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(Journal of Leukocyte Biology. 2002;72:1198-1205.)
© 2002 by Society for Leukocyte Biology

Interleukin-10 differently regulates monocyte chemoattractant protein-1 gene expression depending on the environment in a human monoblastic cell line, UG3

Takashi Ikeda*, Ken Sato*, Naruo Kuwada*, Takuya Matsumura*, Takuya Yamashita*, Fumihiko Kimura*, Kiyohiko Hatake{dagger}, Kazuma Ikeda{ddagger} and Kazuo Motoyoshi*

* Third Department of Internal Medicine, National Defense Medical College, Saitama, Japan;
{dagger} Division of Clinical Chemotherapy, Japanese Foundation for Cancer Research, Tokyo, Japan; and
{ddagger} Division of Blood Transfusion, Okayama University Medical School, Japan

Correspondence: Kazuo Motoyoshi, M.D., Ph.D., Third Department of Internal Medicine, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan. E-mail: motoyosi{at}me.ndmc.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Interleukin (IL)-4, IL-10, and IL-13 affect monocyte/macrophage functions including regulation of cytokine production. We analyzed the regulatory effects of these cytokines on cytokine production using a human monoblastic cell line, UG3. It is interesting that IL-10 up-regulated, whereas IL-4 and IL-13 down-regulated monocyte chemoattractant protein-1 (MCP-1) production by unstimulated UG3 cells. IL-10-induced expression of MCP-1 mRNA occurred without de novo protein synthesis at transcriptional and post-transcriptional levels. The enhancement of binding activity of nuclear factor Sp1 (Sp-1) and signal transducer and activators of transcription (STAT)1 and 3 but not nuclear factor {kappa}B (NF-{kappa}B) was associated with this IL-10-induced MCP-1 expression. Furthermore, IL-10 suppressed lipopolysaccharide (LPS)-induced NF-{kappa}B binding but not Sp-1. The present results suggest IL-10 has two contrasting actions on the MCP-1 production of monocytes/macrophages, between the resting and activated conditions. The combination of activated Sp-1 and STATs is important for IL-10-induced MCP-1 expression in resting monocytes/macrophages, and the inhibition of LPS-induced NF-{kappa}B binding is crucial for down-regulation of MCP-1 by IL-10 in stimulated monocytes/macrophages.

Key Words: monocytes • transcription factors • Sp-1 • STAT


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemokines are identified as chemotactic cytokines and are divided into two families, C-C and C-X-C chemokines. The first two cysteine residues are separated by one amino acid in C-X-C chemokines ({alpha}-chemokine), whereas they are adjacent in C-C chemokine (ß-chemokine) [1 , 2 ]. Most of the C-X-C chemokines recruit neutrophilic and basophilic granulocytes, and C-C chemokines mainly act on monocytes and lymphocytes. Monocyte chemoattractant protein-1 (MCP-1), one of the C-C chemokines, has potent chemotactic activities against monocytes/macrophages (Mo/Mø) [1 , 3 ]. Recent studies confirmed MCP-1 was implicated in atherosclerosis [4 ], inflammatory bowel disease [5 ], glomerulonephritis [6 ], pulmonary fibrosis [7 ], asthma [8 ], rheumatoid arthritis [9 ], and modulation of tumor immunity [10 , 11 ]. MCP-1 is produced by a variety of cell types including Mo/Mø, fibroblasts, vascular endothelial cells, intestinal epithelial cells, and smooth muscle, in response to lipopolysaccharide (LPS), interleukin (IL)-1, tumor necrosis factor {alpha} (TNF-{alpha}), phorbol myristate acetate (PMA), and interferon-{gamma} (IFN-{gamma}) [2 , 3 , 6 , 12 ].

