Abstract
IL-9 is a Th2 cytokine active on various cell types such as T and B lymphocytes, mast cells, and eosinophils, and potentially involved in allergy and asthma. To understand better the molecular mechanisms underlying the activity of this cytokine, we used a cDNA subtraction method to identify genes specifically induced by IL-9 in mouse T cells. One of the IL-9-regulated genes isolated by this approach turned out to encode a 180-amino acid long protein, including a potential signal peptide, and showing 22% amino acid identity with IL-10. This protein, designated IL-10-related T cell-derived inducible factor (IL-TIF), is induced by IL-9 in thymic lymphomas, T cells, and mast cells, and by lectins in freshly isolated splenocytes. Experiments concerning the mechanism regulating IL-TIF expression in T cells indicate that IL-9 induction is rapid (within 1 h), does not require protein synthesis, and depends on the activation of the Janus kinase (JAK)-STAT pathway. In vivo, constitutive expression of IL-TIF was detected by RT-PCR in thymus and brain, suggesting that the role of this new factor is not restricted to the immune system. Transfection of HEK293 cells with the IL-TIF cDNA resulted in the production of a glycosylated protein of about 25 kDa that was found to induce STAT activation in mesangial and neuronal cell lines. Further studies will have to address the possibility that some of the IL-9 activities may be mediated by IL-TIF.
Interleukin-9 is a cytokine produced by activated Th2 lymphocytes and is active on various cell types from the hemopoietic and lymphoid systems. Originally identified as a T cell growth factor, IL-9 also acts on mast cells, B lymphocytes, T cell clones, hemopoietic progenitors, and immature neuronal cell lines (1). In addition, IL-9 seems to be involved in both human and murine tumorigenesis. IL-9 overexpression, indeed, results in a high susceptibility to the development of T cell lymphomas in vivo (2), and constitutive IL-9 expression has been discovered in many human Hodgkin lymphomas (3). More recently, the involvement of IL-9 has been suggested in asthma (4, 5), in line with the effect of this cytokine on IgE production (6), mast cell differentiation (7), and eosinophil activation (8).
IL-9 activities are mediated through a receptor that belongs to the hemopoietin receptor superfamily and associates with γc, also involved in IL-2, IL-4, IL-7, and IL-15 signaling. Upon IL-9 stimulation, the tyrosine kinases JAK3 and JAK1, associated with γc and the IL-9R, respectively, become activated and phosphorylate the IL-9R on a single tyrosine residue. This amino acid is essential for activation of transcription factors STAT1, STAT3, and STAT5 by IL-9, and was shown to be required for most activities of IL-9 investigated so far, such as cell differentiation, regulation of proliferation, and inhibition of corticoid-induced apoptosis (9, 10).
To characterize further the biological activities of IL-9, we tried to identify genes inducible by IL-9 by using a cDNA subtraction approach. In this report, using IL-9-stimulated T lymphoma cells, we isolated a new gene encoding a protein with characteristic features of a cytokine: the presence of an N-terminal hydrophobic signal peptide, a predicted size of 20 kDa, and a 22% amino acid identity with IL-10. In addition, the supernatant of cells transiently transfected by this cDNA was found to induce activation of STAT3 and STAT5 in mesangial and neuronal cell lines.
Materials and Methods
Cell cultures, transfections, and cytokines
BW5147 lymphoma cells were grown in Iscove-Dulbecco’s medium supplemented with 10% FCS, 50 μM 2-ME, 0.55 mM l-arginine, 0.24 mM l-asparagine, and 1.25 mM l-glutamine. MC9 mast cell line was cultured in the same medium supplemented with mIL-3 (100 U/ml from Chinese hamster ovary (CHO) cell supernatants) or mIL-9 (200 U/ml). MES13 mesangial cells were grown in 3:1 mixture of DMEM medium and Ham’s F12 medium supplemented with 14 mM HEPES, 5% FCS, 50 μM 2-ME, 0.55 mM l-arginine, 0.24 mM l-asparagine, and 1.25 mM l-glutamine. The PC12 rat pheochromocytoma cell line was grown in RPMI 1640 containing 10% FCS. HEK293-EBV nuclear Ag (EBNA)3 human embryonic kidney cells and COS-7 were grown in DMEM medium supplemented with 10% FCS. T helper cell clone TS2 (11) was grown in DMEM medium supplemented with 10% FCS, 50 μM 2-ME, 0.55 mM l-arginine, 0.24 mM l-asparagine, and 1.25 mM l-glutamine, and either mIL-9 (200 U/ml) or hIL-2 (200 U/ml). RAW264.7 cells were cultured in DMEM medium supplemented with 5% FCS (Myoclone Super Plus bovine serum; Life Technologies, Grand Island, NY).
