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International Immunology Advance Access originally published online on April 24, 2006
International Immunology 2006 18(6):981-990; doi:10.1093/intimm/dxl034
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© The Japanese Society for Immunology. 2006. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Differential expression of inducible nitric oxide synthase and IL-12 between peritoneal and splenic macrophages stimulated with LPS plus IFN-{gamma} is associated with the activation of extracellular signal-related kinase

Yi-Na Zhu1, Yi-Fu Yang1, Shiro Ono2, Xiang-Gen Zhong1, Yong-Hong Feng1, Yong-Xin Ren1, Jia Ni1, Yun-Feng Fu1, Wei Tang1 and Jian-Ping Zuo1

1 Laboratory of Immunopharmacology, Graduate School of the Chinese Academy of Sciences, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 555 Zuchongzhi Road, Zhangjiang Hi-Tech Park, Shanghai 201203, People's Republic of China
2 Department of Oncology, Osaka University Graduate School of Medicine, 2-2, Yamada-oka, Suita, Osaka 565-0871, Japan

Correspondence to: J.-P. Zuo; E-mail: jpzuo{at}mail.shcnc.ac.cn


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Resident peritoneal macrophages (pM{varphi}) are found deficient in T cell-stimulating capacity compared with the competent splenic macrophages (sM{varphi}). Macrophages (M{varphi})-derived nitric oxide (NO) and IL-12 have been shown to play crucial roles in the interaction between M{varphi} and T cells. To further understand differential functions between pM{varphi} and sM{varphi}, we focused on the production of NO and IL-12 from LPS plus IFN-{gamma}-activated M{varphi}. We demonstrated the differential expression of inducible nitric oxide synthase (iNOS) and IL-12 in pM{varphi} and sM{varphi} with LPS plus IFN-{gamma} stimulation. pM{varphi} produced high level of NO but low level of IL-12, whereas sM{varphi} produced high level of IL-12 but no NO. Furthermore, we demonstrated that there were no differences in IFN-{gamma}-induced signal transducer and activator of transcription-1 activation and consequent interferon regulatory factor-1 and interferon consensus sequence-binding protein up-regulation between pM{varphi} and sM{varphi}. Likewise, p38 mitogen-activated protein kinase was activated by LPS with identical kinetics in both pM{varphi} and sM{varphi}. However, LPS-induced extracellular signal-regulated kinase (ERK) activation was prolonged in pM{varphi} comparing with sM{varphi}. Moreover, we demonstrated, using inhibitor selective for ERK cascade (PD98059), that the prolonged ERK activation contributed a positive signal for iNOS expression and a negative signal for IL-12p40 expression in resident pM{varphi}. In addition, anti-IL-10-neutralizing antibody plus indomethacin could abrogate the inhibitory effects of endogenous IL-10 and prostaglandin E2 on the production of IL-12 by resident pM{varphi} possibly through suppressing ERK activation. Taken together, profound difference in ERK activation may account for differential LPS plus IFN-{gamma} responsiveness between pM{varphi} and sM{varphi}. High production of NO and low production of IL-12 by pM{varphi} may contribute to its deficiency in T cell-stimulating capacity.

Keywords: ERK, iNOS, IL-12, macrophage


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
The versatility and importance of macrophages (M{varphi}) in host defense and homeostasis have long been recognized. Anatomically, M{varphi} isolated from various tissues (splenic, peritoneal, alveolar M{varphi} and Kupffer cells) manifest extreme differences in functional activities (1–5). The most important one is functioning as antigen-presenting cells (APCs) to stimulate T cells response (6). In our study, we found that peritoneal macrophages (pM{varphi}) and splenic macrophages (sM{varphi}) differ in T cell-stimulating capacity. It is consistent with the results of Bamba et al. (7), who showed that resident pM{varphi} is not able to stimulate T cell response to antigen. Therefore, we investigated the differential characteristics involved in the differential function of pM{varphi} and sM{varphi}.

It has been reported that M{varphi}-derived nitric oxide (NO) and IL-12 play crucial roles in the interaction between M{varphi} and T cells. NO has been shown to inhibit T cell responses in several different models (8) and mediate the inhibition of T cell proliferation by pM{varphi} (9). IL-12 is a potent cytokine exerting a number of regulatory effects on T lymphocytes including the facilitation of activated T cells proliferation and IFN-{gamma} production cell (10, 11). We hypothesized that M{varphi}-derived NO and IL-12 may be involved in the deficiency of pM{varphi} in T cell-stimulating capacity compared with the competent sM{varphi}. Thus, we focused on the differential NO and IL-12 production between pM{varphi} and sM{varphi}.