One of the major sources of MCP-1 is Mo/Mø, and their activity is controlled by IFN-{gamma}, IL-4, IL-10, and IL-13. IL-4, IL-10, and IL-13 are secreted from T helper cell type 2 (Th2) lymphocytes [13 ] and also exert various effects on Mo/Mø. They have anti-inflammatory activities for resting and stimulated Mo/Mø that suppress the secretion of mediators, such as IL-1ß, IL-6, IL-8, IL-12, TNF-{alpha}, granulocyte-colony stimulating factor (G-CSF), and prostaglandin E2 (PGE2) [14 15 16 17 18 19 ]. MCP-1 production is also suppressed by IL-4, IL-10, and IL-13 in Mo/Mø stimulated with LPS [20 ]. In the case of unstimulated Mo/Mø, however, the effect of IL-10 against MCP-1 production is controversial. Some previous studies showed IL-10 inhibited MCP-1 production in various cells, including Mo/Mø. The effect is similar to that of other Th2-associated cytokines [21 , 22 ]. However, Yano et al. [20] and Seintz et al. [23 ] reported that IL-10 induces MCP-1 production in human normal monocytes, whereas it is down-regulated by IL-4 and IL-13. There have been some phenomena supporting the induction of monocyte-chemotactic factors by IL-10 in vivo. The infiltration of a relatively large number of macrophages has been documented in human nonsmall lung carcinoma, which produce IL-10 [24 , 25 ]. A transient monocytosis after in vivo administration of IL-10 has also been reported [26 ].

There have been no known studies on the regulatory mechanisms of IL-10-induced MCP-1 expression. The lack of a cell line reproducing these reactions is one of the reasons. Most of the previous human monoblastic cell lines required chemicals such as PMA to differentiate into functional Mo/Mø, which leads to activation of the cells. Therefore, those cells could not be used as models of unstimulated Mo/Mø. Previously, we established a human monoblastic cell line, UG3, which has the ability to release various cytokines such as IL-1ß, IL-6, TNF-{alpha}, IL-8, granulocyte macrophage (GM)-CSF, G-CSF, and M-CSF [27 ]. In the present study, we examined the response of UG3 cells against LPS, IL-4, IL-10, and IL-13 on cytokine production. We were able to reproduce IL-10-induced MCP-1 release from unstimulated UG3 cells, in contrast to down-regulation by IL-4 and IL-13. This is the first cell line that is available as a model to investigate IL-10-induced MCP-1 production. Using this cell line, we confirmed IL-10 enhanced MCP-1 gene expression at transcriptional and post-transcriptional levels and also the involvement of increased binding activity by nuclear factor Sp1 (Sp-1), signal transducer and activators of transcription (STAT)1, and STAT3, but neither activating protein-1 (AP-1) nor nuclear factor {kappa}B (NF-{kappa}B), in this IL-10-induced MCP-1 gene expression. Furthermore, LPS-induced binding activity of NF-{kappa}B, but not Sp-1, was inhibited by IL-10 in a dose-dependent manner. This finding may be the reason for suppression of MCP-1 release from stimulated monocytes by IL-10.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and cell preparation
Recombinant human (rh)IL-3 and GM-CSF were from Kirin Brewery Co. Ltd. (Tokyo, Japan). rhM-CSF was provided by Morinaga Milk Industry Inc. (Tokyo, Japan). UG3 cells were maintained at a density between 4 and 8 x 105/mL in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 5% fetal calf serum (FCS) and IL-3 (5 ng/mL) at 37°C in an atmosphere of 5% CO2 in air. The cells were washed twice with phosphate-buffered saline (PBS), then moved to IMDM containing 5% FCS with GM-CSF (1 ng/mL) or M-CSF (100 ng/mL) for preincubation. All experiments were performed after 2 weeks preincubation in the presence of GM-CSF (1 ng/mL; UG3/GM cells) or M-CSF (100 ng/mL; UG3/M cells). Normal peripheral monocytes were obtained from the peripheral blood of normal volunteers. After gradient centrifugation, monocytes were collected using an eltriator from the mononuclear cell fraction.