BW5147 cells expressing wild-type or mutated hIL-9R were obtained as previously described (9) and cultured in the presence of 1.5 μg/ml puromycin. Two additional hIL-9R mutants, mut6 (activating STAT1 and -3) and mut7 (activating STAT5), were similarly transfected in BW5147 (10). Recombinant mouse and human IL-9 were produced in the baculovirus system in our laboratory and purified as previously described (12). Human recombinant IL-2 (3.5 × 106 U/mg) was provided by Cetus (Chiron Corporation, Amsterdam, The Netherlands). Human recombinant IL-10 (5 × 105 U/mg) was purchased from Peprotech (London, U.K.)
Representational difference analysis
Total RNA was prepared from BW5147 cells stimulated with mIL-9 (200 U/ml) or normal medium for 48 h, using guanidium isothiocyanate lysis and CsCl gradient centrifugation (13). Polyadenylated RNA was purified from total RNA with oligo(dT) cellulose columns (Pharmacia, Uppsala, Sweden). Double-stranded cDNA was generated from 5 μg poly(A)+ RNA using an oligo(dT) primer and the SuperScript Choice System for cDNA synthesis, according to the manufacturer’s recommendations (Life Technologies). Representational difference analysis was performed as described (14, 15).
After three rounds of subtraction, final difference products were digested with DpnII and cloned into the BamHI site of pTZ19R. Double-stranded plasmid DNA was prepared and sequenced with a Thermo-sequenase Sequencing kit (Amersham, Arlington Heights, IL). Sequence comparisons with the GenBank and EMBL databases were performed with the BLAST search program. Oligo(dT)-primed cDNA libraries generated from IL-9-stimulated BW5147 cells were screened with the mouse IL-10-related T cell-derived inducible factor (IL-TIF) DpnII fragment as described (16).
RT-PCR analysis and Southern blotting
Spleen cells (5 × 106/condition) from 6-wk-old BALB/c mice were stimulated 24 h in control medium supplemented or not with LPS (20 μg/ml) or with Con A (1 μg/ml) in the presence of a blocking anti-mIL-9 goat antiserum (1%). Total RNA was isolated using the TRIzol Reagent, according to the manufacturer’s recommendations (Life Technologies). The same method was used to isolate RNA from cytokine-stimulated cell lines (5 × 106 cells/condition). Protein synthesis inhibitor cycloheximide (Sigma, St. Louis, MO) was used at 10 μg/ml. Total RNA was isolated from various organs of 6-wk-old normal FVB/N mice using guanidium isothiocyanate lysis and CsCl gradient centrifugation (13). Reverse transcription was performed on 5 μg total RNA with an oligo(dT) primer. cDNA corresponding to 5 ng of total RNA was amplified for 35 cycles by PCR with specific primers for IL-TIF as follows: sense 5′-CTGCCTGCTTCTCATTGCCCT-3′ (from position 124 of the cDNA sequence), antisense 5′-CAAGTCTACCTCTGGTCTCAT-3′ (from position 784 of the cDNA sequence); for β-actin, sense 5′-ATGGATGACGATATCGCTGC-3′, antisense 5′-GCTGGAAGGTGGACAGTGAG-3′ (18 cycles). Annealing temperatures were 57°C and 60°C for IL-TIF and β-actin, respectively. The post PCR products were analyzed in ethidium bromide-stained 1% agarose gels. In some experiments, specific amplification was confirmed after blotting (ζ-Probe membrane, Bio-Rad, Hercules, CA) by hybridization with internal radioactive probes. The internal probe was 5′-GTCCACGACATCATGCTACTG-3′ for β-actin, and 5′-GACGCAAGCATTTCTCAGAG-3′ for IL-TIF.