Stimulation of M{varphi} by cytokines IFN-{gamma} and/or microbial products such as LPS results in the induction and release of NO and IL-12. NO is generated following the up-regulation of expression of the inducible nitric oxide synthase (iNOS) (12). Similarly, the biological functional IL-12p70 (a heterodimeric cytokine comprising p40 and the constitutively expressed p35 subunit) is regulated by the induction of the p40 subunit (13, 14). Expression of these immunomodulatory proteins appears to be regulated primarily at the level of transcription (15–20).

M{varphi} characteristics heterogeneity may arise as a consequence of heterogeneity in transmembrane and intracellular signaling events, thereby leading to diversity in gene expression (21). IFN-{gamma}-induced signal transducer and activator of transcription (STAT)-1 activation and IFN regulatory factors (IRFs) appear to play key roles in the induction of both of these M{varphi} effectors (17, 22, 23) via the IFN-stimulating response element in iNOS (15) and IL-12 promoter (19). However, the early, receptor proximal signaling mechanisms underlying the regulation of iNOS or IL-12p40 expression by LPS in M{varphi} also played important roles. P38 mitogen-activated protein kinase (MAPK) plays an essential role in the LPS-mediated induction of iNOS (24, 25) and promotes the up-regulation of IL-12 (26). In contrast, extracellular signal-regulated kinase (ERK) contributes positive regulatory signals to the induction of iNOS and act to suppress expression of IL-12p40 (26–29).

In this study, we aimed at the mechanisms that contribute to the heterogeneity of pM{varphi} and sM{varphi} to induce NO and IL-12 in response to LPS plus IFN-{gamma}.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Reagents
Murine recombinant IFN-{gamma} was purchased from PharMingen (San Diego, CA, USA). Ovalbumin (OVA) (grade V), LPS, indomethacin, sulfanilamide and N-[naphthyl] ethyl-enediamine dihydrochloride were Sigma (St Louis, MO, USA) products. PD98059 was purchased from Calbiochem (La Jolla, CA, USA). Anti-mouse IL-10 mAb (JES52A5), anti-mouse CD 40 mAb (HM40-3) and anti-F4/80–PE mAb were purchased from BD PharMingen (San Diego, CA, USA). [3H]thymidine ([3H]TdR) (1 mCi ml–1) was purchased from Shanghai Institute of Atomic Energy.

Mice
Female Balb/c mice (6–8 weeks old) were purchased from Shanghai Experimental Animal Center of Chinese Academy of Sciences. The animals were housed in specific pathogen-free conditions. All mice were allowed to acclimatize in our facility for 1 week before any experiments were started. All experiments were carried out according to the National Institutes of Health Guide for Care and Use of Laboratory Animals, and were approved by the Bioethics Committee of the Shanghai Institute of Materia Medica.

M{varphi} preparations
pM{varphi} were obtained by harvest from peritoneal lavage with cold PBS. Peritoneal exuded cells were centrifuged at 1300 rpm, 4°C, for 5 min and washed with RPMI-1640 medium. Adherent cells (M{varphi} populations) were recovered from the dishes using a rubber policeman after 2-h incubation at 37°C. M{varphi} isolated by this procedure were >90% pure as measured by staining for F4/80 molecule, which has been used widely as a marker for mouse M{varphi} (30).

sM{varphi} were obtained as follows: spleens were fragmented by passage through sterile mesh. Cells were then centrifuged at 1300 rpm, 4°C, for 5 min and erythrocytes were lysed with Tris–NH4Cl. Cells were washed with RPMI-1640 medium for two times. Adherent sM{varphi} were collected by removing the non-adherent cells after 2-h incubation at 37°C. M{varphi} isolated by this procedure were >90% pure as measured by staining for F4/80 molecule.

Anti-OVA-specific immune responses
OVA-primed T cells were purified by using immunomagnetic negative selection to delete B cells and I-A+ APCs as described (31). Balb/c mice were immunized with OVA. After 14 days, lymph node cells from OVA-immunized mice were allowed to react with anti-I-Ad/b mAb and then incubated with magnetic particles bound to goat anti-mouse Ig (Polysciences, Inc., Eppelheim, Germany). A T cell population depleted of anti-I-Ad/b-labeled and surface Ig+ cells was obtained by removing cell-bound magnetic particles with a rare earth magnet (Polysciences, Inc.). Purity of the resulting T cell populations was examined by flow cytometry and found to be consistently >95%.

OVA-primed T cells (4 x 105 per well) were co-cultured with M{varphi} (1 x 105 per well) which were obtained from normal mice in 96-well flat-bottomed tissue culture plates in the presence of OVA (100 µg ml–1). Supernatants were harvested at indicated times to measure IFN-{gamma} levels. For proliferation, cells were pulsed with 1 µCi per well [3H]TdR for 18 h before harvest and assessed for [3H]TdR incorporation.