Detection of cytokines in the culture supernatant
After 2 weeks preincubation, the cells were harvested using a cell scraper (Sumitomo Bakelite Co. Ltd., Tokyo, Japan), and were washed twice with PBS and once with IMDM supplemented with 5% FCS. Then, cells were resuspended in IMDM containing 5% FCS without any cytokines at the final cell concentration of 5 x 105/mL. At this time, reagents were added into the culture medium to give the final concentration as indicated. Six hours after incubation with the effectors at 37°C, each culture supernatant was analyzed by enzyme-linked immunosorbent assay (ELISA) with commercially available kits according to the manufacturer’s instructions.The kits used for analysis were as follows: IL-1ß [minimal detectable concentration (MDC), 3.9 pg/mL], IL-6 (MDC, 3.1 pg/mL), IL-8 (MDC, 31.3 pg/mL), TNF-{alpha} (MDC, 15.6 pg/mL), MCP-1 (MDC, 31.2 pg/mL), G-CSF (MDC, 39.1 pg/mL), M-CSF (MDC, 31.3 pg/mL), and GM-CSF (MDC, 7.8 pg/mL), which were all supplied by R&D Systems, Inc. (Minneapolis, MN), and PGE2 (MDC, 10 pg/mL), which was obtained through PerSeptive Biosystems, Inc. (Framingham, MA).

RNA isolation and Northern blot analysis
Total RNA was extracted by the guanidium isothiocyanate procedure from UG3/GM cells incubated in the presence or absence of the agents for the indicated times. The denatured RNA was electrophoresed in 1% agarose gel containing 2.2 mol/L formaldehyde, transferred to a nylon membrane (Hybond-N, Amersham Japan, Tokyo), and hybridized with the 32P-labeled probe. The membranes were autoradiographed with X-ray film at -70°C with an intensifying screen. In some experiments, autoradiography was generated on a BAS2000 (Fuji, Tokyo, Japan). To prepare the MCP-1 probe, cDNA was obtained from RNA isolated as described above by the reverse transcriptase-polymerase chain reaction method using primer pairs, MCP-1s: 5'-ACTCTCGCCTCCAGCATGAAAGTC-3' and MCP-1as: 5'-TGCAAAGACCCTCAAAACATCCCA-3'. This amplified cDNA was inserted into the plasmid (Original TA Cloning® kit, Invitrogen Corp., Carlsbad, CA) according to the manufacturer’s instructions. The plasmid DNA was purified by equilibrium centrifugation in CsCl-ethidium bromide gradients and was then digested with EcoRI using the DNA probe for MCP-1 after sequencing. The ß-actin DNA probe was obtained from pHFß A-3'ut plasmid by digestion with EcoRI and BamHI.

Determination of the involvement of de novo protein synthesis in IL-10-induced MCP-1 mRNA expression
To determine the necessity of de novo protein synthesis in IL-10-induced MCP-1 mRNA expression, we examined the effects of a protein synthesis inhibitor, cycloheximide. Cells were prepared as described above and preincubated for 30 min in the absence or presence of 2 µg/mL cycloheximide. Then, IL-10 was added to the medium to reach 25 ng/mL as the final concentration. After 2 h incubation with or without IL-10 in the presence or absence of cycloheximide, total RNA was isolated and analyzed by Northern blotting as described above.

Transcriptional run-on assay
In brief, UG3/GM cells (5x107 cells) were incubated in the IMDM supplemented with IL-10 (50 ng/mL) for the indicated times. Nuclei were isolated by lysing the cells in an ice-cold hypotonic buffer: 10 mmol/L Tris-HCl (pH 7.4), 10 mmol/L KCl, 3 mmol/L MgCl2, and 0.5% Nonidet P-40. After washing with the same buffer, nuclei were resuspended in nuclear storage buffer: 40% glycerol, 50 mmol/L Tris-HCl (pH 8.3), 5 mmol/L MgCl2, 0.1 mmol/L EDTA. Then, nuclei were incubated for 30 min at 30°C in a reaction buffer containing 150 mmol/L KCl, 2.5 mmol/L MgCl2, 5 mmol/L Tris-HCl (pH 8.0), 0.25 mmol/L adenosine 5'-triphosphate (ATP), 0.25 mmol/L guanosine 5'-triphosphate, 0.25 mmol/L cytidine 5'-triphosphate, and 200 µCi {alpha}-32P-uridine 5'-triphosphate (3000 Ci/mmol), and the reaction was terminated by the addition of DNase I. The reaction mixture was digested with proteinase K (400 µg/mL) and was followed by phenol/chloroform extraction. Nuclear RNAs were pelleted using isopropanol and passed through a Bio-Spin® chromatography column (Bio-Rad Laboratories, Hercules, CA). As hybridization substances, plasmid DNAs were denatured and spotted onto nylon membrane using a slot blot apparatus (BIO-DOT SF, Bio-Rad Laboratories). Newly elongated nuclear RNAs were hybridized to the membrane in the solution containing 1 x 107 cpm/mL 32P-labeled RNA at 42°C for 3 days. The membranes were autoradiographed with X-ray film at -70°C with an intensifying screen.

