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The Journal of Immunology, 2004, 172: 2147-2154.
Copyright © 2004 by The American Association of Immunologists

Novel Regulatory Mechanisms of CD40-Induced Prostanoid Synthesis by IL-4 and IL-10 in Human Monocytes

Yasushi Inoue, Takeshi Otsuka1, Hiroaki Niiro, Shuji Nagano, Yojirou Arinobu, Eiichi Ogami, Mitsuteru Akahoshi, Katsuhisa Miyake, Ichiro Ninomiya, Sakiko Shimizu, Hitoshi Nakashima and Mine Harada

Department of Medicine and Biosystemic Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Interleukins IL-4 and IL-10 are considered to be central regulators for the limitation and eventual termination of inflammatory responses in vivo, based on their potent anti-inflammatory effects toward LPS-stimulated monocytes/macrophages and neutrophils. However, their role in T cell-dependent inflammatory responses has not been fully elucidated. In this study, we investigated the effects of both cytokines on the production of PGE2, a key molecule of various inflammatory conditions, in CD40-stimulated human peripheral blood monocytes. CD40 ligation of monocytes induced the synthesis of a significant amount of PGE2 via inducible expression of the cyclooxygenase (COX)-2 gene. Both IL-10 and IL-4 significantly inhibited PGE2 production and COX-2 expression in CD40-stimulated monocytes. Using specific inhibitors for extracellular signal-related kinase (ERK) and p38 mitogen-activated protein kinase (MAPK), we found that both kinase pathways are involved in CD40-induced COX-2 expression. CD40 ligation also resulted in the activation of NF-{kappa}B. Additional experiments exhibited that CD40 clearly induced the activation of the upstream kinases MAPK/ERK kinase 1/2, MAPK kinase 3/6, and I-{kappa}B in monocytes. IL-10 significantly inhibited CD40-induced activation of the ERK, p38 MAPK, and NF-{kappa}B pathways; however, inhibition by IL-4 was limited to the ERK pathway in monocytes. Neither IL-10 nor IL-4 affected the recruitment of TNFR-associated factors 2 and 3 to CD40 in monocytes. Collectively, IL-10 and IL-4 use novel regulatory mechanisms for CD40-induced prostanoid synthesis in monocytes, thus suggesting a potential role for these cytokines in regulating T cell-induced inflammatory responses, including autoimmune diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CD40 is a member of the TNFR family and is expressed on various kinds of cells in vivo (1, 2, 3). Activated T cells are a major source of CD40 ligand (CD40L),2 and the significance of the CD40L-CD40 interaction to B cells is clearly underscored by the observation that disruption of the CD40L gene results in no germinal center formation (4, 5, 6). Intriguingly, in addition to this phenotype, these mice have defective macrophage effector functions (7), suggesting a pivotal role for the CD40L-CD40 system in T cell-dependent macrophage activation in vivo. For example, in rheumatoid arthritis (RA) patients, synovial macrophages express functional CD40, and functional CD40L is expressed on the surrounding synovial fluid T cells (8, 9). CD40L expression on CD4-positive T cells in RA patients correlates with the disease activity (10), and the blockade of this pathway causes alleviation of arthritis in mice (11). Although the outcome of CD40 ligation in B cells has been studied extensively, observations in monocytes have been relatively limited to date. This is probably due to the fact that monocytes, in general, express lower levels of CD40 on their cell surfaces compared with B cells. Thus, an experimental system with high sensitivity may be required to study the biological effects of CD40 ligation on monocytes. It has previously been shown that CD40 ligation of monocytes results in the secretion of proinflammatory cytokines and chemokines such as IL-1, IL-6, IL-8, IL-10, IL-12, TNF-{alpha}, and macrophage-inflammatory protein-1a (1, 2, 6). In addition to these factors, monocytes produce a number of other proinflammatory mediators, which are crucial for the progression of inflammation.

PGs are lipid mediators that play a pivotal role in various physiological processes. In addition, these mediators are believed to be involved in a number of pathological inflammatory conditions such as arthritis (12). Cyclooxygenase (COX) is a key regulatory enzyme catalyzing the limiting step of prostanoid synthesis. The COX enzyme exists as at least two isoforms: COX-1, a constitutively expressed enzyme that is expressed in a wide spectrum of tissues (13), and COX-2, an inducible enzyme that is up-regulated in response to extracellular stimuli, including LPS and cytokines (14, 15, 16). Many investigators, including us, have previously shown that LPS stimulation of monocytes results in the production of a large amount of PGE2, and that this process depends on the inducible expression of the COX-2 enzyme (15, 16, 17). We also took this observation one step further and showed that two mitogen-activated protein kinases (MAPKs), namely extracellular signal-related kinase (ERK) and p38 MAPK, are required for LPS-induced COX-2 expression in monocytes (18). In contrast, little is known to date about T cell (cell to cell contact)-dependent prostanoid synthesis in monocytes.