Transient transfection for production of recombinant IL-TIF protein
For transient transfection of an IL-TIF expression plasmid in the HEK293-EBNA and COS-7 cell lines, the IL-TIF cDNA was cloned, respectively, into pCEP-4 plasmid (Invitrogen, Groningen, The Netherlands) under the control of the CMV promoter and into pEF-BOS plasmid (17), under the control of the EF-1α promoter. Cells were seeded in 6-well plates (Nunc, Roskilde, Denmark) at 3 105 cells/well 1 day before transfection. Transfections were conducted using Lipofectamine method (Life Technologies), according to the manufacturer’s recommendation with 2 μg of plasmid DNA. After transfection, cells were incubated in 1.5 ml of normal medium for 3 days for maximal production of recombinant IL-TIF. For radioactive labeling of IL-TIF, transfected cells were incubated in 0.6 ml of methionin-free medium supplemented with 60 μCi 35S-labeled methionin for 24 h, and 12.5 μl of supernatant were analyzed by SDS-PAGE in a 14% acrylamide gel, followed by autoradiography for 1 day.
Detection of IL-TIF by Western blot analysis
A synthetic peptide beginning with an NH2-terminal cysteine residue followed by the mIL-TIF sequence 40–61 (CKLEVSNFQQPYIVNRTFMLAK) was conjugated to maleimide-activated keyhole limpet hemocyanin (KLH) (inject-maleimide activated carrier proteins; Pierce, Rockford, IL). Two rabbits were immunized with 150 μg of peptide-KLH conjugate in CFA by multiple-site intradermal injection. At 3-wk intervals, the rabbits were boosted with 150 μg of peptide-conjugated in IFA by multisite subcutaneous injections. Fifteen days after the second boost, the animals were test bled.
Ten microliters from supernatant of HEK293 cells transfected with IL-TIF were mixed with Laemmli sample buffer and boiled for 5 min. Proteins were separated on a precast NOVEX SDS-PAGE polyacrylamide gel (14%). After transfer, the polyvinylidene difluoride (PVDF) membrane (Amersham) was blocked in 5% nonfat dry milk, washed, and probed with rabbit polyclonal IL-TIF antiserum (1/500) and with HRP-linked anti-rabbit Ab (1/5000; Amersham). An ECL detection kit (Amersham) was used for expression of chemiluminescence.
STAT activation assays
Nuclear extracts were prepared as described previously (9). Briefly, cells (5 × 106) were stimulated for 10 min with transfected 293-EBNA cell supernatants (1%), washed with PBS, and resuspended in 1 ml ice cold hypotonic buffer “A” for 15 min (10 mM HEPES buffer (pH 7.5), containing 10 mM KCl, 1 mM MgCl2, 5% glycerol, 0.5 mM EDTA, 0.1 mM EGTA, 0.5 mM DTT, 1 mM Pefabloc (Boehringer Mannheim), 10 μg/ml aprotinin, and 5 mM NaF). The cells were lysed by adding 65 μl Nonidet P-40 10% and vortexing. Nuclei were pelleted (30 s at 14,000 rpm) and extracted for 30 min in 100 μl hypertonic buffer “B” (buffer “A” supplemented with HEPES (20 mM), glycerol (20%) and NaCl (420 mM)). Nuclear debris was removed through a 2-min centrifugation. Analysis of DNA binding activity was performed as described (18) using a 32P-labeled oligonucleotide probe corresponding to the 3′ 18 bp of the γ response region (GRR) of the FcγRI gene: upper strand, 5′-ATGTATTTCCCAGAAA-3′; lower strand, 5′-CCTTTTCTGGGAAATAC-3′.
Supershifts were performed by adding the following Abs to the mixture of nuclear extracts and labeled DNA probe: 0.75 μg of anti-STAT1 Ab (catalog no. G16920, Transduction Laboratories, Lexington, KY), 1 μg anti-STAT3 Ab (clone ST3-5G7, Zymed Laboratories, San Francisco, CA), or 1 μg anti-STAT5b Ab (sc no. 835-X, Santa Cruz).