Gel shift assay
UG3 cells were cultured in the presence or absence of LPS (10 ng/mL) and/or various concentration of IL-10 for 1 h. After washing twice with PBS, the cells were lysed in the extraction buffer containing 20 mmol/L HEPES, 20% glycerol, 10 mmol/L NaCl, 0.2 mmol/L EDTA, 1.5 mmol/L MgCl2, 0.1% Triton X-100, 1 mmol/L orthovanadate, protease inhibitors, 1 mmol/L dithiothreiotol, 10 µg/mL phenylmethylsulfonyl fluoride, leupeptin, pepstatin, and aprotinin. Released nuclei were confirmed with trypan blue dye exclusion under a microscope and were resuspended in the extraction buffer as stated above. Then, NaCl was added to the sample, giving 0.4 mol/L as the final concentration. Extracted nuclear protein was frozen immediately and stored at -80°C until use for the assays. The protein concentration was measured by Bradford methods. The sequence of double-stranded DNA probes (Santa Cruz Biotechnology, Santa Cruz, CA) was as follows: Sp-1, 5'-AGTTGAGGGGACTTTCCCAGGC-3'; NF-{kappa}B, 5'-CGCTTGATGACTCAGCCGGAA-3'; AP-1, 5'-GGTCCCGGGCGGCGTCGGT-3'; STAT1, 5'-ATTCCTGTAAG-3'; and STAT3, 5'-TTCTGGGAATT-3'. These probes were labeled with {gamma}-32P-ATP using T4 polynucleotide kinase. For binding reactions, nuclear extracts containing 10–20 µg protein were incubated with 32P-labeled probes in the binding buffer (12 mmol/L HEPES, pH 7.9, 60 mmol/L NaCl, 4 mmol/L MgCl2, 1 mmol/L EDTA, 1 mmol/L dithiothreiotol, and 14% glycerol) containing 4 µg polydeoxyinosinic-deoxycytidylic acid at room temperature for 30 min. The reaction products were analyzed by electrophoresis in a 5% polyacrylamide gel.

Statistical analyses
Statistical analyses were performed with the paired t-test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of LPS on cytokine production by UG3 cells
Based on previous findings [27 ], UG3 cells preincubated for 2 weeks in the presence of GM-CSF (1 ng/ml; UG3/GM) or M-CSF (100 ng/ml; UG3/M) were used as the model of monocytes or macrophages, respectively. The concentrations of the mediators produced by UG3/GM or UG3/M cells are summarized in Table 1 . LPS enhanced production of IL-1ß, IL-6, TNF-{alpha}, IL-8, MCP-1, G-CSF, and PGE2 by UG3/GM and UG3/M cells. LPS-induced enhancement of the production of IL-1ß, IL-6, TNF-{alpha}, and G-CSF by UG3/GM cells tended to be strong compared with that by UG3/M cells. In contrast, enhanced IL-8, MCP-1, and PGE2 secretion by LPS was stronger in UG3/M cells than in UG3/GM cells.