In response to the excessive ongoing inflammatory responses, the body also triggers anti-inflammatory mechanisms to limit unfavorable outcomes in many ways. In this regard, IL-10 and IL-4, originally described as soluble factors preferentially synthesized in Th2 cells (19, 20), appear to be prototypic anti-inflammatory cytokines that limit inflammatory responses. Many investigators, including us, have previously shown that both cytokines are capable of inhibiting the expression of a wide range of proinflammatory cytokines in LPS-stimulated monocytes/macrophages (17, 18, 21). We have previously demonstrated that IL-10 and IL-4 significantly blocked LPS-induced PGE2 production via down-regulation of COX-2 induction in monocytes (17, 18, 22). The significant role of IL-10 in the regulation of prostanoids in vivo was further shown by other authors using IL-10-deficient mice (23). However, these studies were mostly performed using LPS-stimulated cells. Therefore, we cannot simply deduce from these reports that IL-10 and IL-4 are central regulators under different inflammatory conditions in vivo. Given the previous observations that IL-10 and IL-4 cooperatively alleviate the progression of arthritis models in mice (24, 25, 26), both cytokines might exert a potent regulatory effect on the T cell-dependent inflammatory responses.

In the present study, we first investigated prostanoid synthesis in monocytes following CD40 ligation, and its regulation by IL-10 and IL-4. Furthermore, we took these observations one step further and investigated the underlying molecular mechanisms.


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

LPS was purchased from Difco (Detroit, MI). NS-398, a specific inhibitor of COX-2, was purchased from Alexis (San Diego, CA). PD98059, a specific inhibitor of MAPK/ERK kinase 1 (MEK1), was purchased from Cell Signaling Technology (Beverly, MA). SB203580, a specific inhibitor of p38 MAPK, was kindly provided by SmithKline Beecham Pharmaceuticals (King of Prussia, PA). Human rIL-10 was kindly provided by K. Moore (DNAX, Palo Alto, CA). Human rIL-4 was kindly provided by Schering-Plough (Bloomfield, NJ). Soluble CD40L (sCD40L) was obtained from Alexis. Rabbit polyclonal Abs against control or phospho-specific ERK1/2, MEK 1/2, p38 MAPK, MAPK kinase 3/6 (MKK3/6), and I-{kappa}B were purchased from Cell Signaling Technology. Curcumin was obtained from Sigma-Aldrich (St. Louis, MO). An anti-COX-2 mAb was purchased from Transduction Laboratories (Lexington, KY). An anti-human CD40 mAb (BE-1) was purchased from Ancell (Minneapolis, MN). Anti-human TNFR-associated factor 2 (TRAF2) (H-249) and anti-human TRAF3 polyclonal Abs (H-122) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Isolation and culture of human monocytes

Human monocytes were isolated and cultured, as previously described (17, 18, 27). Buffy coats from healthy donors were provided by the Fukuoka Red Cross Blood Center (Fukuoka, Japan). Briefly, PBMCs were separated by a Ficoll-Hypaque method. Subsequently, monocytes were isolated from PBMCs by immunologically positive selection using anti-CD14 immunomagnetic beads (Miltenyi Biotec, Auburn, CA). The isolated cells were >96% nonspecific esterase positive and >95% viable, as determined by trypan blue dye exclusion. Then monocytes were cultured in RPMI 1640 medium with 10% FBS (Life Technologies) at 37°C in a humidified atmosphere with 5% CO2 in air. In all experiments, monocytes were >90% viable after treatment with CD40 in combination with either cytokines or inhibitors.

Enzyme immunoassay (EIA) for PGE2, thromboxane B2 (TXB2), and leukotriene B4 (LTB4), and ELISA for TNF-{alpha}

Determinations of PGE2, TXB2, LTB4, and TNF-{alpha} levels in the culture supernatants of monocytes (3 x 105 cells) were conducted using commercially available kits for PGE2, TXB2, LTB4 EIA (Cayman Chemicals, Ann Arbor, MI), and TNF-{alpha} ELISA (BioSource International, Camarillo, CA), respectively. The culture supernatants were collected at 24 h after stimulation, in which we observed the maximal prostanoid production in monocytes (data not shown).

Analysis of COX-2 protein expression

Monocytes (4 x 106 cells) were treated at 37°C with or without PD98059, SB203580, curcumin, IL-4, or IL-10 for 2 h before stimulation with 100 ng/ml sCD40L. The cells were collected at 18 h after stimulation, in which we observed the maximal COX-2 expression in monocytes (data not shown). The cell pellets were lysed in 50 µl of SDS sample buffer (62.5 mM of Tris-HCl, pH 6.8, 10% glycerol, 2% (w/v) SDS, 50 mM of DTT, 0.1% (w/v) BPB) at 25°C. The cell lysates were then sonicated for 15 s and centrifuged at 15,000 x g for 15 min. The resulting cell lysates (2 x 106 cells/lane) were separated by 12.5% SDS-PAGE and transferred onto a polyvinylidene difluoride membrane. The membrane was blocked with PBS containing 5% skim milk and 0.1% Tween 20, and then incubated with 0.25 µg/ml mouse anti-COX-2 mAb at 25°C for 2 h. The membrane was subsequently incubated with a peroxidase-linked species-specific F(ab')2 from anti-mouse sheep Ig (1/1000 dilution), and analyzed using the Amersham ECL system (Amersham, Arlington Heights, IL).