NO quantification
pM{varphi} (1 x 105 per well) and sM{varphi} (1 x 105 per well) were cultured in triplicate in 96-well plates and stimulated with 2 ng ml–1 IFN-{gamma} plus 1 µg ml–1 LPS for 24 h. The production of NO was determined by assaying culture supernatant for NO2–, a stable reaction product of NO. Briefly, 100 µl of culture supernatant was mixed with an equal volume of Griess reagent (1% sulfanilamide and 0.1% N-[naphthyl] ethyl-enediamine dihydrochloride in 2.5% H3PO4) at room temperature for 10 min. Absorbance was measured at 540 nm in a microplate reader. NO2– concentration was calculated from a NaNO2 standard curve.

Prostaglandin E2 determination
pM{varphi} (1 x 106 per well) and sM{varphi} (1 x 106 per well) were cultured in six-well plates for 2 h without stimulation. Prostaglandin E2 (PGE2) production in the supernatants was assayed by the radioimmunoassay (RIA) method using a 3H-PGE2-RIA kit (Beijing Dongya Institute) according to the manufacturer's protocol.

Cytokines measurement
IL-10, IL-12 and IFN-{gamma} productions in the culture supernatants were detected by standard sandwich ELISA. The OptELIA IL-10, IL-12p40, IL-12p70 and IFN-{gamma} mouse set were obtained from BD PharMingen and were performed according to the manufacturer's instructions.

Real-time quantitative reverse transcription–PCR
pM{varphi} (1 x 106 per well) and sM{varphi} (1 x 106 per well) were stimulated with 2 ng ml–1 IFN-{gamma} plus 1 µg ml–1 LPS in six-well plates for indicated periods, and lysed using Trizol reagent (GIBCO). Total RNA was isolated from each cell preparation and reverse transcribed into cDNA. Relative quantitation with real-time PCR was performed with SYBR Green PCR Reagents (Qiagen, Valencia, CA, USA) and a Continuous Fluorescence Detection System (MJ Research, USA), according to the manufacturer's instructions. The mRNA levels were normalized to those of ß-actin.

Western blotting
Stimulated cells (1 x 107 per sample) were lysed in lysis buffer (25 mM Tris buffer, pH 7.4, containing 150 mM NaCl, 1% Nonidet P-40, 1 mM EDTA, 10 mM NaF, 1 mM dithiothreitol, 50 µg ml–1 each of leupeptin, aprotinin and phenylmethylsulphonylfluoride) by incubation on ice for 30 min. Lysates were then centrifuged at 13 000 x g at 4°C for 10 min, and the supernatants were transferred to fresh tubes and stored at –80°C until required. Proteins (10 µg per lane) were electrophoresed in a 10% polyacrylamide gel and transferred to nitrocellulose transfer and immobilization membranes (Schleicher & Schuell). The membranes were treated with 5% non-fat milk for 1 h to block non-specific binding, rinsed and incubated with a panel of rabbit polyclonal antibodies against phospho-ERK1/2, phospho-p38, ERK1/2, p38, phospho-STAT1 S727, phospho-STAT1 Y701 and STAT1 (Cell Signaling Technology) overnight at 4°C. The membranes were then treated with a 1:2000 dilution of HRP-conjugated anti-rabbit IgG (H + L) for 1 h. Immune complexes were detected with a chemiluminescence substrate (Pierce) and exposed to Kodak X-ray film (Kodak).


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
The deficiency of pM{varphi} in T cell-stimulating capacity
Thioglycollate-elicited pM{varphi} have long been used as APC (32, 33). Whether resident pM{varphi} also could be used as APC to stimulate T cells response was now investigated. T cell-stimulating capacity of resident pM{varphi} was compared with sM{varphi} in the T cell and M{varphi} co-culture assay as follows: pM{varphi} or sM{varphi} were co-cultured with OVA-primed T cells in the presence of 100 µg ml–1 OVA antigen. The 24-h culture supernatants were analyzed for the presence of IFN-{gamma}, and the T cell proliferation was measured by [3H]TdR incorporation in 72-h culture. As shown in Fig. 1, pM{varphi} failed to induce proliferation and IFN-{gamma} production in OVA-primed T cells with antigen OVA stimulation in vitro. In contrast, sM{varphi} induced a well proliferation response and a large amount of IFN-{gamma} production in OVA-primed T cells. It suggested that pM{varphi} is deficient in T cell-stimulating capacity.


Figure 1
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  		Fig. 1 The deficiency of pM{varphi} in T cell-stimulating capacity. Resident pM{varphi} and sM{varphi} were prepared as described in Methods. OVA-primed T cells (4 x 105 per well) were cultured with M{varphi} (1 x 105 per well) in the presence of 100 µg ml–1 OVA in 96-well culture plates for 72 h to assess [3H]thymidine incorporation (A) or for 24 h to determine IFN-{gamma} production (B). Results are expressed as mean (±SD). Three experiments were performed with similar results.