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  		Table 1. Effects of LPS, IL-4, IL-10, and IL-13 on Cytokine Production by UG3 Cells

 
Effects of IL-4, IL-10, and IL-13 on MCP-1 production by UG3 cells
IL-4, IL-10, and IL-13 inhibited LPS-induced production of all mediators by UG3/GM and UG3/M cells (Table 1) . Suppression was strongest by IL-10 and weakest by IL-13 among all the mediators analyzed, in UG3/GM and UG3/M cells. Suppression of mediator secretion by IL-13 reached a similar degree as IL-4 at 50 ng/ml but did not reach the level of IL-10 (data not shown). IL-4, IL-10, and IL-13 suppressed the production of IL-6, TNF-{alpha}, IL-8, and PGE2 by UG3/GM and UG3/M cells without LPS stimulation (data not shown). However, IL-10 up-regulated MCP-1 release by both types of unstimulated UG3 cells, whereas IL-4 and IL-13 down-regulated it (Table 2 ). MCP-1 regulation by Th2-associated cytokines may differ with the cell type or their conditions even in the same cell type, monocytes. As the increase in MCP-1 production by UG3/GM cells following IL-10 stimulation was much stronger than that by UG3/M cells, UG3/GM cells were used in the experiments of MCP-1 gene expression. We confirmed this IL-10-induced MCP-1 production using normal monocytes in the absence of LPS stimulation, which was similar to previous findings [20 , 23 ] (Table 2) .


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  		Table 2. Effects of IL-4, IL-10, and IL-13 on MCP-1 Production by Normal Monocytes and Unstimulated UG3 Cells

 
Dose- and time-dependency and involvement of de novo protein synthesis in IL-10-induced MCP-1 production
UG3/GM cells constitutively produced low levels of MCP-1 without any stimuli at the protein and mRNA levels (Figs. 1 and 2 ). IL-10 stimulated the accumulation of MCP-1 protein (Fig. 1) and mRNA (Fig. 2) , in UG3/GM cells dose-dependently. The levels of MCP-1 mRNA were rapidly increased at 1 h after addition of IL-10 and reached maximum at 2 h (Fig. 3 ). At 4 h after the stimulation, it had returned to the prestimulation level. Next, we analyzed whether IL-10 directly induced MCP-1 mRNA expression using cycloheximide, an inhibitor of protein synthesis. Cycloheximide alone induced MCP-1 mRNA expression. IL-10-induced MCP-1 mRNA expression was further augmented by cycloheximide (Fig. 4 ). These results confirmed that IL-10 increased accumulation of MCP-1 mRNA without de novo protein synthesis. Cycloheximide-induced MCP-1 mRNA expression suggests the involvement of de novo protein synthesis in mRNA destabilization or enhancement of gene transcription. We also analyzed the involvement of cyclooxygenase or PGE2 in the IL-10-induced increase in MCP-1 mRNA. However, there were no significant differences in IL-10-induced MCP-1 mRNA expressions with or without indomethacin (data not shown). Based on these results, we analyzed the transcriptional and post-transcriptional regulation of IL-10-induced MCP-1 mRNA expression.



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  		Figure 1. Dose-dependent effects of IL-10 on MCP-1 production by unstimulated UG3/GM cells. The concentration of MCP-1 released into culture supernatant by UG3/GM cells (2x105 cells/mL) was measured with ELISA after 6 h incubation with various concentration of IL-10. Error bars indicate the standard deviation of the MCP-1 concentration. *P < 0.01 and **P < 0.05 compared with MCP-1 production by untreated cells. Results are representative of three independent experiments.

 


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  		Figure 2. Dose-dependent effects of IL-10 on the levels of MCP-1 mRNA. UG3/GM cells were incubated for 2 h with various concentrations of IL-10. Total RNA (25 µg/lane) obtained from the cells was analyzed by Northern blotting. Results are representative of three independent experiments.

 


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  		Figure 3. Time-dependent effects of IL-10 on MCP-1 mRNA expression. After incubation with IL-10 (50 ng/mL) for the indicated durations, the level of MCP-1 mRNA in UG3/GM cells was analyzed by Northern blotting. Total RNA blotted in each lane was 25 µg. Results are representative of three independent experiments.