For Western blot analysis, cells (4 × 105) were stimulated for 10 min with 293-EBNA cell supernatants (2%), or with hIL-10 (10 ng/ml), before lysis in 80 μl of SDS sample buffer (Bio-Rad). Five microliters of the samples were separated on a precast NOVEX SDS-PAGE polyacrylamide gel (14%). After transfer, the nitrocellulose membrane (Hybond C, Amersham) was blocked in 5% nonfat dry milk, washed, and probed with rabbit polyclonal anti-phospho-STAT3 Abs (1/10,000, from New England Biolabs, Beverly, MA) and with HRP-linked anti-rabbit Ab (1/5000; Amersham). An ECL detection kit (Amersham) was used for expression of chemiluminescence. The blots were subsequently reprobed with anti-β actin Abs (1/10,000; from Sigma) to confirm equal loading of the different lanes.
Results
Identification of a new IL-9-induced gene in BW5147 lymphoma
Based on the hypothesis that biological activities of this cytokine result from its ability to activate specific target genes, we performed a representational difference analysis of gene expression on BW5147 lymphoma cells that were stimulated with IL-9 for 24 h. Previously described activities of IL-9 on these cells include protection against dexamethasone-induced apoptosis and induction of differentiation genes such as Granzyme A and Ly6A/E (19). BW5147 cells were stimulated with IL-9 or control medium for 24 h before mRNA extraction. The isolation of IL-9-induced transcripts was performed by using the representational difference analysis technique as described by Hubank and colleagues (14). Oligo(dT)-primed cDNAs were synthesized, digested with DpnII, and used to generate the respective amplicons. After three rounds of subtractive hybridization, the third difference product was cloned, and 24 clones were sequenced. Three different IL-9-induced transcripts were identified. One of these clones consisted of a 225-nucleotide DpnII fragment with no homology with previously described genes. The full-length sequence was obtained by screening of an oligo(dT)-primed cDNA library from IL-9-stimulated BW5147 cells. The largest clone isolated contained 1124 nucleotides including a 537-bp open reading frame that encodes a 179-amino acid protein.
Further analysis of the predicted amino acid sequence showed the presence of an hydrophobic N-terminal sequence suggestive of a signal peptide for protein secretion. In addition, protein homology searches in the GenBank databases with the BlastP program detected a 22% amino acid identity with mouse and human IL-10. No significant homology was found with any other cytokine. This protein was designated IL-TIFα (for IL-lo-related T cell-derived Inducible factor α) because preliminary results from genomic sequencing indicate the existence of at least another gene encoding a highly related protein. IL-TIFβ differs only from IL-TIFα by 3 amino acid changes. The amino acid alignment of IL-TIFα, IL-TIFβ, and IL-10 is shown in Fig. 1⇓.
Alignment of murine IL-TIFα, IL-TIFβ, and IL-10 protein sequences. Residues sharing identity between IL-TIF and IL-10 are boxed. European Molecular Biology Laboratory (EMBL) Nucleotide Sequence Database accession number of the murine IL-TIFα cDNA is AJ249491; for IL-TIFβ cDNA, AJ249492.
Expression of IL-TIF mRNA
First, the kinetics of IL-TIF expression in BW5147 cells was studied by RT-PCR after IL-9 stimulation for various periods of time. RT-PCR was performed with oligonucleotides common to both forms of IL-TIF. As shown in Fig. 2⇓, IL-TIF mRNA was up-regulated within 30 min of IL-9 stimulation, and maximal expression was reached at 3 h of stimulation. This expression was stable until at least 48 h of IL-9 stimulation. Analysis of IL-TIF expression in various cell lines revealed that IL-TIF was also induced in mast cells upon IL-9, but not IL-3 stimulation, as well as in T helper clones upon IL-9, but not IL-2 stimulation (Fig. 3⇓A). Expression of IL-TIF was also detected in Con A-activated spleen cells. Noticeably, neutralizing anti-IL-9 Abs failed to inhibit this induction (Fig. 3⇓B), suggesting either that Con A can directly induce IL-TIF expression or that other cytokines can also mediate this process.
