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CUTTING EDGE |
* Division of Therapeutic Proteins, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892;
Department of Biochemistry and Molecular Biology, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, NJ 07103;
Protein Structure Section, Macromolecular Crystallography Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702;
Ludwig Institute for Cancer Research, and the Experimental Medicine Unit, Christian de Duve Institute for Cellular Pathology, Université de Louvain, Brussels, Belgium
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) comprise the core of the IFN family. The IL-10 family is composed of six cellular cytokines (IL-10, IL-19, IL-20, IL-22, IL-24, and IL-26) and several viral homologs (1, 2, 3, 4, 5). Three cytokines of the recently discovered IFN-
family (6, 7) demonstrate limited homology to cytokines from both the type I IFN and IL-10 families (6, 7). A single type II IFN, IFN-
, has diverged from other CRF2 cytokines, although its tertiary and quaternary structure is similar to that of IL-10. Several CRF2 ligands, including IL-26, were only recently identified, and their functions are just beginning to be characterized. Nevertheless, the functional receptor complexes for all CRF2 ligands except IL-26 have been defined.
Originally designated as AK155, IL-26 was cloned as a protein expressed by herpesvirus saimiri-transformed T cells (8). IL-26 mRNA was also detected in several T cell lines, including human T cell leukemia virus-transformed cell lines. IL-26 mRNA is expressed in activated NK cells and T cells with enhanced expression upon type 1 polarization (9). IL-26 is also produced by activated memory but not by naive CD4+ T cells, independently of costimulation (9). Although these data suggest that IL-26 may influence certain aspects of the immune response, the major function(s) of IL-26 remains to be defined. As a first step in defining IL-26 activities, we identified a receptor complex specific for IL-26 and characterized the signal transduction events induced by this cytokine. A combination of two receptor subunits, IL-20R1 (CRF2-8) and IL-10R2 (CRF2-4), is required to form the functional IL-26R complex. Although it was previously shown that IL-20R1 can dimerize with IL-20R2 to form the receptor for IL-19, IL-20, and IL-24, and IL-10R2 can dimerize with either IL-10R1, IL-22R1, or IFN-R1 to form receptors for IL-10, IL-22, and IFN-, respectively, this is the first demonstration that IL-20R1 and IL-10R2 can also dimerize to generate the receptor for IL-26. IL-26-mediated signaling through this receptor complex induced activation of two members of the STAT family, STAT1 and STAT3.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Expression vectors for IL-10R2 and the IL-22R1/R1chimeric receptor were described (10, 11). The cDNA fragment encoding the IL-20R1 extracellular domain was amplified from a library containing cDNA isolated from human placenta (catalog no. HL4025AH; Clontech, Palo Alto, CA) with primers 5'-GCCGGATCCATGCGGCCGCTGCCGCTGCCG-3' and either 5'-ATCGCTAGCCATTTAGCCTTGAACTCTGATG-3' or 5'-CGACTAGTTTTAGCCTTGAACTCTGATG-3'. PCR products were digested with BamHI restriction endonuclease and cloned into BamHI and EcoRV sites of the pcDEF3 vector, generating plasmids pEF2-IL-20R1EC/Nhe and pEF2-IL-20R1EC/Spe, respectively. Plasmid pEF2-IL-20R1/R1 was created by recloning IFN-R1 intracellular domain from plasmid pEF3-IL-10R1/R1 (10) to plasmid pEF2-IL-20R1EC/Nhe with NheI and BssHII restriction endonucleases. The PCR product encoding the transmembrane and intracellular domains of IL-20R1 was generated with primers 5'-GTGGCTAGCCTGGTATGTTTTGCCCAT-3' and 5'-GCGAATTCGTCTGGCAAACATTTATTGA-3' and cloned into NheI and EcoRI sites of plasmids pEF2-IL-20R1EC/Nhe or pEF2-FL-IL-10R1/CRF2-12 (6), resulting in plasmids pEF2-IL-20R1 and pEF2-FL-IL-10R1/IL-20R1. The PCR product encoding the Fc fragment of human IgG1 was generated with primers 5'-CGACTAGTGACAAAACTCACACAT-3' and 5'-GCCGAATTCATTTACCCGGAGACAG-3' and human PBMC cDNA, and cloned into SpeI and EcoRI sites of plasmid pEF2-IL-20R1EC/Spe, resulting in plasmid pEF2-IL-20R1-Fc. Plasmid pEF2-IL-10R2-Fc was created by cloning PCR product encoding the extracellular domain of IL-10R2 into KpnI and SpeI sites of plasmid pEF2-IL-20R1-Fc, resulting in plasmid pEF2-IL-10R2-Fc. Plasmids encoding soluble IL-20R1 and IL-10R2 were generated by introducing a stop codon at the end of the extracellular domains of the receptors by PCR.