 


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  		Figure 4. Effects of protein synthesis inhibitor on the expression of MCP-1 mRNA after incubation with IL-10. UG3/GM cells were preincubated in the presence or absence of cycloheximide (CHX; 2 µg/mL) for 30 min and then further incubated with or without IL-10 (50 ng/mL) for 2 h. Total RNA (20 µg/mL) was prepared and blotted as described in Materials and Methods after incubation. Results are representative of three independent experiments.

 
Transcriptional and post-transcriptional regulation of IL-10-induced MCP-1 mRNA expression
We performed the transcriptional run-on assay and half-life assay to clarify the mechanisms of IL-10-induced MCP-1 mRNA accumulation. Transcriptional run-on assay confirmed that the MCP-1 gene was constitutively transcribed at low levels in unstimulated UG3/GM cells. UG3/GM cells exposed to IL-10 for 1 h showed an 11.2-fold increased transcriptional rate of MCP-1 (Fig. 5 ). The half-life of steady-state MCP-1 mRNA in unstimulated cells was not greater than 30 min, whereas that in the cells stimulated with IL-10 was ~90 min (Fig. 6 ). This result showed that IL-10 enhanced the stability of MCP-1 transcripts.



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  		Figure 5. Transcriptional activity of MCP-1 mRNA in the presence or absence of IL-10. UG3/GM cells were cultured in the presence or absence of IL-10 (50 ng/mL) for 1 h, and nuclei were isolated as described in Materials and Methods. Newly elongated RNAs were hybridized to the plasmid containing MCP-1 (5 µg), ß-actin (3 µg), or pUC19 (5 µg) as a control to detect nonspecific hybridization. Results are representative of two independent experiments.

 


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  		Figure 6. Stability of steady-state MCP-1 mRNA exposed to IL-10. UG3/GM cells were cultured in the presence or absence of IL-10 (50 ng/mL) for 2 h, and then actinomycin D (5 µg/mL) was added to the cultures for the indicated durations. Total RNA was analyzed by Northern blotting. The value of RNA blotted in each lane was 30 µg or 20 µg in the cells incubated in the presence or absence of IL-10, respectively. The radioactivity compared with that of ß-actin is shown as a graph (lower). The cells before the incubation with actinomycin D were assumed to have 100% relative radioactivity. Results are representative of three independent experiments.

 
Effect of IL-10 on LPS-induced binding activity of transcription factors of resting and stimulated UG3/GM cells
The human MCP-1 5'-flanking region has several cis-acting, transcription-regulatory elements, including the IFN-{gamma}-activated site, {kappa}B-binding sites, PMA-responsive elements, and the GC box as a binding site of STATs, NF-{kappa}B, AP-1, and Sp-1, respectively [12 , 28 29 30 ]. We performed a gel-shift assay to examine the effects of IL-10 on the binding activity of these transcription factors. Binding of nuclear protein to the probe was observed to be low for all probes, even in unstimulated cells. Similar to previous findings [31 , 32 ], the binding activity for NF-{kappa}B was increased after stimulation with LPS, and IL-10 inhibited this LPS-induced binding to NF-{kappa}B in a dose-dependent manner (Fig. 7A ). No change was shown in the binding activity for NF-{kappa}B after stimulation with IL-10 alone (Fig. 7A) . As for Sp-1, LPS enhanced binding activity to some extent, and IL-10 markedly enhanced it in a dose-dependent manner (Fig. 7B) . In addition, we confirmed that IL-10 enhanced binding of nuclear protein for STAT1 (Fig. 8A ) and STAT3 (Fig. 8B) but not AP-1 (data not shown) in UG3/GM cells. The shifted band was shown in the reaction with nuclear protein obtained from IL-10-induced cells, which were similar to the positive controls. We used nuclear extracts from cells treated with IFN-{gamma} and IL-6 as positive control in STAT1 and STAT3, respectively. Those bands disappeared by incubation with the cold probe (Fig. 8) .