Analysis of MAPK phosphorylation

Monocytes (4 x 106 cells) were treated at 37°C with IL-4 or IL-10 for 2 h before stimulation with 100 ng/ml sCD40L. After various time intervals, the cell pellets were lysed on ice for 30 min with 40 µl of lysis buffer (1% Triton X-100, 50 mM of HEPES, pH 7.4, 150 mM of NaCl, 30 mM of sodium pyrophosphate, 50 mM of NaF, 1 mM of Na3VO4, 1 mM of PMSF). After centrifugation, supernatants were subsequently mixed with an equal amount of 2x SDS sample buffer. The resulting cell lysates (2 x 106 cells/lane) were subjected to Western blot analysis using rabbit polyclonal control or phospho-specific MAPK (ERK1/2, p38 MAPK, MEK1/2, MKK3/6) Ab (1/1000 dilution). The membrane was subsequently incubated with a peroxidase-linked species-specific F(ab')2 from anti-rabbit donkey Ig (1/2000 dilution), and analyzed using the Amersham ECL system.

Immunoprecipitation

The procedure of immunoprecipitation was previously described (27). Monocytes (5 x 106 cells/lane) were treated with IL-4 or IL-10 for 2 h before stimulation with sCD40L for 15 min. The cell pellets were lysed in the lysis buffer (1% Triton X-100, 50 mM of Tris-HCl, pH 7.5, 150 mM of NaCl, 5 mM of EDTA, 10 mM of NaF, 2 mM of Na3VO4, 2 mM of PMSF, 0.05 mg/ml aprotinin, 2 mM of N-ethylmaleimide), and cell lysates were then immunoprecipitated with 30 µl of protein G plus protein A-agarose preconjugated (Oncogene Research Products, San Diego, CA) and 2 µg of anti-human CD40 mAb overnight at 4°C. The beads were washed four times and boiled with 1x SDS sample buffer. Precipitated proteins from 5 x 106 monocytes were subjected to Western blot analysis using anti-TRAF2 Ab, anti-TRAF3 Ab, or anti-human CD40 mAb (1/1000 dilution). The membrane was subsequently incubated with a peroxidase-linked species-specific F(ab')2 from anti-rabbit donkey Ig (1/2000 dilution) or anti-mouse donkey Ig (1/2000 dilution), and analyzed using the Amersham ECL system.

Extraction of nuclear proteins and EMSA

The procedures for extraction of nuclear proteins and EMSA have previously been described (27). Briefly, human monocytes (5 x 106 cells) were treated at 37°C with or without IL-4 or IL-10 for 2 h before stimulation by 100 ng/ml sCD40L and then lysed to extract nuclear proteins. Nuclear extracts (2 µg) were mixed with binding buffer (20 mM of HEPES-NaOH, pH 7.9, 2 mM of EDTA, 100 mM of NaCl, 10% glycerol, 0.2% Nonidet P-40), poly(dI-dC), and a 32P-labeled oligonucleotide probe. The mixtures were incubated at room temperature for 30 min. The reaction mixtures were loaded onto a 4% polyacrylamide gel and electrophoresed with 0.25x Tris base, boric acid, EDTA (TBE) running buffer. The gel was then dried onto Whatman 3MM paper (Whatman, Clifton, NJ). The DNA/protein complexes were visualized by autoradiography. The sequence of the oligonucleotide probe used to detect the DNA-binding activities of NF-{kappa}B was: NF-{kappa}B, 5'-GCT CAT GGG TTT CTC CAC CAA G-3' (28) (the NF-{kappa}B binding site is underlined).

Isolation of total RNA from human monocytes and RT-PCR assay

Monocytes (5 x 105 cells) obtained from informed healthy volunteers were treated at 37°C with or without IL-4 or IL-10 for 2 h before stimulation by 100 ng/ml sCD40L. After stimulation for 6 h, total RNA from 5 x 105 monocytes was extracted by using the guanidinium isothiocyanate/phenol extraction method (Isogen; Nippon Gene, Tokyo, Japan), quantified by measuring the absorbance at 260 nm, and stored at -80°C until further analysis. RNA (1 µg) preparation was used for first-strand cDNA synthesis (RNA PCR kit; PE Biosystems, Urayasu, Japan). The reaction was performed according to the manufacturer’s recommendations. Briefly, the reverse transcriptase reaction was conducted at 42°C for 15 min and the reaction was terminated by heating at 99°C for 5 min, followed by rapid chilling on ice.