The nucleotide sequences of the modified regions of all constructs were verified in their entirety by DNA sequencing.
Cells, reagents, EMSA, and Western and Northern blotting
COS-1 cells, an SV40-transformed fibroblast-like simian CV-1 cell line, colorectal adenocarcinoma HT-29 cells, colon carcinoma COLO-205 cells, human embryonic kidney 293 cells, and breast adenocarcinoma MCF7 cells were maintained and propagated as recommended by American Type Culture Collection (Manassas, VA). Cells were transfected as described (6, 10, 12).
To evaluate STAT activation, cells were treated for 15 min at 37°C with various cytokines as indicated in the text and used for either EMSA experiments with IFN- activation sequence (GAS) probe or for Western blotting as described (6, 10). IL-26 and neutralizing IL-10R2 and IL-26 Abs were purchased from R&D Systems (Minneapolis, MN). IL-10, IL-19, IL-20, and IL-22 were from PeproTech (Rocky Hill, NJ). IL-24, IL-22 binding protein (IL-22BP), soluble and Fc fusion receptors were produced in COS cells. IL-22R1 Ab has been described (13). To produce IL-20R1 Abs, P815 mastocytoma cells were transfected with the human IL-20R1 cDNA cloned into the pEF-BOS plasmid before injection into DBA/2 mice. After rejection of the tumors, the sera of these mice had high titers of neutralizing IL-20R1 Abs.
Two blots containing RNA derived from various normal tissues (Human Brain MTN Blot II; catalog no. 7755-1; Clontech; and Human Fetal mRNA Blot II; catalog no. D2802-50; Invitrogen, San Diego, CA) were hybridized with radiolabeled cDNA specific for IL-20R1 or IL-10R2 as described (11, 14).
IL-26 binding assay
ELISAs were performed as follows. Greiner (Nurtingen, Germany) Hybind plates were coated overnight at 4°C with 12.5 µg/ml either BSA or IL-26 in sodium carbonate (pH 9.5). The plates were then incubated 3 h at 22°C with 100 µl of COS cell-conditioned medium (mock) or COS cell supernatants containing Fc fusion receptors with or without soluble (s)IL-20R1. Receptor-Fc binding was measured with the use of goat anti-human IgG polyclonal Ab coupled to peroxidase as described (13). Relative binding was determined by dividing values obtained for specific binding to IL-26 by those for unspecific binding to BSA.
Size-exclusion chromatography was performed as described (15) with sIL-20R1 (15) and IL-26 produced in E. coli cells with the use of maltose-binding protein fusion protein and purification system (pMAL-p2x; New England Biolabs, Beverly, MA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Functional CRF2 receptor complexes are heterodimers. Each subunit plays its unique role within a given receptor complex. A receptor with longer intracellular domain, denoted the R1 type receptor subunit, orchestrates recruitment of various signaling proteins and, therefore, determines the specificity of cytokine signaling. The R2 type receptor chain has a short intracellular domain that mainly does not influence the specificity of signaling. Nevertheless, both R1 and R2 receptors associate with Janus (Jak) tyrosine kinases, which trigger intracellular signal transduction events following ligand binding (3, 4).
The phylogenetic tree of the family of IL-10-related cytokines demonstrates that the family can be further divided into two groups (Fig. 1). IL-19, IL-20, and IL-24 belong to one group, whereas IL-26 along with IL-10 and IL-22 form a distinct group. The members of each group share stronger homology within the same group than with members of the other group. In addition, IL-19, IL-20, and IL-24 use the common R2 type receptor chain, IL-20R2, for signaling, suggesting that the cytokines of the other group, IL-10, IL-22, and IL-26, may also share a common R2 chain as a part of their specific receptor complexes. In fact, IL-10R2 is a shared receptor chain for the IL-10, IL-22, and IFN- receptor complexes. This observation suggested that IL-10R2 may also be the second chain for the IL-26 receptor complex.
