Published online before print June 24, 2004
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University of Virginia Health System, Departments of Medicine, Digestive Health Center of Excellence and Microbiology, Charlottesville
1 Correspondence: University of Virginia Health System, PO Box 800708, Charlottesville, VA 22908-0708. E-mail: mfs3k{at}virginia.edu
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Key Words: lipopolysacharide • cytokine • macrophage
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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IL-1Ra is member of the IL-1 gene family, which despite binding to the IL-1R with approximately equal affinities as IL-1
and IL-1ß, has no known agonist activities [9
, 10
]. Thus, IL-1Ra represents a naturally occurring means through which the proinflammatory action of IL-1 can be modulated. The term IL-1Ra actually refers to three closely related proteins. The first form to be described, secretory or sIL-1Ra, was cloned from immunoglobulin G-stimulated human monocytes and encodes a protein of 177 amino acids, including a 25 amino acid hydrophobic leader sequence, which is subsequently cleaved, resulting in a secreted 152 amino acid mature protein. An alternative form of IL-1Ra, intracellular or icIL-1Ra, was cloned from an adherent monocyte cDNA library [11
]. This structural variant is created when an alternative first exon is spliced into an internal acceptor site in the first exon of the sIL-1Ra RNA within the region encoding for the secretory leader sequence, resulting in a protein that is identical to the mature sIL-1Ra protein except for seven additional amino acids at the amino terminal end. A third isoform of IL-1Ra, termed icIL-1RaII, is derived from an alternative translation initiation at the second ATG of the sIL-1Ra or icIL-1Ra mRNA [12
].
We and others have demonstrated that treatment of macrophages with microbial products such as lipopolysaccharide (LPS) or peptidoglycan results in an increase in sIL-1Ra gene expression and protein secretion. However, unlike other cytokines, such as IL-1ß and TNF-, whose LPS-induced expression is inhibited by IL-10, the expression of IL-1Ra is actually enhanced. Such a response is generally consistent with the anti-inflammatory roles of IL-1Ra and IL-10. In the current study, we have explored the molecular mechanisms responsible for the IL-10-induced enhancement of IL-1Ra gene regulation in macrophages. A previous study by Lang et al. [13
] demonstrated that signal transducer and activator of transcription (STAT)3 was essential for all observed effects of IL-10 on LPS-induced cytokine gene expression. Consistent with this finding, we now demonstrate that the proximal region of the sIL-1Ra promoter contains a functional STAT3-binding site, which is essential for its response to IL-10. Additionally, we have determined that endogenous IL-10, produced in response to LPS stimulation, results in the activation of STAT3 and the enhancement of IL-1Ra gene expression in macrophages.
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Cells and cell culture
The RAW 264.7 murine macrophage cell line was obtained from American Type Culture Colleciton (Manassas, VA) and cultured in RPMI + 10% fetal bovine serum (FBS; Hyclone, Logan, UT). L929 cells were a gift of David Richies (National Jewish, Denver, CO) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) + 10% FBS. Bone marrow-derived macrophages (BMDM) were generated from C57BL/6J and IL-10–/– (on the C57BL/6J background) mice obtained from Jackson Laboratories (Bar Harbor, ME). Bone marrow cells from the femurs of 8- to 16-week-old mice were cultured on petri dishes for 5 days in DMEM supplemented with 10% FBS and 20% L929 cell-conditioned media (LCCM; as a source of macrophage-colony stimulating factor). Half of the media was replaced on Day 3 and completely changed on Day 4. On Day 5, adherent macrophages were harvested from the plates and split into six-well tissue-culture dishes at 
1 x 106 cells per well and cultured overnight in the absence of LCCM prior to stimulation as necessary.