 
Differential expression of iNOS and IL-12 between pM{varphi} and sM{varphi}
Resident pM{varphi} inhibit T cell proliferation via a NO-dependent mechanism (9). M{varphi}-derived IL-12 stimulates IFN-{gamma} synthesis and proliferation in activated T cells (10, 11). Therefore, we hypothesized that M{varphi}-derived effectors, NO and IL-12 may be involved in the mechanisms responsible for differential T cell-stimulating capacity of pM{varphi} and sM{varphi}. To determine the ability of pM{varphi} and sM{varphi} to produce NO, cells were stimulated with LPS plus IFN-{gamma} for 24 h. The NO2– production was dramatically increased over basal level (~1.8 µM) in supernatants from stimulated pM{varphi}. While in supernatants of sM{varphi}, NO2– was held at the basal level (Fig. 2A). M{varphi} generate NO from L-arginine through iNOS, which is regulated transcriptionally. The iNOS mRNA expression is marked increased by LPS plus IFN-{gamma} stimulation in pM{varphi} but to a low extent in sM{varphi} (Fig. 2C). It suggests that pM{varphi} well respond to LPS plus IFN-{gamma} to generate NO.


Figure 2
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  		Fig. 2 Different induction of NO and IL-12 from pM{varphi} and sM{varphi}. Resident pM{varphi} and sM{varphi} were stimulated with IFN-{gamma} (2 ng ml–1) plus LPS (1 µg ml–1) and triplicate samples were cultured in 96-well plates. After 24-h incubation, culture supernatants were collected, and NO2– was measured by Griess reaction (A) and IL-12p40 was measured by ELISA (B). Results are expressed as mean (±SD). (C) pM{varphi} and sM{varphi} were stimulated with IFN-{gamma} (2 ng ml–1) plus LPS (1 µg ml–1) and triplicate samples were cultured in 6-well plates for 6 h, and total RNA was extracted. iNOS and IL-12p40 mRNA expression were analyzed by real-time RT–PCR. Results are expressed as mean (±SD)-fold increase in specific mRNA level compared with non-stimulated pM{varphi}. pM{varphi} and sM{varphi} were stimulated with IFN-{gamma} (2 ng ml–1) plus anti-CD40 mAb (10 µg ml–1) and triplicate samples were cultured in 96-well plates. After 24-h incubation, culture supernatants were collected, and NO2– was measured by Griess reaction (D) and IL-12p40 was measured by ELISA (E). Results are expressed as mean (±SD). Three experiments were performed with similar results.

 
Whether pM{varphi} and sM{varphi} could produce IL-12 was similarly investigated. Cells were stimulated with LPS plus IFN-{gamma} for 24 h. In contrast to the production of NO, IL-12p40 was little in stimulated pM{varphi} but dramatically up-regulated in stimulated sM{varphi} (Fig. 2B). IL-12p40 mRNA expression was in parallel with the protein level (Fig. 2C). The results indicated that sM{varphi} well response to LPS plus IFN-{gamma}-induced IL-12p40 production.

In addition to T cell-independent stimulation (e.g. LPS), the CD40 engagement with cognate T cell CD40L is an important T cell-dependent stimulus that trigger M{varphi} activation, including inducing NO (34, 35) and IL-12 production (36, 37). Again, we observed the differential production of NO and IL-12 between pM{varphi} and sM{varphi} induced by agonistic anti-CD40 mAb plus IFN-{gamma}. pM{varphi} generated NO but not IL-12p40 (Fig. 2D), while sM{varphi} produced only IL-12p40 (Fig. 2E).

Identical activation STAT-1 between pM{varphi} and sM{varphi}
Above results suggested that pM{varphi} and sM{varphi} differ in the responsiveness to LPS plus IFN-{gamma} stimulation. Thus, the underlying signaling events were then investigated. Both IRF-1–/– and ICSBP–/– mice are deficient in NO and IL-12 production, suggesting that they are critical transcription factors in the regulation of iNOS (38, 39) and IL-12p40 production (23, 40, 41). These transcription factors are regulated by IFN-{gamma}-induced STAT-1 activation. STAT-1 is phosphorylated in response to IFN-{gamma} on both Y701 and S727, and phosphorylation at both of these residues is required for maximal transcriptional activity of STAT-1 (42). As shown in Fig.3A, there were comparable levels in serine and tyrosine phosphorylation of STAT-1 between pM{varphi} and sM{varphi} stimulated with LPS plus IFN-{gamma} for 30 min. STAT-1 activation leads to up-regulation of interferon consensus sequence-binding protein (ICSBP) and IRF-1 expression (43, 44). Therefore, we also compared the expression of these transcription factors by reverse transcription (RT)–PCR. In consistent with STAT-1 activation, there were no relative changes in IRF-1 and ICSBP mRNA expression between pM{varphi} and sM{varphi} (Fig. 3B).