Kinetics of IL-TIF mRNA up-regulation by IL-9. BW5147 cells were cultured in the absence or in the presence of 200 U/ml IL-9 for the indicated periods of time. Total RNA was extracted, and RT-PCR amplification was performed with oligonucleotides specific for IL-TIF or β-actin. A Southern blot of the PCR products was performed, and filters were hybridized with IL-TIF or β-actin internal oligonucleotides.
Inducibility of IL-TIF expression. A, The indicated IL-9-responsive cells were cultured in medium containing saturating concentrations of the indicated cytokines: 10 days in the presence of 100 U/ml IL-2 or 200 U/ml IL-9 for T helper cell clone TS2, 10 days in the presence of 200 U/ml IL-3 or 200 U/ml IL-9 for mast cell line MC9. Total RNA was extracted, and RT-PCR amplification was performed with oligonucleotides specific for IL-TIF or β-actin. After blotting of the PCR products, filters were hybridized with IL-TIF or β-actin internal oligonucleotides. B, Spleen cells (5 × 106) were stimulated 24 h with 20 μg/ml LPS or with 1 μg Con A in the presence of a 1% blocking anti-mIL-9 goat antiserum. Total RNA was extracted, and RT-PCR amplification was performed with oligonucleotides specific for IL-TIF or β-actin.
IL-TIF expression was also analyzed in vivo by RT-PCR. Constitutive IL-TIF expression was reproducibly detected in thymus and at lower level in brain (Fig. 4⇓). In vivo expression of IL-TIF was not modified by IL-9 overexpression in transgenic animals (data not shown), probably resulting from the need of preactivation to induce IL-9 responsiveness of nontransformed cells (1).
Tissue distribution of IL-TIF mRNA. Total RNA was isolated from various organs of 6-wk-old normal FVB/N mice using guanidinium isothiocyanate lysis and CsCl gradient centrifugation. The RT-PCR amplification was performed with oligonucleotides specific for IL-TIF. A Southern blot of the PCR products was performed, and filters were hybridized with an IL-TIF-specific internal oligonucleotide. Similar results were obtained from FVB/N mice overexpressing the IL-9 gene (data not shown).
Mechanisms of IL-TIF induction by IL-9
To determine whether IL-9 directly up-regulates IL-TIF expression or whether this process requires protein synthesis, BW5147 cells were stimulated with IL-9 in the presence of cycloheximide. As shown in Fig. 5⇓, cycloheximide did not affect IL-TIF expression, as assessed by RT-PCR analysis, indicating that new protein synthesis is not required for this IL-9 activity. To analyze further the mechanisms of IL-TIF up-regulation in response to IL-9, we took advantage of BW5147 transfectant cells expressing mutated forms of the human IL-9 receptor (hIL-9R). When cells were transfected with the wild-type hIL-9R, hIL-9 induced a similar IL-TIF expression to that induced by mIL-9 in the parental cells (Fig. 6⇓). By contrast, hIL-9 was inactive in cells expressing a mutated form of hIL-9R (phe116) in which tyrosine residue 116 was replaced by a phenylalanine, resulting in the inability of the receptor to activate STAT transcription factors. To determine the respective role of the different STAT proteins activated by IL-9 (STAT1, -3, and -5), other hIL-9R mutants that specifically activate STAT5 (mut7) or STAT1 and -3 (mut6) were stably transfected in BW5147. As shown in Fig. 6⇓, STAT5 is not necessary for IL-TIF expression, since this gene is still induced in BWmut6 cells (where STAT5 is not activated in response to hIL-9). In addition, STAT5 is not sufficient for IL-TIF expression, since this gene is not significantly up-regulated in BWmut7 (where STAT5 is the only STAT activated by hIL-9).
Protein synthesis is not required for IL-TIF up-regulation by IL-9. Total RNA was extracted from BW5147 cells stimulated for 24 h with control medium or medium containing 200 U/ml IL-9, and with or without 10 μg/ml cycloheximide for 4.5 h. Total RNA was extracted, and RT-PCR amplification was performed with oligonucleotides specific for IL-TIF or β-actin. PCR products were analyzed by agarose gel electrophoresis.