Plasmid DNAs
The 294-bp human (h)sIL-1Ra promoter/luciferase reporter construct was described previously [15
, 16
]. A recombinant polymerase chain reaction (PCR) approach was used to generate the mutated STAT-binding element (mSBE) promoter construct using the wild-type (WT) hIL-1Ra promoter as a template. To generate a mutation in the putative SBE, primers mutated interferon (IFN)-stimulated response element (mISRE)2-A (5'-AGGGTTTCTCTCgaattcGATGCGAGGAGGGT-3') and mISRE2-B (the reverse complement of A) were used to mutate 6 bp within the putative SBE, thus creating a new EcoRI site. PCR reaction 1 was performed with mISRE2-B and a KpnI-ended oligonucleotide 296F, specific for bases –296 to –276. A second reaction was performed with mISRE2-A and a HindIII-ended oligonucleotide corresponding to bases +12 to + 27 of the hIL-1Ra gene [methylation-specific PCR 1 (MSPCR1)]. The resulting PCR products were purified, and equal molar amounts were mixed and used as templates in a third PCR reaction using the two outside primers (296F and MSPCR1). The final PCR product was digested with KpnI and HindIII and cloned into pA3Luc. Nucleotide sequences were confirmed by automated sequencing at the University of Virginia Biomolecular Research Facilty (Charlottesville).
The dominant-negative STAT3 (Y705A) expression plasmid was a kind gift of Larry Pfeffer (University of Tennessee Health Science Center, Memphis). All plasmid DNAs were purified using the endotoxin-free prep kit from Clontech (Palo Alto, CA).
Northern blot analysis
Total RNA was purified using the Trizol reagent (Invitrogen, Carlsbad, CA). For Northern blot, 20 µg RNA was electrophoresed through a formaldehyde-containing 1.5% agarose gel and transferred onto nitrocellulose membranes. Membranes were prehybridized for 4 h at 42°C in a solution containing 50% formamide, 5x saline sodium citrate, 5x Denhardt’s solution, 50 mM KH2PO4, and 250 µg/ml salmon sperm DNA and were then hybridized overnight at 42°C with a radiolabeled cDNA probe for mouse IL-1Ra {labeled with [-32P]-deoxycytidine 5'-triphosphate using the Nick translation system (Invitrogen)}. After autoradiography, the IL-1Ra probe was stripped from the blot, and the filter was reprobed with a 32P-labeled glyceraldehyde 3-phosphate dehydrogenase (GAPDH) probe and autoradiographed.
Transfections and luciferase assays
RAW 264.7 cells were transfected using the calcium phosphate procedure as described previously [17
]. In addition to the indicated sIL-1Ra promoter/luciferase reporter plasmid, all cells were cotransfected with 0.5 µg renilla luciferase reporter plasmid (phRLTK; Promega, Madison, WI) as an internal control for transfection efficiency. Following stimulation as indicated, cultures were lysed, luciferase activities were determined using the dual luciferase kit from Promega, and all activities normalized to the activity of the cotransfected renilla plasmid. Data are expressed as means ± SD of triplicates. All transfection experiments were performed at least three times.
Quantitative reverse transcriptase (RT)-PCR
Total RNA was purified using the Trizol reagent (Invitrogen). RT of 0.5 µg total cellular RNA was performed in a final volume of 20 µl containing 5x first-strand buffer, 1 mM each deoxy-unspecified nucleoside 5'-triphosphate, 20 U placental RNase inhibitor, 5 µM random hexamer, and 9 U Moloney murine leukemia virus RT (Invitrogen). After incubation at 37°C for 45 min, the samples were heated for 5 min at 92°C to end the reaction and stored at –20°C until PCR use. cDNA (2 µl) was subjected to real-time, quantitative PCR using the MJ Research Opticon system with SYBR Green I (Molecular Probes, Eugene, OR) as a fluorescent reporter. sIL-1Ra, IL-10, and hypoxanthine guanine phosphoribosyl transferase (HPRT) cDNAs were amplified in separate reactions. Duplicate PCR reactions were performed for each sample, and the average threshold cycle number was determined using the Opticon software. Levels of sIL-1Ra or IL-10 expression normalized to HPRT levels were determined using the formula 2(Rt–Et), where Rt is the threshold cycle for the reference gene (HPRT), and Et is the threshold cycle for the experimental gene (
CT method). Data are thus expressed as arbitrary units. Sequences for the oligonucleotides used are provided in Table 1
.