Primer design for competitive PCR assay and generation of DNA competitor

The specific primers used are as follows: for COX-2 (29), 5'-TTC AAA TGA GAT TGT GGG AAA ATT GCT-3' (forward), 5'-AGA TCA TCT CTG CCT GAG TAT CTT-3' (reverse), predicted size of fragment 301 bp; for GAPDH (30), 5'-CCA TGG AGA AGG CTG GGG- 3' (forward), 5'-CAA AGT TGT CAT GGA TGA CC-3' (reverse), predicted size of fragment 195 bp. Composite primers were engineered to contain sequences that amplified the cDNA fragment with the gene-specific primer sequences flanking their 5' ends. The DNA competitors were designed so that the PCR product from the cDNA could be separated from that of its competitor, and were generated using reagents supplied in a commercial kit (Competitive DNA Construction Kit; Takara Shuzo, Otsu, Japan). Briefly, a 30-cycle PCR was conducted on {lambda}DNA using the corresponding primers for the target sequence. Concentrations of the DNA competitors were then measured using a spectrophotometer and adjusted so that each stock concentration was 2 x 1011 copies/µl.

Competitive PCR assay

A series of competitive PCR was set up using 3- or 10-fold dilutions of the DNA competitors with a constant amount of the first-strand cDNA and performed in 20-µl PCR mixtures. PCR was performed using a program of 30 cycles (GAPDH) or 35 cycles (COX-2) of 94°C for 30 s, 60°C for 30 s, and 72°C for 1 min, with a final 10-min extension at 72°C. The amplified products were subjected to electrophoresis on a 3% agarose gel.

Quantitative real-time PCR and Taqman probes and primers

The primers and probes used in the real-time PCR of COX-1, COX-2, and GAPDH mRNA were designed using the Primer Express program and synthesized (Applied Biosystems, Tokyo, Japan) as follows: for COX-1, 5'-CAGAGACCCAACAGCAGTGATG-3' (forward), 5'-GCTGCCTACAGAGGTCCTGAGA-3' (reverse), and 5'-FAM-TTCAGCAAGCAGCCCTCCACTCCA-TAMRA-3'; for COX-2, 5'-GAATCATTCACCAGGCAAATTG-3' (forward), 5'-CATAAAGCGTTTGCGGTACTCA-3' (reverse), and 5'-FAM-TTCCTACCACCAGCAACCCTGCCA-TAMRA-3'; for GAPDH, 5'-CCACATCGCTCAGACACCAT-3' (forward), 5'-CCAGGCGCCCAATACG-3' (reverse), 5'-FAM-AAGGTGAAGGTCGGAGTCAACGGATTTG-TAMRA-3'. Monocytes (5 x 105 cells) obtained from informed healthy volunteers were treated at 37°C with or without IL-4 or IL-10 for 2 h before stimulation by 100 ng/ml sCD40L. After stimulation for 6 h, total RNA was isolated and cDNA (1 µg/20 µl) was synthesized, as described above. Each PCR system (50 µl) contained 1 µl of cDNA, 25 µl of common 2x TaqMan Universal PCR Master Mix (Applied Biosystems), 10 µM of gene-specific primers, and 5 µM of TaqMan probe. TaqMan-PCRs were performed according to the manufacturer’s instructions (Applied Biosystems). Sequence-specific amplification was detected with an increased fluorescent signal of 5-[(N-(3'-diphenylphosphinyl-4'-methoxycarbonyl) phenylcarbonyl) aminoacetamido] fluorescein (FAM) during the amplification cycles using an ABI prism 7700 sequence detection system (PerkinElmer, Yokohama, Japan). Relative expression levels were calculated by the relative standard curve method, as outlined in the manufacturer’s technical bulletin. A standard curve was generated using the fluorescent data from the 5-fold serial dilutions of total RNA of the highest expression sample. GAPDH primers served as an internal control to ensure the efficiency of the reverse transcription and the amount of RNA used in each reaction, and the concentrations of GAPDH cDNA in each sample were used to normalize the gene-specific concentrations.

Densitometric and statistical analysis

Densitometric analysis of the signal intensities was conducted using the Scion Image {beta} 4.0.2 program (Scion, Frederick, MD). Student’s t test was used to compare control and experimental groups. Values of p > 0.05 were considered not significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
First, we tested whether CD40 ligation could induce PGE2 production by monocytes. We found that unstimulated monocytes constitutively produced a small amount of PGE2, while CD40-stimulated monocytes produced a dramatically larger amount of PGE2, which is known to be synthesized by COX-2 (Fig. 1A). Although neither IL-4 nor IL-10 affected the basal PGE2 production by monocytes, both cytokines showed the similar significant (p < 0.01) inhibition of CD40-induced PGE2 production by monocytes, as illustrated in Fig. 1A. In addition, CD40 induced the generation of TXB2, the other COX-2-dependent product, which was again inhibited by IL-4 and IL-10 (Fig. 1B). In contrast, the production of LTB4, a COX-2-independent product, was not affected by CD40, IL-4, or IL-10 (Fig. 1C).