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R1 intracellular domain enables chimeric IL-22R1/IFN-R1 (IL-22R1/R1) and IL-20R1/IFN-R1 (IL-20R1/R1) chains to activate STAT1 upon ligand binding and induce various IFN--specific biological activities such as up-regulation of MHC class I Ag expression (11). Thus, to investigate whether IL-20R1 or IL-22R1 plays a functional role in the IL-26 receptor complex, each chimeric receptor was transiently expressed in monkey (COS) cells or human embryonic kidney 293 cells. Stable transfectants were also obtained for the human colorectal adenocarcinoma cell line HT29 and the breast adenocarcinoma cell line MCF7. In all cell lines expressing IL-20R1/R1, IL-26 activated STAT1 DNA-binding complexes as determined by EMSA, whereas IL-26 failed to induce STAT1 activation in parental cell lines or in cells expressing IL-22R1/R1 (Fig. 2A and data not shown). In addition, in COS cells, IL-26-induced transcription of the luciferase reporter gene driven by a minimal TK promoter containing three copies of a GAS element was dependent on the expression of IL-20R1. IL-10, IL-19, IL-20, IL-22, and IL-24 did not induce STAT activation in parental MCF7 cells or MCF7 cells transfected with IL-20R1/R1 (Fig. 2A). We also demonstrated that expression of the intact IL-20R1 in MCF7 cells enables IL-26 to induce activation of STAT1 and STAT3 as determined by EMSA (Fig. 2A). Western blotting revealed that IL-26 predominantly activated STAT3 in these cells (Fig. 2B).
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Interaction between IL-26 and its receptors was further tested by modified ELISA and size-exclusion chromatography. IL-20R1-Fc fusion protein was able to bind to the IL-26-coated plate, whereas IL-10R2-Fc was not capable of binding (Fig. 2C). However, in the presence of the sIL-20R1, we detected low-level binding of IL-10R2-Fc to the plate, suggesting that IL-10R2-Fc protein interacted with the complex composed of IL-26 and sIL-20R1. Size-exclusion chromatography demonstrated that IL-26 and sIL-20R1 were fractionated together (Fig. 2D), further confirming that IL-26 and IL-20R1 do interact in solution.
Expression of the IL-20R1 and IL-10R2 genes in normal tissues
IL-10R2 is ubiquitously expressed (9, 18). Thus, the expression of IL-20R1 should be a factor limiting responsiveness of cells to IL-26. A high level of IL-20R1 mRNA expression was previously detected in skin, testis, heart, placenta, salivary gland, and prostate by RT-PCR (19). Moderate expression was observed in brain, lung, stomach, pancreas, ovary, uterus, thyroid, and adrenal glands. Barely detectable expression was seen in kidney, liver, colon, muscle, and small intestine. Interestingly, IL-20R1 expression was found to be elevated in psoriatic skin (19). Receptor expression was also detected in keratinocytes, endothelial cells, and immune cells in psoriatic lesions.
We also evaluated several tissues for IL-20R1 expression by Northern blotting. Two distinct IL-20R1 transcripts (2.2 and 4 kb), were detected in skin and lung tissues (Fig. 3A). Although RT-PCR detected IL-20R1 expression in adult heart (19), expression of this gene was undetectable by Northern blotting in fetal heart (Fig. 3A), which could indicate the different pattern of expression between adult and fetal tissues. Database searches demonstrate that many IL-20R1 expressed sequence tags were derived from various brain cDNA libraries (UniGene no. Hs.21814). Indeed, Northern blotting revealed that cerebellum has a very high level of IL-20R1 gene expression (Fig. 3B). Lower levels were seen in medullar and spinal cord. In contrast, only a very low level of IL-10R2 mRNA expression was detected in certain brain compartments (Fig. 3B). Thus, IL-20R1 may have a unique function in brain tissues.