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Table 1. RT-PCR Primer Sequences
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DNA affinity isolation of STAT3
A 34-bp double-stranded and biotinylated oligonucleotide corresponding to bases –125 to –92 of the hsIL-1Ra promoter was bound to streptavidin-coated magnetic beads (Dynal, Great Neck, NY). Nuclear extracts from macrophages were prepared as described previously [18
]. Binding reactions were performed by diluting 50 µg nuclear extract in binding buffer (12.5 mM HEPES, pH 7.9, 31.25 mM KCl, 0.625 mM dithiothreitol, 3.125% glycerol, 125 µg/ml bovine serum ablumin) to achieve a final salt concentration of 30 mM NaCl and 28 mM KCl and were incubated with 50 µl magnetic beads/immobilized DNA for 60 min at 4°C. Beads were washed three times with 100 µl 0.5x binding buffer. After the final wash, the beads were collected and resuspended in 100 µl SDS sample buffer, loaded onto a SDS-polyacrylamide gel electrophoresis (PAGE), and analyzed for STAT3 by Western blot as described above. For oligonucleotide competitions, diluted nuclear extracts were preincubated with the desired double-stranded oligonucleotide for 30 min at 4°C prior to the addition of immobilized DNA.
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Figure 1. IL-10 enhances LPS-induced IL-1Ra mRNA and promoter activity. Northern blot analysis of IL-1Ra mRNA expression. RAW 264.7 murine macrophages were stimulated with 1 µg/ml LPS, 5 ng/ml IL-10, or the combination for 4 h. Total RNA was isolated, blotted, and probed as described in Materials and Methods. C, Control.
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Figure 2. Dominant-negative STAT3 blocks IL-10-induced sIL-1Ra promoter activity. RAW 264.7 cells were transiently transfected with the hsIL-1Ra/luciferase reporter plasmid and 2 µg empty vector control DNA or an expression plasmid for dominant-negative STAT3 (Y705A). Cultures were stimulated with LPS, IL-10, or the combination for 8 h prior to assay for luciferase activity. Firefly luciferase was normalized to the activity of the cotransfected phRLTK, as described in Materials and Methods. Results are means ± SEM of three experiments. *, P < 0.05, compared with LPS alone by Student’s t-test.
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Examination of the DNA sequence within this proximal 148-bp promoter fragment indicated a potential SBE between –101 and –110: TTTTGGAAAA. Notably, this sequence is perfectly conserved between the human and mouse genes. A biotinylated, double-stranded oligonucleotide corresponding to bases –125 to –92 of the IL-1Ra promoter was bound to streptavidin-coated magnetic beads and used as a probe to isolate DNA-binding proteins from nuclear extracts of RAW 264.7 cells. The pulled-down proteins were then electrophoretically separated by SDS-PAGE and probed for STAT3 by Western blot. As demonstrated in Figure 3A , STAT3 binding to the IL-1Ra promoter was indeed induced following stimulation with IL-10 or LPS + IL-10 for 30 min. Preincubation of nuclear extracts from IL-10-stimulated RAW 264.7 cells with a 30-bp oligonucleotide surrounding this sequence resulted in a decrease of STAT3 binding to the biotinylated promoter fragment (Fig. 3B) . In contrast, an oligonucleotide containing a 7-bp mutation within the core SBE (TTTTGGAAA changed to TcgaattcA) did not block binding to the –148 fragment. These results therefore demonstrated that the proximal sIL-1Ra promoter region contained a specific binding site for IL-10-induced STAT3. We have termed this promoter element SBE3.
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Figure 3. IL-10 induces STAT3 binding to the sIL-1Ra promoter. (A) Nuclear extracts were prepared from RAW 264.7 macrophages stimulated with 5 ng/ml IL-10, 1 µg/ml LPS, or the combination for 45 min. Extracts were reacted with a biotinylated sIL-1Ra promoter fragment and bound proteins subjected to Western blotting as described Materials and Methods. (B) Nuclear extracts from IL-10-stimulated cells were reacted with the biotinylated sIL-1Ra promoter fragment as in A in the absence or presence of a 25-fold molar excess of a 30-bp double-stranded oligonucleotide corresponding to the putative STAT3-binding site. Mut, Site-specific mutation within the STAT3-binding site. Results are representative of three separate experiments.