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  		FIGURE 1. Effects of IL-4 and IL-10 on PGE2 production in CD40-stimulated monocytes. Human monocytes (3 x 105 cells/well) were treated for 2 h with the indicated concentrations of IL-4 or IL-10, and then cultured for 24 h with or without 100 ng/ml sCD40L. EIA for PGE2 (A), TXB2 (B), and LTB4 (C) were performed, as described in Materials and Methods. Results are expressed as the mean ± SD of triplicate cultures. Similar results were obtained in three separate experiments. **, p < 0.01 (compared with sCD40L only).

 
Next, we examined whether or not CD40-induced PGE2 production depended on COX-2, a crucial enzyme catalyzing a limiting step of prostanoid synthesis. As shown in Fig. 2, NS-398, a specific COX-2 inhibitor, significantly (p < 0.05) blocked CD40-induced, but not constitutive (data not shown), PGE2 production by monocytes in a dose-dependent manner, suggesting that the COX-2 enzyme is actively involved in this process.



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  		FIGURE 2. COX-2 enzyme is involved in CD40-induced PGE2 production by monocytes. Human monocytes (3 x 105 cells/well) were treated for 1 h with the indicated concentrations of NS-398, and then cultured for 24 h with or without 100 ng/ml sCD40L. Culture supernatants were assayed for PGE2 by EIA. Results are expressed as the mean ± SD of triplicate cultures. Similar results were obtained in three separate experiments. *, p < 0.05 (compared with sCD40L only).

 
In response to various stimuli, the amount of COX-2 protein is significantly increased in monocytes (14, 15, 16). The expression of COX-2 protein in CD40-stimulated monocytes was also markedly induced after 18 h. IL-4 (85% inhibition, p < 0.01) and IL-10 (97% inhibition, p < 0.01) significantly inhibited this expression (Fig. 3). To further confirm whether both cytokines regulated COX-2 expression at the upstream of the protein level, we examined COX-2 mRNA expression using a competitive PCR assay. As shown in Fig. 4 (A and B), the amount of COX-2 mRNA in CD40-stimulated monocytes was ~30-fold more (6 x 105 copies) than that in unstimulated monocytes (2 x 104 copies). Notably, COX-2 mRNA expression was inhibited by IL-4 (6 x 104 copies) and IL-10 (2 x 104 copies). These findings were further confirmed by using a quantitative real-time PCR (Fig. 4C). CD40 ligation of monocytes induced COX-2 mRNA expression (>21-fold), which was significantly inhibited by IL-4 and IL-10 (IL-4, p < 0.01; IL-10, p < 0.01). We also examined COX-1 mRNA expression; however, we could not quantify it probably due to low expression levels in monocytes even after CD40 ligation (data not shown).



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  		FIGURE 3. Effects of IL-4 and IL-10 on CD40-induced COX-2 protein expression. Human monocytes (4 x 106cells) were treated for 2 h with or without IL-4 (10 ng/ml) or IL-10 (10 ng/ml), and then stimulated with sCD40L (100 ng/ml) for 18 h. After stimulation, cell lysates (2 x 106 cells/lane) were collected and used to determine the expression of COX-2 protein by Western blot analysis (A). The densitometric analyses were conducted from three separate experiments, and the mean (±SD) fold inductions compared with control samples are indicated (B). **, p < 0.01 (compared with CD40 only).

 


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  		FIGURE 4. IL-4 and IL-10 regulate CD40-induced COX-2 mRNA expression in monocytes. Human monocytes (5 x 105 cells) from healthy donors were treated for 2 h with or without IL-4 (10 ng/ml) or IL-10 (10 ng/ml), and then stimulated for 6 h with sCD40L (100 ng/ml). After stimulation, total RNA was extracted and then first-strand cDNA was synthesized by RT-PCR. A, To quantify the amount of COX-2 mRNA, each sample was diluted to be adjusted by quantifying the amount of total mRNA by competitive PCR using the internal standard (GAPDH) competitor. Finally, competitive PCR for COX-2 was performed using serial 3-fold dilutions of competitors (indicated), and the amplified products were subjected to electrophoresis on a 3% agarose gel. Larger sized bands are PCR products derived from competitive fragments, of which concentrations are indicated above the bands. B, The fold induction compared with a control, analyzed using a competitive PCR, is indicated. C, COX-2 mRNA expression analyzed using a real-time PCR. The relative mRNA amounts of COX-2 vs GAPDH are expressed as the mean ± SD of triplicate samples. Similar results were obtained in three separate experiments. **, p < 0.01 (compared with CD40 only).

 
To determine whether MAPK pathways are involved in CD40-induced PGE2 production and COX-2 expression in monocytes, we tested the effects of specific inhibitors for MEK1/2 (PD98059) and p38 MAPK (SB203580) in CD40-stimulated monocytes. We have previously shown the specificity of these inhibitors in monocytes (18). Both inhibitors significantly (p < 0.01) inhibited PGE2 production (Fig. 5A) as well as COX-2 protein expression (p < 0.01) (Fig. 5, B and C), suggesting that the ERK and p38 MAPK pathways are involved in CD40-induced prostanoid synthesis.