Figure 3
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  		Fig. 3 LPS plus IFN-{gamma} induced STAT-1 activation and consequent synthesis of IRF-1 and ICSBP from pM{varphi} and sM{varphi}. (A) Resident pM{varphi} and sM{varphi} were stimulated with IFN-{gamma} (2 ng ml–1) plus LPS (1 µg ml–1) for 30 min. Whole-cell extracts were analyzed by western blot for STAT-1 phosphorylation and blotting of STAT-1 protein was done to ensure equal protein loading. (B) pM{varphi} and sM{varphi} were stimulated with IFN-{gamma} (2 ng ml–1) plus LPS (1 µg ml–1) for 90 min. Total RNA was extracted, and IRF-1 and ICSBP mRNA expression were analyzed by real-time RT–PCR. Results are expressed as mean (±SD)-fold increase in specific mRNA level compared with non-stimulated pM{varphi}. Three experiments were performed with similar results.

 
Different activation of ERK between pM{varphi} and sM{varphi}
To investigate whether the ERK and p38 MAPK pathways are involved in LPS signal transduction in pM{varphi} and sM{varphi}, we examined the activation of the two MAPKs by detecting their dually phosphorylated (Tyr/Thr) forms by western blotting using specific anti-phosphokinase antibodies. There was no basal activation of ERK in unstimulated pM{varphi} and sM{varphi}. LPS strongly induced a rapid increase of ERK activation in both of M{varphi}, which peaked at 15 min but remained elevated for at least 60 min in pM{varphi} (Fig. 4). The ERK activation was shorter in sM{varphi}, which remained <30 min. Likewise, p38 MAPK was activated with maximum activity achieved at 15 min and subsided by 60 min in both pM{varphi} and sM{varphi} (Fig. 4). These results clearly showed that LPS induced the prolonged ERK activation in pM{varphi} while the same kinetics of p38 MAPK activation in both M{varphi}.


Figure 4
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  		Fig. 4 LPS plus IFN-{gamma} induced ERK and p38 MAPK activation in pM{varphi} and sM{varphi}. (A) Resident pM{varphi} and sM{varphi} were stimulated with IFN-{gamma} (2 ng ml–1) plus LPS (1 µg ml–1) for indicated periods. Whole-cell lysates were resolved by SDS-PAGE, followed by immunoblotting using a set of antibodies that recognized activated (dually phosphorylated on Tyr/Thr) ERK and p38 MAP kinase expression. Equivalent protein loading was determined by reprobing the appropriate blots with anti-total ERK, anti-total p38 MAP kinase antibodies. (B) Densitometer scans of pERK/ERK, pp38/p38 ratio. Results are expressed as mean (±SD)-fold increase in specific protein level compared with non-stimulated pM{varphi}. Three experiments were performed with similar results.

 
Role of ERK on iNOS expression in pM{varphi}
Involvement of the ERK MAPK cascade in LPS-mediated iNOS regulation has been reported in thioglycollate-elicited pM{varphi} (26). We investigated whether it also plays a role in iNOS induction in resident pM{varphi}. A selective and potent inhibitor of the ERK MAPK cascade, PD98059, mediates its effects by binding to and inactivating the ERK-specific MAPK kinase. PD98059 (40 µM) completely abrogated ERK activation (dual phosphorylation) (Fig. 5A). To investigate whether ERK was involved in the LPS plus IFN-{gamma}-stimulated induction of iNOS, we used PD98059 to test whether it affects LPS plus IFN-{gamma}-stimulated NO2– production. The results showed that PD98059 (5–40 µM) dose dependently inhibited NO2– induction in LPS plus IFN-{gamma}-stimulated pM{varphi} (Fig. 5B). At the concentrations used, the compound did not affect cell viability (data not shown). These results were confirmed by RT–PCR of iNOS mRNA expression, which showed that PD98059 (20 µM) markedly decreased LPS plus IFN-{gamma}-induced iNOS mRNA expression (Fig. 5C). Taken together, these results suggested that ERK activation plays a positive role in resident pM{varphi} for iNOS induction.