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Figure 4. Mutation of the STAT3-binding site at –110 abolishes IL-10-induced sIL-1Ra promoter activity. RAW 264.7 cells were transiently transfected with the WT hsIL-1Ra/luciferase reporter or one that contained a mSBE. Cultures were stimulated with 1 µg/ml LPS (solid bars) in the absence or presence of 5 ng/ml IL-10 for 8 h prior to assay for luciferase activity. Results are the means ± SEM of three separate experiments. *, P < 0.05, compared with LPS alone by Student’s t-test.
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Figure 5. IL-10 mRNA expression in RAW 264.7 cells and BMDM. Total RNA was isolated from cells stimulated for the indicated time with 1 µg/ml LPS and analyzed for IL-10 mRNA expression by quantitative RT-PCR as described in Materials and Methods. Results are from a representative experiment of three separate performed with each cell type.
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4 h following stimulation, sIL-1Ra mRNA expression from IL-10–/– BMDM was clearly lower than that observed in the WT macrophages. The differences in sIL-1Ra expression between the two strains increased with time, suggesting that endogenously produced sIL-10 plays an important role in LPS-induced sIL-1Ra gene expression. This result was reproducible in three separate experiments, although the absolute levels of sIL-1Ra mRNA varied somewhat between experiments. In all experiments, stimulation of IL-10–/– macrophages with LPS in the presence of exogenously added IL-10 resulted in a restoration of the response to mRNA levels comparable with or greater than those observed in the WT macrophages (Fig. 6B) .
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Figure 6. Role of endogenously produced IL-10 in the LPS-induced expression of sIL-1Ra mRNA in BMDM, which from C57BL/6 or IL-10–/– mice, were stimulated with 100 ng/ml LPS for the indicated time prior to harvest and assay for sIL-1Ra mRNA expression by quantitative RT-PCR as described in Materials and Methods. (A) IL-10–/– macrophages had decreased levels of sIL-1Ra mRNA compared with WT macrophages following LPS stimulation. (B) Costimulation of IL-10–/– macrophages with 5 ng/ml IL-10 restored the LPS-induced sIL-1Ra mRNA expression to WT levels. Results are typical of three separate experiments.
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mRNA expression in the WT versus IL-10–/– macrophages. Although the levels of sIL-1Ra mRNA were decreased in the IL-10–/– macrophages, levels of TNF- mRNA were elevated consistent with previous studies [3
, 21
] (data not shown). Thus, the decreased sIL-1Ra mRNA expression observed in the LPS-stimulated IL-10–/– macrophages was likely a result of the loss of IL-10 production and not a defect in response to LPS.
LPS-induced STAT3 activation in WT versus IL-10–/– macrophages
As demonstrated above, the sIL-1Ra promoter contains a STAT3-binding site, which was required for the response of this gene to IL-10. As IL-10 produced by BMDM appeared to contribute to the LPS-induced IL-1Ra gene expression, we assessed the activation of STAT3 in response to LPS. BMDM from WT or IL-10–/– mice were stimulated with LPS, IL-10, or IL-6 for varying times, and STAT3 phosphorylation on tyrosine 705 was assessed as a marker of STAT3 activation. As shown in Figure 7
, IL-10 and IL-6 induced a rapid phosphorylation of STAT3 in WT and IL-10–/– macrophages. Stimulation of WT macrophages with LPS resulted in a delayed induction of STAT3 phosphorylation, which was evident by 1 h and continued for at least 3 h. This timing was consistent with that observed for the LPS-induced expression of IL-10 in the WT macrophages. In contrast, the induction of STAT3 phosphorylation in the IL-10–/– macrophages was delayed and severely blunted compared with the WT macrophages. This same pattern of STAT3 phosphorylation was observed in three separate experiments. Consistent with our findings of poor IL-10 production by LPS-activated RAW 264.7 cells, LPS-induced STAT3 activation was not observed in this cell line (data not shown).