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  		FIGURE 5. ERK and p38 MAPK kinases are involved in CD40-induced PGE2 production and COX-2 expression in monocytes. Human monocytes (3 x 105 cells/well) were treated for 1 h with PD98059 (50 µM) or SB203580 (1 µM) and then cultured for 24 h with or without 100 ng/ml sCD40L. The concentrations of these inhibitors were determined to give a maximal inhibition of activation of kinases and be less influential to cell viability in our preliminary experiments (data not shown). Culture supernatants were assayed for PGE2 by EIA (A). Results are expressed as the mean ± SD of triplicate cultures. Monocytes (4 x 106cells) were treated for 2 h with or without IL-4 (10 ng/ml) or IL-10 (10 ng/ml), and then stimulated for 18 h with sCD40L (100 ng/ml). After stimulation, cell lysates (2 x 106 cells/lane) were collected and used to determine the expression of COX-2 protein by Western blot analysis (B). The densitometric analyses were conducted based on three separate experiments, and the mean (±SD) fold inductions compared with control samples are indicated (C). **, p < 0.01 (compared with CD40 only).

 
We next examined the effects of IL-4 and IL-10 on the activation of the ERK and p38 MAPK pathways in CD40-stimulated monocytes. CD40 ligation caused significant ERK phosphorylation, which was inhibited by IL-4 (79% inhibition, p < 0.01) and IL-10 (84% inhibition, p < 0.01) (Fig. 6A). CD40 ligation also led to the phosphorylation of MEK1/2, an upstream kinase required for ERK phosphorylation. MEK1/2 phosphorylation was also inhibited by IL-4 (79% inhibition, p < 0.05) and IL-10 (90% inhibition, p < 0.01) (Fig. 6B). Moreover, CD40 ligation induced phosphorylation of p38 MAPK and MKK3/6, an upstream kinase required for p38 MAPK phosphorylation (Fig. 6, C and D). IL-10 partially, but significantly inhibited the phosphorylation of p38 MAPK and MKK3/6 (60%, p < 0.05 and 49%, p < 0.05), while IL-4 had no effect on either kinase (Fig. 6, C and D). These results suggest that IL-10 regulates CD40-induced activation of the ERK and p38 MAPK pathways, while IL-4 regulates that of the ERK, but not the p38 MAPK pathway.



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  		FIGURE 6. Effects of IL-4 and IL-10 on the activation of the ERK and p38 MAPK pathways in CD40-stimulated monocytes. Human monocytes (4 x 106cells) were treated for 2 h with or without IL-4 (10 ng/ml) or IL-10 (10 ng/ml), and then stimulated for 1 h (A and B) or 10 min (C and D) with sCD40L (100 ng/ml). After stimulation, cell lysates (2 x 106 cells/lane) were collected and used to determine the phosphorylation of ERK1/2, MEK1/2, p38MAPK, or MKK3/6 by Western blot analysis. The densitometric analyses were conducted based on three separate experiments, and the mean (±SD) fold inductions compared with control samples are indicated. *, p < 0.05; **, p < 0.01 (compared with CD40 only).

 
To date, there have been several reports demonstrating that CD40, like other TNFR family members, not only induces MAPK activation, but also induces NF-{kappa}B activation. We thus investigated whether the NF-{kappa}B pathway is involved in CD40-induced PGE2 production in monocytes by using an inhibitor of NF-{kappa}B, curcumin (31, 32). PGE2 production in monocytes was significantly (p < 0.01) inhibited in a dose-dependent manner, and complete inhibition was achieved in the presence of 50 µM of curcumin (Fig. 7). We next examined the effects of IL-4 and IL-10 on CD40-induced NF-{kappa}B activation in monocytes using an EMSA. CD40 ligation resulted in strong NF-{kappa}B activation in monocytes. IL-10 significantly (p < 0.05) blocked this NF-{kappa}B activation, whereas IL-4 showed little suppressive effect (Fig. 8A). Consistent with the EMSA data above, I-{kappa}B was markedly phosphorylated after CD40 ligation, and IL-10 inhibited its phosphorylation (70% inhibition, p < 0.05). However, IL-4 failed to show such an inhibitory effect (Fig. 8B). Collectively, these results suggested that IL-4 and IL-10 exert differential effects on the NF-{kappa}B pathway, namely IL-10 inhibited CD40-induced NF-{kappa}B activation in monocytes partly via the inhibitory effect on I-{kappa}B phosphorylation.



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  		FIGURE 7. NF-{kappa}B pathway is involved in CD40-induced PGE2 production in monocytes. Human monocytes (3 x 105 cells/well) were prestimulated for 1 h with the indicated concentrations of curcumin, and then cultured for 24 h with or without 100 ng/ml sCD40L. Culture supernatants were assayed for PGE2 by EIA. Results are expressed as the mean ± SD of triplicate cultures. Similar results were obtained in three separate experiments. **, p < 0.01 (compared with sCD40L only).