Figure 5
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  		Fig. 5 Effect of PD98059 on iNOS expression induced by LPS plus IFN-{gamma} in pM{varphi}. (A) PD98059 inhibited the induced ERK activation. Resident pM{varphi} were pre-treated with PD98059 (40 µM) or medium alone for 1 h and then stimulated with IFN-{gamma} (2 ng ml–1) plus LPS (1 µg ml–1) for 30 min. Following preparation of cell lysates, 30 µg of each sample was resolved by SDS-PAGE and immunoblotted as described in Fig. 4. (B) Triplicate samples were cultured in 96-well plates and incubated with the indicated concentrations of PD98059 or medium alone for 1 h before stimulation with IFN-{gamma} (2 ng ml–1) plus LPS (1 µg ml–1). After further 24-h incubation, culture supernatants were collected, and NO2– was measured by Griess reaction. Results are expressed as mean (±SD). (C) pM{varphi} pre-treated with PD98059 (20 µM) or medium alone for 1 h were stimulated with IFN-{gamma} (2 ng ml–1) plus LPS (1 µg ml–1) for 6 h, and total RNA was extracted. iNOS mRNA expression was analyzed by real-time RT–PCR. Results are expressed as mean (±SD)-fold increase in specific mRNA level compared with non-stimulated pM{varphi}. Three experiments were performed with similar results.

 
Role of ERK on IL-12p40 expression in pM{varphi}
Earlier studies demonstrated that ERK activation suppressed LPS-mediated IL-12p40 expression in thioglycollate-elicited pM{varphi} (26). To assess the relative roles of ERK in LPS plus IFN-{gamma}-mediated IL-12 production in resident pM{varphi}, we investigated whether PD98059 affected the LPS plus IFN-{gamma}-stimulated IL-12p40 production in pM{varphi}. PD98059 enhanced the production of IL-12p40 in a dose-dependent manner (5–40 µM) (Fig. 6A), suggesting that ERK plays a negative regulatory role in pM{varphi} for LPS plus IFN-{gamma}-mediated IL-12p40 production. RT–PCR analysis also revealed that PD98059 enhanced IL-12p40 mRNA expression (Fig. 6B). Thus, these results demonstrated that, ERK plays a negative role in the regulation of signals leading to the induction of IL-12p40 in contrast to the positive effect on iNOS induction in resident pM{varphi}.


Figure 6
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  		Fig. 6 Effect of PD98059 on IL-12p40 expression induced by LPS plus IFN-{gamma} in pM{varphi}. (A) Triplicate samples were cultured in 96-well plates and incubated with the indicated concentrations of PD98059 or medium alone for 1 h before stimulation with IFN-{gamma} (2 ng ml–1) plus LPS (1 µg ml–1). After further 24-h incubation, culture supernatants were collected, and IL-12p40 was measured by ELISA. Results are expressed as mean (±SD). (B) pM{varphi} pre-treated with PD98059 (20 µM) or medium alone for 1 h were stimulated with IFN-{gamma} (2 ng ml–1) plus LPS (1 µg ml–1) for indicated periods, and total RNA was extracted. IL-12p40 mRNA expression was analyzed by real-time RT–PCR. Results are expressed as mean (±SD)-fold increase in specific mRNA level compared with non-stimulated pM{varphi}. Three experiments were performed with similar results.

 
ERK involved in the promotion of IL-12 production by anti-IL-10 mAb and indomethacin-treated resident pM{varphi}
IL-10 and PGE2 have been reported to be produced from LPS-stimulated M{varphi} (45, 46). pM{varphi} produced a large amount of IL-10 and PGE2 comparing with sM{varphi} in the culture without additional stimulation (Fig. 7A). To determine whether the impairment of IL-12 production in LPS plus IFN-{gamma}-stimulated pM{varphi} was reflected by endogenous immunosuppressive factors, IL-10 and PGE2, we measured the regulatory effect of anti-IL-10 mAb and indomethacin on IL-12. IL-12p40 was nearly undetectable in either unstimulated or LPS plus IFN-{gamma}-stimulated pM{varphi} culture supernatants (Fig. 7B). Indomethacin, a COX inhibitor which shuts off PGE2 synthesis, caused a significant increase of IL-12p40 production in pM{varphi} with LPS plus IFN-{gamma} stimulation. Anti-IL-10-neutralizing mAb pre-treatment also increased IL-12p40 production in pM{varphi} culture supernatants (Fig. 7B). pM{varphi} pre-treated with anti-IL-10 mAb plus indomethacin almost completely restored the deficient of IL-12p40 and biological functional form IL-12p70 productions in response to LPS plus IFN-{gamma} stimulation (Fig. 7B and C).