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Figure 7. LPS induced STAT3 phosphorylation in WT and IL-10–/– macrophages. Equal numbers of BMDM from C57BL/6 (upper) or IL-10–/– (lower) were stimulated with 100 ng/ml LPS, 5 ng/ml IL-10, or 5 ng/ml IL-6 for the indicated time (minutes). Cells were lysed and analyzed for STAT3 tyrosine 705 phosphorylation (pSTAT3) as described in Materials and Methods. Results are typical and from a representative experiment of three performed.
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Figure 8. LPS-induced STAT3 activation requires de novo IL-10 production. (A) Treatment of BMDM with cycloheximide (CXH) abolished LPS-induced STAT3 phosphorylation (pSTAT3). BMDM were incubated with 10 µg/ml cycloheximide for 15 min prior to stimulation with 100 ng/ml LPS (2 h) or 5 ng/ml IL-10 (15 min). Lysates were analyzed for STAT3 Y705 phosphorylation as described in Materials and Methods. (B) IL-10-neutralizing antibodies inhibited LPS-induced STAT3 phosphorylation. C57BL/6 BMDM were incubated with neutralizing antibodies to IL-10 (IL-10; 1:50 dilution) concurrently with 100 ng/ml LPS for 1 h prior to assay for STAT3 Y705 phosphorylation. Results shown are from representative experiments of four performed.
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LPS-induced STAT3 binding to the IL-1Ra promoter
Finally, to determine if the LPS-activated STAT3 is in fact capable of binding DNA and potentially regulating the expression of the sIL-1Ra gene, we prepared nuclear extracts from C57BL/6J BMDM stimulated with IL-10 or LPS and examined STAT3 binding to a fragment of the sIL-1Ra promoter as described above. As shown in the STAT3 Western blot in Figure 9
, exogenous IL-10 rapidly (15 min) induced STAT3 binding as did LPS stimulation (2 h). Taken together, these experiments demonstrate that stimulation of macrophages with LPS results in the phosphorylation and DNA binding of STAT3 via an autocrine mechanism involving the de novo production of IL-10.
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Figure 9. LPS induced STAT3 binding to the hsIL-1Ra promoter. Nuclear extracts were prepared from C57BL/6 BMDM stimulated with 5 ng/ml IL-10 (15 min) or 100 ng/ml LPS (2 h). Extracts were reacted with the biotinylated hsIL-1Ra promoter fragment and analyzed for STAT3 binding by Western blot as in Figure 3
. This experiment was repeated four times, and the results shown are from one representative experiment.
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and IL-6 [26
, 27
]. Although IL-10 is known predominantly for its ability to inhibit proinflammatory cytokine production, early on, it was also appreciated that it played a positive role in the regulation of another anti-inflammatory cytokine, IL-1Ra [7 , 8 ]. The balance between the production of IL-1 and IL-1Ra has been shown to play an important role in the severity of and/or susceptibility to a broad variety of infectious and chronic inflammatory diseases [28 ]. In a mouse model of collagen-induced arthritis, IL-1Ra KO mice develop a much more severe disease than do their WT littermates, and overexpression of IL-1Ra results in a significant reduction in incidence and severity [29 ]. Likewise, in several animal models of inflammatory bowel disease, neutralization of IL-1Ra has been demonstrated to exacerbate disease, and treatment with IL-1Ra attenuates the disease process [30 31 32 33 ]. Notably, mice deficient in IL-10 [23 , 34 ] and macrophage/neutrophil-specific STAT3 expression [35 ] spontaneously develop a severe enterocolitis as a result of increased proinflammatory cytokine production and perhaps, in part, owing to a decreased production of IL-1Ra as our current studies demonstrate.