 


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  		FIGURE 8. Effects of IL-4 and IL-10 on CD40-induced NF-{kappa}B activation in monocytes. Human monocytes were treated for 2 h with or without IL-4 (10 ng/ml) or IL-10 (10 ng/ml), and then stimulated for 10 min (A) or 1 h (B) with sCD40L (100 ng/ml). After stimulation, nuclear proteins were extracted and assayed for NF-{kappa}B DNA-binding activity by EMSA (A), and cell lysates were collected and used to determine the phosphorylation of I-{kappa}Ba by Western blot analysis (B). The densitometric analyses were conducted based on three separate experiments, and the mean (±SD) fold inductions compared with control samples are indicated (B). *, p < 0.05 (compared with CD40 only).

 
Finally, we examined whether IL-4 or IL-10 affects the recruitment of TRAF proteins to CD40 in monocytes (Fig. 9). A previous study showed that TRAF2 and TRAF3 are recruited to CD40 in B cells upon CD40 ligation (33). Before stimulation, both TRAF2 and TRAF3 associated with CD40 to some extent in monocytes. CD40 ligation facilitated further recruitment of TRAF2 and TRAF3 to CD40 at 15 min, in which we observed the maximal TRAF recruitment to CD40 in our preliminary experiments (data not shown). IL-4 and IL-10 had no effect on the recruitment of TRAF2 and TRAF3 to CD40, suggesting that each cytokine regulates CD40 signaling at the downstream levels of TRAF recruitment to CD40.



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  		FIGURE 9. Effects of IL-4 and IL-10 on the recruitment of TRAF2 and TRAF3 to CD40 in monocytes upon CD40 ligation. Human monocytes (5 x 106 cells/lane) were treated for 2 h with IL-4 (10 ng/ml) or IL-10 (10 ng/ml), and then stimulated for 15 min with or without 100 ng/ml sCD40L. After stimulation, cell lysates were immunoprecipitated with anti-human CD40 mAb. Precipitated proteins were immunoblotted for TRAF2, TRAF3, and CD40. The densitometric analyses were conducted based on three separate experiments, and the mean (±SD) fold inductions compared with unstimulated samples are indicated.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Activation of monocytes/macrophages is a crucial event for the development of the inflammatory response in vivo because these cells are capable of synthesizing a wide array of proinflammatory mediators in response to a number of soluble factors such as LPS, cytokines, and mitogens. LPS is the most extensively studied inducer of the synthesis of proinflammatory cytokines and prostanoids in monocytes/macrophages (34, 35). In addition to soluble factors, it is becoming appreciated that monocytes/macrophages can also be strongly activated through direct interaction with other cells, such as activated T cells. In fact, the CD40L-CD40 system appears to be important for this function. Previous studies have shown that CD40 ligation induces the production of proinflammatory cytokines in monocytes (1, 2). In this study, we have shown that CD40 ligation on monocytes significantly induces the synthesis of PGE2, a pivotal proinflammatory mediator. Moreover, this process is dependent on CD40-induced COX-2 expression in monocytes. Consistent with our findings, Zhang et al. and Cao et al. (36, 37) demonstrated that CD40 ligation induced PGE2 production in lung and orbital fibroblasts via the induction of COX-2 expression. Zhang et al. showed that CD40 ligation alone induced only minimal COX-2 mRNA expression, and further up-regulation was achieved by the pretreatment of these cells with IFN-{gamma}. This might be partly due to the up-regulation of CD40 expression on the cell surface by IFN-{gamma} because we have found that the same is true in monocytes in our system (data not shown).

We also showed that IL-10 and IL-4 strongly inhibited CD40-induced PGE2 production in monocytes via down-regulation of COX-2 expression at the gene level. This trend is similar to our observations of LPS-induced COX-2 expression in monocytes, as previously described (17, 18). In contrast, the effects of both cytokines appeared to be different depending on the molecules induced by CD40 ligation. Both cytokines inhibited IL-1 production to a similar extent (38). Interestingly, IL-10 inhibited, but IL-4 enhanced, CD40-induced IL-12 production, while both cytokines significantly blocked LPS/IFN-{gamma}-induced IL-12 production in macrophages (39). We found that IL-10 strongly, but IL-4 only partially, inhibited CD40-induced TNF-{alpha} production in monocytes (data not shown). Among a wide range of proinflammatory mediators, prostanoid synthesis might be a common target for regulatory roles of IL-10 and IL-4 in vivo.