Figure 7
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  		Fig. 7 Effect of anti-IL-10 mAb and indomethacin on IL-12 production and ERK activation in pM{varphi}. (A) Resident pM{varphi} and sM{varphi} were cultured without stimulation for 2 h. Culture supernatants were collected and the level of IL-10 was measured by ELISA and PGE2 production was determined by RIA. Results are expressed as mean (±SD). Resident pM{varphi} pre-treated with anti-IL-10 mAb (10 µg ml–1), indomethacin (1 µM) or both for 2 h were stimulated with IFN-{gamma} (2 ng ml–1) plus LPS (1 µg ml–1). Culture supernatants were collected at 24 h, and the level of IL-12p40 (B), IL-12p70 (C) was measured by ELISA. Results are expressed as mean (±SD). (D) Cell lysates were harvested after stimulated with IFN-{gamma} (2 ng ml–1) plus LPS (1 µg ml–1) for indicated time. Western blot analysis of ERK activation was conducted as described in Fig. 4. (E) Comparison of IL-12p40 production from pM{varphi}, anti-IL-10 mAb plus indomethacin pre-treated pM{varphi}, PD98059 pre-treated pM{varphi} and sM{varphi} induced by IFN-{gamma} plus LPS stimulation. pM{varphi}, pM{varphi} pre-treated with anti-IL-10 mAb (10 µg ml–1) plus indomethacin (1 µM) for 2 h, pM{varphi} pre-treated with PD98059 (20 µM) for 1 h and sM{varphi} were stimulated with IFN-{gamma} (2 ng ml–1) plus LPS (1 µg ml–1). Culture supernatants were collected at 24 h, and the level of IL-12p40 was measured by ELISA. Results are expressed as mean (±SD). Three experiments were performed with similar results.

 
ERK mediates the suppression of IL-12 production (26) and IL-10 could up-regulate ERK activation (47, 48). Therefore, we investigated whether anti-IL-10 mAb plus indomethacin promoted IL-12 production by de-activating ERK in LPS plus IFN-{gamma}-stimulated pM{varphi}. The results showed that the treatment of pM{varphi} with anti-IL-10 mAb, indomethacin or anti-IL-10 mAb plus indomethacin significantly inhibited the activation of ERK in comparison with untreated pM{varphi} 30–60 min after LPS plus IFN-{gamma} stimulation (Fig. 7D). The results also supported our hypothesis that ERK may be involved in the impairment of IL-12p40 expression by endogenous IL-10 and PGE2 in pM{varphi}.

The IL-12p40 production of PD98059-treated pM{varphi} is lower than that of untreated sM{varphi} (Fig. 7E). Moreover, PD98059-treated pM{varphi} produced less IL-12p40 than pM{varphi} treated with anti-IL-10 mAb + indomethacin (Fig. 7E). It suggests that the promotion of IL-12p40 production by anti-IL-10 mAb + indomethacin involved other ERK-independent suppression mechanisms in addition to ERK-dependent mechanisms. Similarly, ERK-independent suppression mechanisms may account for the differential expression of IL-12p40 between pM{varphi} and sM{varphi}.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The results presented in this paper show that ERK activation is differentially induced in pM{varphi} and sM{varphi} with LPS plus IFN-{gamma} stimulation or anti-CD40 plus IFN-{gamma} stimulation and may account for differential regulation of effector responses (iNOS and IL-12) in pM{varphi} and sM{varphi}. Prolonged ERK activation not only contributes to the marked iNOS induction in pM{varphi} comparing with sM{varphi}, taken that ERK plays a positive role in iNOS expression, but also underlay the impairment in IL-12 induction in pM{varphi} comparing with sM{varphi}, taken that ERK plays a negative role in IL-12p40 expression.

Previous studies on the role of ERK in induction of iNOS and IL-12p40 have produced conflicting results, suggesting that the regulation of such gene expression varies in a receptor and cell type-dependent manner (49–51). Feng et al. (26) demonstrated that ERK contributes positive regulatory signals to the induction of iNOS and act to suppress expression of IL-12p40 in thioglycollate-elicited pM{varphi}. However, pM{varphi} elicited by non-specific inflammatory agents (e.g. thioglycollate and proteose peptone) are immature, inflammatory macrophages recruited from the circulating and marginal pool (52–54). How inflammatory agents affect the activation state and contribute to heterogeneity of M{varphi} has not been well studied. Regarding this, we choose to yield mature, resident M{varphi} from normal mouse peritoneal cavity, which possess physiologic characteristics. Using resident pM{varphi}, we confirm the differential regulation effect of ERK in iNOS and IL-12p40 expression as Feng et al. (26) described. In this paper, we have only studied the early events in LPS plus IFN-{gamma} signaling, including IFN-{gamma}-induced STAT-1 activation and consequent IRF-1 and ICSBP up-regulation, LPS-induced MAPK activation. Differences in transcription factors NF-{kappa}B (p50–p50, p50–c-rel and p50–p65 complexes) and IRFs (IRF-1, IRF-2 and ICSBP) activation and their binding to the promoter of iNOS and IL-12p40 between pM{varphi} and sM{varphi} were not referred to in this study. The suppression mechanism of IL-12p40 production by ERK is not clarified very well. Suppression of IRF-1 in the thioglycollate-elicited pM{varphi} stimulated by IFN-{gamma} plus LPS was reported (29) but this is not the case in this paper (Fig. 3B). LPS plus IFN-{gamma} stimulate the formation of multiple complexes at Ets site (which comprises complexes containing IRF-1) (19). LPS plus IFN-{gamma} similarly stimulate the binding of IRF-like complexes to the IRF site in the iNOS promoter (15). It seems that IRF-1 binding plays positive roles in both IL-12p40 and iNOS transcription. However, LPS plus IFN-{gamma}-stimulated IL-12p40 mRNA expression (Fig. 6B) is up-regulated and iNOS mRNA expression is down-regulated in PD98059 pre-treated M{varphi} (Fig. 5C). It suggests that IRF-1 protein expression may not contribute to both the suppression mechanism of IL-12p40 production by ERK and the promotion mechanism of NO production by ERK. Additional experiments should be executed to find out some nuclear complexes (whose components are known or unclear) binding to both IL-12p40 and iNOS promoter and contributed to both the suppression mechanism of IL-12p40 production by ERK and the promotion mechanism of NO production by ERK.