IL-TIF up-regulation by IL-9 in cells expressing mutated hIL-9R. BW5147 cells transfected with the wild-type human IL-9R (BWh9R) or mutants of this receptor partially (mut6 and mut7) or totally defective in STAT activation (phe116) were stimulated with 500 U/ml human IL-9 for 24 h. Total RNA was extracted, and RT-PCR amplification was performed with oligonucleotides specific for IL-TIF or β-actin. PCR products were analyzed by agarose gel electrophoresis.
Identification of IL-TIF-responsive cell lines
Production of recombinant murine IL-TIF protein was performed by transient transfection of HEK293 cells. 35S-methionin labeling showed that, upon transfection with the IL-TIF cDNA, HEK293 cells produced a heterogeneous 23- to 30-kDa protein that was recognized by Abs raised against an N-terminal peptide of the IL-TIF sequence (Fig. 7⇓). This heterogeneity most likely results from glycosylation of the protein, which contains four putative N-linked glycosylation sites.
Production of recombinant IL-TIF protein in HEK293 cells. A, The IL-TIF cDNA cloned into pCEP4 in the sense (S) or antisense (AS) direction was transfected in the HEK293-EBNA cells. Labeling of newly synthesized protein was achieved by incorporation of 35S-labeled methionin for 24 h. Supernatants were analyzed by SDS-PAGE in a 14% acrylamide gel followed by overnight autoradiography. B, Western blot analysis of supernatants from HEK293 cells transfected with the same expression vectors. IL-TIF protein was detected using rabbit polyclonal Abs raised against aa 40–61 from the IL-TIFα sequence.
Such HEK293 cells supernatants were used to assess the putative biological activities of IL-TIF on various cell types. In these experiments, IL-TIF failed to reproduce in vitro biological activities of IL-9 such as proliferation of T cells, mast cells, or inhibition of corticoid-induced apoptosis (data not shown), indicating that these activities of IL-9 are not mediated by a IL-TIF autocrine loop. The possibility that IL-TIF interacts with the same receptor as IL-10 was investigated by stimulating RAW264 macrophages cell line or freshly isolated peritoneal cells with IL-TIF or IL-10. After 5 to 10 min of stimulation, we analyzed STAT3 activation by Western blot using an Ab specific for phosphorylated STAT3. As shown in Fig. 8⇓, IL-10, but not IL-TIF, strongly activated STAT3 in these cells. In addition, IL-TIF did not induce the proliferation of the IL-10 responsive mast cell line MC-9 (data not shown), indicating that IL-TIF does not activate cells through the IL-10 receptor.
IL-TIF does not activate STAT3 in macrophages. RAW264 cells and freshly isolated peritoneal cells from DBA/2 mice were stimulated for 10 min with hIL-10, mIL-TIF (2% 293-EBNA cell supernatant), or with a supernatant from mock-transfected cells. Cell lysates were analyzed by Western blot hybridization using anti-phospho-STAT3 Abs as described in Materials and Methods. The membrane was subsequently reprobed with anti-β actin Abs to confirm similar loading in the different lanes.
Further search for IL-TIF-responsive cell types was based on the hypothesis that IL-TIF, like most cytokines, would induce activation of STAT transcription factors in target cells. Using an EMSA with the FcγRI-derived GAS sequence, which binds to all STAT factors, we screened a number of in vitro cell lines for the nuclear translocation of STAT transcription factors after 15 min of IL-TIF stimulation. Using this assay, we identified two IL-TIF-responsive cell lines: a murine kidney mesangial cell (MES13) and a rat pheochromocytoma cell (PC12) (Fig. 9⇓A). Further characterization of STAT factors that are activated in response to IL-TIF in MES13 cells was achieved by supershift experiments with Abs specific for STAT-1, -3, or -5. As shown in Fig. 9⇓B, anti-STAT1 Abs had no effect on the GRR retardation complex. By contrast, anti-STAT3 Abs supershifted most of the signal detected, and the weak remaining complexes were supershifted in by anti-STAT5, indicating that STAT3 and, to a lesser extend, STAT5 are the major STAT transcription factors activated by IL-TIF in this kidney mesangial cell line.