In this study, we investigated the underlying molecular mechanisms for prostanoid regulation by IL-10 and IL-4. In particular, we focused on the MAPK and NF-{kappa}B pathways because these pathways had been suggested to play a key role in inflammatory responses (40, 41). By using a specific inhibitor of each pathway, it was suggested that the ERK and p38 MAPK pathways are involved in CD40-induced COX-2 expression in monocytes. Subsequent experiments indicated that CD40 ligation induced the phosphorylation of ERK1/2 and its upstream kinase MEK1/2 in monocytes, consistent with recent studies (42, 43). CD40 ligation also induced the phosphorylation of p38 MAPK and its upstream kinase MKK3/6 in monocytes. Similar observations were reported using B and monocyte-derived dendritic cells (44, 45). In contrast to our current findings, Suttles et al. (42) found CD40 ligation induced activation of ERK, but not p38 MAPK in monocytes. This discrepancy might be due to a difference in the means of CD40 stimulation, because plasma membrane products from activated CD4 T cells were used in their study, while the pure soluble CD40 ligand was used in our study (42).

Intriguingly, IL-10 and IL-4 exerted differential effects on the activation of ERK and p38 MAPK in CD40-stimulated monocytes. Both cytokines significantly inhibited CD40-induced ERK activation, consistent with the previous study (42). In addition, IL-10 and IL-4 inhibited phosphorylation of MEK1/2 to a similar level. In contrast, IL-10, but not IL-4, inhibited activation of p38 MAPK and MKK3/6. These results are in agreement with our previous studies on LPS-stimulated monocytes/macrophages (18). Collectively, these observations strongly suggest that both cytokines differentially regulate the activation of kinases upstream of MEK1/2 and MKK3/6. One of the earliest events in CD40 signaling involves the recruitment of TRAF proteins to CD40 (33). Intriguingly, neither IL-10 nor IL-4 affected the recruitment of TRAF2 and TRAF3 to CD40 in monocytes. This suggests that IL-10 and IL-4 regulate CD40 signaling pathways at the downstream levels of TRAF recruitment to CD40. Further work is still necessary to define which level of CD40 signaling pathways is target for IL-10 and IL-4. STAT3 and STAT6 play a crucial role in the signaling pathways for IL-10 and IL-4, respectively (46, 47). The mechanisms by which STAT activation by these cytokines leads to their inhibitory effects on the MAPK pathways remain to be clarified.

In addition to the MAPK pathway, CD40 ligation eventually leads to NF-{kappa}B activation (2, 48, 49). We indicated that the NF-{kappa}B pathway is also involved in CD40-induced COX-2 expression in monocytes, and IL-10 remarkably inhibited CD40-induced NF-{kappa}B DNA-binding activity, consistent with previous studies using different stimuli (50, 51, 52). In addition to the inhibition of I-{kappa}B phosphorylation, there may be other mechanisms involved in IL-10-mediated inhibition of NF-{kappa}B (50). In contrast, IL-4 had a less pronounced inhibitory effect on CD40-induced NF-{kappa}B activation and I-{kappa}B phosphorylation in monocytes in this study. Given that IL-4 partially inhibited CD40-induced TNF-{alpha} production in monocytes (data not shown), NF-{kappa}B activation might be a more dominant signaling pathway for the control of TNF-{alpha} production. It has been shown that IL-4 inhibits NF-{kappa}B-dependent gene expression via the activation of STAT6 in macrophages (53, 54, 55). Thus, we cannot rule out the possibility that IL-4 might directly regulate the transcriptional activity of NF-{kappa}B-regulated genes.

The results in this study strongly support the hypothesis that IL-4 and IL-10 are central regulators of prostanoid synthesis, even when T cell-dependent inflammation is dominant. In light of the fact that CD40L expression is often observed in activated T cells surrounding inflamed lesions in a number of autoimmune disorders, both cytokines seem more likely to provide a beneficial therapeutic application for these conditions. Indeed, both cytokines clearly ameliorate the progression of autoimmune disease models in mice such as RA and experimental autoimmune encephalomyelitis (56, 57, 58, 59). It is thus possible that these cytokines may work in concert with suboptimal doses of other anti-inflammatory drugs to produce a superior therapeutic outcome with fewer adverse effects. Obviously, further investigations are necessary to unravel regulatory mechanisms of IL-10 and IL-4, which will help us to develop improved methods of using these cytokines in various clinical situations.


   Acknowledgments
 
We thank Dr. Thomas M. Yankee for helpful discussion and for proofreading the English used in this manuscript.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Takeshi Otsuka, Department of Medicine and Biosystemic Science, Graduate School of Medical Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka, 812-8582, Japan. E-mail address: takeshi{at}intmed1.med.kyushu-u.ac.jp Back

2 Abbreviations used in this paper: CD40L, CD40 ligand; COX, cyclooxygenase; EIA, enzyme immunoassay; ERK, extracellular signal-related kinase; LTB4, leukotriene B4; MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK kinase; MKK, MAPK kinase; RA, rheumatoid arthritis; sCD40L, soluble CD40L; TRAF, TNFR-associated factor; TXB2, thromboxane B2. Back

Received for publication February 20, 2003. Accepted for publication November 19, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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