Then, we further investigated the possible mechanism responsible for the impairment in IL-12 production by resident pM{varphi} in response to LPS plus IFN-{gamma}. IL-10 and PGE2 could be produced by LPS-stimulated M{varphi} (45, 46). IL-10 has been reported to up-regulate ERK activation (47, 48); however, there was no information about the effect of PGE2 on the ERK activation in M{varphi}. Through our study, we suppose that PGE2 may act on ERK activation in the fashion similar with IL-10 (Fig. 7D). However, endogenous IL-10 and PGE2 inhibit IL-12 expression may partly through prolonging ERK activation in pM{varphi} and additional ERK-independent suppression mechanisms may exist because IL-12p40 production of PD98059-treated pM{varphi} is lower than that of pM{varphi} treated with anti-IL-10 mAb + indomethacin (Fig. 7E). Also, the differential expression of IL-12p40 between pM{varphi} and sM{varphi} may involve other ERK-independent suppression mechanisms in addition to ERK-dependent mechanisms given that the IL-12p40 production of PD98059-treated pM{varphi} is lower than that of sM{varphi} (Fig. 7E). LPS-induced activation of proteasome, I kappa B and NF-kappa B independent of protein kinase C, protein kinase A, ERK and p38 MAPK signal pathways regulate the IL-12 expression in M{varphi} (55). ERK-independent suppressive mechanisms involving IL-12p40 production in pM{varphi} need further studies.

Bamba et al. (7) showed that resident pM{varphi} is not able to stimulate T cell response to antigen, results consist with our finding in this study. Before co-culture, resident pM{varphi} express CD80, CD86 and CD40, but do not express high level of MHC-II. This is one of the reasons underlying the deficiency of pM{varphi} in T cells-stimulating capacity. In addition, taken that NO is the key mediator to inhibit T cells proliferation by pM{varphi} (9), together with the fact that pM{varphi} secrete large amounts of NO when activated while sM{varphi} do not produce NO, we conclude that pM{varphi}-derived NO may be the second key mediator. The third, sM{varphi}-derived IL-12 induce production of IFN-{gamma} from T cells (10, 11), which induces further activation of sM{varphi} and elicits more IL-12 from sM{varphi}. Compared with sM{varphi}, pM{varphi} produce little IL-12 and therefore are deficient in this positive feedback.

In conclusion, endogenous IL-10 and PGE2 may partly contribute to the prolonged ERK activation from pM{varphi} in response to LPS plus IFN-{gamma}; the prolonged ERK activation partly contribute to produce large amounts of NO but little IL-12 from pM{varphi}; high level of NO and low level of IL-12 may partly contribute to the deficiency in T cell-stimulating capacity of pM{varphi}.


   Acknowledgements
 
This work was supported by the grant of the Knowledge Innovation Program of Chinese Academy of Sciences (No. KSCX2-SW-202) and Shanghai Science and Technology Committee (No. 03DZ19228).


   Abbreviations
 
APC, antigen-presenting cell
ERK, extracellular signal-regulated kinase
[3H]TdR, [3H]thymidine
ICSBP, interferon consensus sequence-binding protein
iNOS, inducible nitric oxide synthase
IRF, interferon regulatory factor
MAPK, mitogen-activated protein kinase
M{varphi}, macrophage
NO, nitric oxide
OVA, ovalbumin
PGE2, prostaglandin E2
pM{varphi}, peritoneal macrophage
sM{varphi}, splenic macrophage
STAT, signal transducer and activator of transcription
RIA, radioimmunoassay

   Notes
 
Transmitting editor: T. Hamaoka

Received 20 December 2005, accepted 31 March 2006.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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