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  		Figure 7. Effects of IL-10 on the binding activity of transcription factors. Gel-shift assay was performed to analyze the change in the binding activity for the NF-{kappa}B (A) and Sp-1 (B) by LPS and/or IL-10. The 32P-labeled probe was incubated with nuclear extracts obtained from UG3/GM cells stimulated with or without LPS (10 ng/mL) and/or IL-10 at various concentrations for 1 h. The arrows indicate specific DNA-protein complexes. Results are representative of three independent experiments.

 


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  		Figure 8. Effects of IL-10 on the binding activity of STATs. Gel-shift assay was performed to analyze the binding activity for STAT1 (A) and STAT3 (B) by LPS and/or IL-10. The 32P-labeled probe was incubated with nuclear extracts obtained from UG3/GM cells stimulated with or without LPS (10 ng/mL) and/or IL-10 (100 ng/mL) for 1 h. As the positive control, nuclear extracts from cells treated with IFN-{gamma} and IL-6 were used in STAT1 and STAT3, respectively. The arrows indicate specific DNA-protein complexes. Results are representative of three independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
LPS induces Mo/Mø to produce various cytokines, such as IL-1, IL-6, TNF-{alpha}, IL-8, MCP-1, GM-CSF, and G-CSF [33 ], and UG3 cells could reproduce these responses. Unlike other factors, MCP-1 production from Mo/Mø has been controversial. Although some studies reported that LPS could not stimulate MCP-1 production from Mo/Mø [34 , 35 ], others reported stimulation of MCP-1 production [20 , 36 ] and also down-regulation of constitutive MCP-1 release by LPS treatment [12 , 37 ]. Yano et al. [20 ] reported that the responsiveness of normal Mo/Mø to LPS varied markedly among donors. The difference in cell source and environment may be the reason for those discrepancies. In the present study, LPS stimulated MCP-1 secretion from UG3/GM and UG3/M cells.

Similar to previous findings [14 15 16 17 18 19 ], IL-4, IL-10, and IL-13 suppressed the production of IL-6, TNF-{alpha}, IL-8, and PGE2 by UG3/GM and UG3/M cells (data not shown). However, IL-10 up-regulated MCP-1 release by both types of unstimulated UG3 cells, whereas IL-4 and IL-13 down-regulated it (Table 2) . The present results are compatible with those of previous studies using normal peripheral blood monocytes or alveolar macrophages [20 , 23 ]. Recently, Kucharzik et al. [22 ] reported that MCP-1 synthesis in intestinal epithelial cells and Caco-2 cells was suppressed by IL-10, regardless of the kind of concomitant stimulation. Furthermore, Flesch et al. [38 ] reported that IL-4 is an activator, and IL-10 is a deactivator for Listeria monocytogenes-infected monocytes. MCP-1 regulation by Th2-associated cytokines may differ with the cell type or the conditions within cells of the same type, Mo/Mø. UG3 is the first cell line that reproduced these reactions to Th2-associated cytokines.

The present results showed that IL-10 stimulated the accumulation of MCP-1 protein and mRNA dose-dependently. Furthermore, IL-10-induced MCP-1 mRNA expression was independent of de novo protein synthesis. The transcriptional regulation is one of the important mechanisms for the accumulation of mRNA, and the present results suggested that IL-10 markedly increased the rate of MCP-1 transcription. Stabilization of mRNA is also important for an increase in protein synthesis, especially for cytokines because of the short lives of RNAs. As shown in Figure 6 , IL-10 enhanced stability of MCP-1 transcripts. These findings demonstrated that IL-10-induced expression of MCP-1 mRNA occurred without de novo protein synthesis at transcriptional and post-transcriptional levels.

MCP-1 expression in monocytes, glioblastoma, fibrosarcoma, and leiomyosarcoma cells is controlled by an NF-{kappa}B-binding site that appears to be associated with IL-1ß-, TNF-{alpha}-, LPS-, and PMA-specific regulation [29 , 31 , 39 ]. A promoter proximal Sp-1 site is required for TNF-induced MCP-1 expression in murine monocytes [39 ] or basal transcriptional activity in various cell types [29 ]. Other studies suggested that AP-1 could be a major regulator of PMA-induced MCP-1 transcription [28 , 40 ]. IFN-{gamma}-induced MCP-1 expression requires IFN-{gamma}-activated site-binding activity involving STAT1 in astrocytoma cells [30 ]. STAT1 and STAT3 are also well known to be involved in the signaling pathway from IL-10 receptor in Mo/Mø [41 ].