In these studies, we described the molecular mechanisms, whereby IL-10 leads to an increase in sIL-1Ra gene expression in macrophages. Previous studies in human neutrophils indicated that IL-10 up-regulated LPS-induced IL-1Ra production [36
] and that IL-10 and IL-4 synergized with TNF- to induce IL-1Ra expression [6
, 37
]. In both studies, IL-10 was shown to result in an increase in IL-1Ra mRNA stability, which led to an accumulation of message and increased protein production. Our current studies thus extend these findings by demonstrating that IL-10 can also enhance the de novo transcription of the sIL-1Ra gene, at least in macrophages. The effect of IL-10, provided exogenously or endogenously as a consequence of LPS stimulation, resulted in the activation of STAT3 and its binding to a site within the proximal region of the hsIL-1Ra promoter. We identified the STAT3-binding site as the DNA sequence TTTTGGAAA located between –101 and –110 upstream of the transcriptional start site in the hsIL-1Ra promoter; this sequence is perfectly conserved in the mouse, rat, and hIL-1Ra genes. A previous study by Lang et al. [13
] demonstrated, using macrophage-specific STAT3 KO, that the expression of STAT3 was necessary for the IL-10-induced effects on all genes examined. Our studies described herein provide the first clear evidence that IL-10-activated STAT3 acts directly on the promoter for the sIL-1Ra gene to induce its activation.
It is intriguing that the effect of STAT3 activation was evident only when macrophages were costimulated with LPS, indicating that IL-10-induced STAT3 activation alone is insufficient to induce gene transcription. Consistent with our report, Crepaldi et al. [37
] also noted that IL-10-induced STAT3 activation was insufficient for inducing IL-1Ra gene expression in the absence of a second signal (e.g., TNF-). In contrast, Jenkins et al. [8
] and Williams et al. [25
] demonstrated that IL-10 alone could induce IL-1Ra expression from peripheral blood monocytes. The reason for this discrepancy is unclear, but we hypothesize that adherence of the monoyctes and/or the purification procedure may have provided the initial priming signal required for IL-10-induced IL-1Ra production. The nature of this second signal is under investigation, but it appears to be unrelated to the binding of PU.1 to two sites described previously within the proximal promoter region [18
]. Mutation of those two binding sites, although severely impairing the response to LPS, did not affect the ability of IL-10 to enhance the activation of the promoter in the presence of LPS (data not shown). One possible clue to the nature of this putative priming signal is the LPS-induced activation of c-jun. Zhang et al. [38
] described a direct protein–protein interaction between STAT3 and c-jun that was necessary for the IL-6-induced expression of the 2-macroglobulin gene. Thus, it is possible that STAT3 and c-jun cooperate to activate transcriptionally the sIL-1Ra gene. This possibility is currently being explored.
Recent studies by Benkhart et al. [39
] demonstrated that the LPS-induced expression of the IL-10 gene was critically dependent on a STAT3-binding site within the promoter. In those studies, they also demonstrated that LPS stimulation of macrophages led to the induction of STAT3 phosphorylation and DNA-binding activity. This effect was observed following a 4-h stimulation with LPS, a time that is consistent with the LPS-induced STAT3 activation, which we have observed in our studies (Fig. 8)
. Benkhart et al. [39
] did not identify the mechanism responsible for the LPS-induced STAT3 activation but speculated that it may be a result of the production of other cytokines including IL-6, IFN-, and IL-10. In those studies, antibody neutralization of autocrine IL-6 did not inhibit IL-10 gene transactivation. Our studies comparing LPS-induced STAT3 activation in WT and IL-10 KO macrophages and with the use of IL-10-neutralizing antibodies have pointed to a clear link between the autocrine production of IL-10 and STAT3 activation. We have not assessed whether neutralization of endogenously produced IL-10 will also effect IL-10 production in these cells.
In summary, the present study has demonstrated that IL-10, acting by inducing the activation of STAT3 and its binding to a specific site within the proximal promoter region, can induce the expression of the sIL-1Ra. Additionally, we demonstrated a positive role for endogenously produced IL-10 in the response of the sIL-1Ra gene in LPS-stimulated macrophages. Finally, LPS was capable of inducing a delayed activation of STAT3 that was dependent on de novo protein synthesis and the production of IL-10. These results serve to provide additional insight into the mechanisms behind one of the important cytokine networks involved in the innate immune response to infection.
Received October 30, 2003; revised May 7, 2004; accepted May 26, 2004.
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] to induce IL-1ra production by human neutrophils Cytokine 8,147-151[CrossRef][Medline]
B/PU.1/GA-binding protein-binding site J. Biol. Chem. 273,24272-24279- and interferon -induced genes by suppressing tyrosine phosphorylation of STAT1 Blood 93,1456-1463
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