 activation in human macrophages and DCs. Additionally, we show that PI3K p110α positively controls LPS-induced IL-12 production through the different mechanisms of MD-macrophages and MD-DCs. We first clarified prominent and selective roles for PI3K 110β in regulating IL-12 production induced by LPS and defined the molecular mechanism underlying the regulation of IL-12 production by PI3K p110β in human APCs. These findings have been masked by conventional approaches, such as the use of global PI3K inhibitors and knockout mice. IL-12 plays a key role in the generation of Th1 vs Th2 responses. Therefore, identifying new signaling cascades for controlling IL-12 production may have important implications for the elucidation of the mechanism of human disease states involving dysregulation of the immune response, such as malignant tumors, infections, and allergic and autoimmune diseases.

The authors thank Noriaki Sunaga, Kyoichi Kaira, and Noriko Yanagitani (Gunma University) for valuable discussion.

The authors have no financial conflicts of interest.

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 in part by Grant 19790680 from the Ministry of Education, Culture, Sports, Science and Technology, Japan (to M.U.).

3

Abbreviations used in this paper: DC, dendritic cell; MD-DC, monocyte-derived DC; MD-macrophage, monocyte-derived macrophage; siRNA, small interfering RNA.

4

The online version of this article contains supplemental material.

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