The present results suggested IL-10-induced MCP-1 expression involved the activation of STATs and Sp-1, but neither NF-{kappa}B nor AP-1 did. Previous studies confirmed IL-10 triggered the tyrosine phosphorylation of STAT1 and STAT3 via the Janus tryrosine kinase (Jak)-STAT pathway [42 , 43 ]. However, there are no reports of IL-10 signaling via Sp-1 binding. It was suggested that the Sp-1-binding site proximal to the transcription initiation site of the MCP-1 gene is mainly required for basal transcriptional activity of MCP-1 [29 ]. Recently, Ping et al. [44 ] reported that the lack of Sp-1-binding activity resulted in the inaction of the MCP-1 promoter assembly, although the binding of NF-{kappa}B is unaffected. Although further studies are essential, the present results suggest the possibility of complicated interactions between Sp-1 and STATs lead to this IL-10-induced MCP-1 expression in unstimulated Mo/Mø. When Mo/Mø are stimulated by LPS, IL-10 may down-regulate MCP-1 production according to the inhibition of NF-{kappa}B-binding activity, although IL-10 enhanced Sp-1 and STATs binding activity. The difference between the combination of activated or deactivated transcription factors could be the reason for the contrasting actions of IL-10 between UG3 cells with and without LPS stimulation.

IL-4, IL-10, and IL-13 exhibit contrasting actions on some aspects of Mo/Mø function, in addition to MCP-1 production. IL-10 inhibits the major histocompatibility complex class II expression and antigen presentation capacity, whereas IL-4 and IL-13 enhance them [16 ]. Moreover, IL-10 stimulates the expression of the p75 TNF receptor in monocytes stimulated with LPS, in contrast to the suppression by IL-4 [45 ]. IL-4 and IL-13 share the common {gamma} chain of the IL-2 receptor in T cells [15 ], but IL-10 has its own unique receptor [46 ]. The difference in the receptors may be one of the reasons for these contrasting findings among IL-10, IL-4, and IL-13. In fact, the signal from IL-4 or IL-13 receptors is transmitted to STAT6 but not to other STATs or Sp-1 [47 ]. Furthermore, IL-10 triggers the tyrosine phosphorylation of STAT1 and STAT3 via the Jak-STAT pathway [42 , 43 ].

The IL-10-induced signal pathways or interactions with other cytokines are still controversial. In fact, it was reported that IL-10 does not affect IFN-{gamma}-induced tyrosine phosphorylation of STAT1 or STAT1-binding capacity to the IFN-{gamma} response element [48 ]. However, other studies showed the ability of IL-10 to suppress IFN-induced tyrosine phosphorylation of STATs [49 ]. Cassatella et al. [50 ] confirmed the existence of an IL-10-induced CIS3/suppressor of cytokine signaling 3 mRNA expression pathway, which is independent of STATs in human neutrophils. Furthermore, Ping et al. [39 ] demonstrated the importance of a novel dimethylsulfate-hypersensitive sequence in addition to the {kappa}B site and GC box in TNF-induced MCP-1 expression in murine monocytes. Even the existence of novel regulatory regions in IL-10-induced MCP-1 expression in Mo/Mø may be possible. UG3 is the first cell line that was confirmed to reproduce such varied physiological phenomena, including the reactions to Th2-associated cytokines [27 , 51 ]. UG3 cells thus provide features to qualify them as a useful model to further investigate the regulatory mechanism of cytokine production and signal transduction of these cytokines on Mo/Mø.


   ACKNOWLEDGEMENTS
 
We thank Dr. Jesse Dallin for helpful suggestions to prepare this manuscript.

Received August 25, 2001; revised July 30, 2002; accepted August 9, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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