STAT activation in IL-TIF-responsive cell lines. A, Nuclear extracts were prepared from MES13 and PC12 cells stimulated for 15 min with mIL-TIF (1% 293-EBNA cell supernatant) or with a supernatant from mock-transfected cells. Gel shift assays were performed as described in Materials and Methods, with the FcγRI-derived GRR probe. Competition with cold GRR oligonucleotide was performed with a 100× excess of the cold probe. B, EMSA performed with MES13 as in (A) with addition of anti-STAT-1, -3, and -5 Abs to supershift STAT-containing complexes.
Discussion
In this report, we describe the identification and cloning of IL-TIF, a new murine cytokine showing a weak but significant amino acid identity with IL-10. This gene was found to be expressed in T cells, upon activation by IL-9 or Con A, in mast cells, thymus, and brain. The overall sequence identity between IL-TIF and IL-10 is 22% at the amino acid level, in the same range as that between murine IL-1α and IL-1β (23% amino acid identity). In addition, most of the identical residues between IL-TIF and IL-10 are located in the C-terminal half of the protein, which has been found to be critical for IL-10 activity, raising the hypothesis that IL-TIF could exert some biological activities related to those of IL-10. However, HEK293-derived IL-TIF failed to activate STAT3 in mouse macrophages or to induce the proliferation of IL-10-responsive MC9 cells, indicating that these factors do not share the same receptor complex. As previously observed for IL-10, STAT3 seems to be the main activated STAT protein activated by IL-TIF. Further analyses of the IL-TIF receptor complex are definitely needed to assess the extent of redundancy and common features within IL-TIF and IL-10 signaling.
The observation that IL-TIF is expressed in activated T cells and, at a steady state level, in organs such as thymus and brain suggests that this factor exhibits pleiotropic activities, within and outside the immune system. In this respect, the fact that PC12 cells, often used as a model for neuronal cells, respond to IL-TIF stimulation, points to a role for this new cytokine in neurological processes. The other IL-TIF-responsive cell line identified so far was derived from kidney mesangial cells, a cell type that shares several features with macrophages, including the ability to phagocytose, produce inflammatory cytokines, release eicosanoids, and generate inducible NO synthase (20, 21). Considering the important role of IL-10 in regulation of monocyte-macrophage activation, the activity of IL-TIF on this cell type definitely deserves extensive evaluation.
The original goal of this study was to identify genes that mediate the biological activities of IL-9. In vitro, IL-TIF failed to reproduce activities such as induction of proliferation of T helper clones, mast cells, or inhibition of corticoid-induced apoptosis, indicating that these processes are not mediated by autocrine IL-TIF production. In vivo, transgenic mice that overexpress IL-9 do not show enhanced constitutive expression of IL-TIF in the organs tested. However, this possibility still has to be evaluated in various physiopathological situations where a role for IL-9 has been suggested, including asthma (4, 5, 22), and nematode infections (23). The availability of recombinant IL-TIF protein and of Abs against this cytokine should allow to address these questions in the near future.
Footnotes
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↵1 This work was supported in part by the Belgian Federal Service for Scientific, Technical and Cultural Affairs; by the Actions de Recherche Concertées from the Communauté Française de Belgique—Direction de la Recherche Scientifique; and by the Opération Télévie. J.C.R. is a research associate with the Fonds National de la Recherche Scientifique, Belgium.
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↵2 Address correspondence and reprint requests to Dr. Jean-Christophe Renauld, Ludwig Institute for Cancer Research, Avenue Hippocrate, 74, B-1200 Brussels, Belgium. E-mail address: renauld{at}licr.ucl.ac.be
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↵3 Abbreviations used in this paper: EBNA, EBV nuclear Ag; IL-TIF, IL-10-related T cell-derived inducible factor; GRR, γ response region.
- Received September 24, 1999.
- Accepted December 7, 1999.
- Copyright © 2000 by The American Association of Immunologists
References
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