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To define which STATs are activated by signaling through the IL-20R1 chain, we examined the activation of STATs in HT-29 cells that were stably transfected with a chimeric receptor, FL-IL-10R1/IL-20R1, composed of the FLAG epitope-tagged extracellular domain of the IL-10R1 chain linked to the intracellular domain of IL-20R1 (Fig. 4A). This chimeric receptor is able to bind IL-10 and dimerize with IL-10R2, the shared second chain for the IL-10, IL-22, IFN-, and IL-26 receptor complexes, to transduce a signal. Thus, IL-10-mediated signaling through the FL-IL-10R1/IL-20R1 chimeric receptor mimics signaling through the native IL-26 receptor complex. The use of the chimeric receptor allowed us to dissect signaling events activated through the IL-20R1 intracellular domain. This also eliminated the possibility that IL-26 may cross-react with receptors other than the IL-20R1 and IL-10R2 complex. Thus, the STAT proteins that are activated by IL-10 in these cells represent those that are normally recruited to the full-length IL-20R1 intracellular domain. STAT activation was measured by immunoprecipitating specific STAT proteins from whole-cell lysates, and then Western blotting with phosphotyrosine-specific Abs.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As demonstrated in this study, IL-20R1 can couple with IL-10R2 to form the IL-26 receptor complex (Fig. 5). The complex is specific for IL-26, because other IL-10-related cytokines, IL-10, IL-19, IL-20, IL-22, and IL-24, do not signal through the IL-26 receptor complex. IL-26 activates the Jak/STAT signaling pathway, resulting in rapid tyrosine phosphorylation of STAT1 and STAT3 in cells that express endogenous IL-20R1 and IL-10R2 (Fig. 5). IL-10R2 is broadly expressed on a wide variety of tissues, whereas only a limited number of tissues express IL-20R1. Therefore, the ability to respond to IL-26 is largely determined by expression of IL-20R1. IL-10R2 also functions as a receptor subunit for the IL-10, IL-22, and IFN- receptor complexes (6, 7, 10, 11, 21). Whereas IL-20R1 binds IL-26 directly, IL-10R2 does not. Nevertheless, IL-10R2 Ab completely blocked IL-26-induced signal transduction, providing strong direct evidence that IL-10R2 is required to assemble the functional IL-26 receptor complex. Thus, it seems that a common functional role of IL-10R2 is well conserved in all of the receptor complexes that use IL-10R2 as a receptor subunit. IL-10R2 does not interact with cytokines directly; nevertheless, its engagement is required to assemble functional receptor complexes for IL-10, IL-22, IFN-, and IL-26, where each of these cytokines specifically interacts with its own unique receptor chain. Our characterization of the functional IL-26 receptor complex provides another interesting example of receptor sharing by CRF2 cytokines. Although unique receptor complexes can be formed through the shared use of a limited number of transmembrane proteins, the full biological significance of class II cytokine receptor sharing remains to be determined.
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A second general consequence of receptor sharing may be that CRF2 cytokines can cross-regulate their activities by competing for receptor components. It is interesting to speculate that IL-10, a cytokine with strong anti-inflammatory activity, could sequester IL-10R2 and thereby regulate the functions of other CRF2 cytokines that require the IL-10R2 chain for signaling. Among these cytokines, IL-22 demonstrates proinflammatory activities, and inhibition of its function at the receptor level would be consistent with IL-10’s predominant anti-inflammatory function.
It was recently demonstrated that IL-20 stimulates hemopoiesis, with a specificity toward multipotential progenitors (22). Based on partial receptor sharing between IL-20 and IL-26, it is interesting to speculate that IL-26 may have a similar activity. With the IL-10 family of cytokines and their receptors defined, it should now be possible to dissect their ligand-specific, shared, and mutually regulatory functions.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Sergei V. Kotenko, Department of Biochemistry and Molecular Biology, Room E-641, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, 185 South Orange Avenue, Newark, NJ 07103. E-mail address: kotenkse{at}umdnj.edu
3 Abbreviations used in this paper: CRF, cytokine receptor family; GAS, IFN- activation sequence; IL-22BP, IL-22 binding protein; s, soluble; Jak, Janus kinase.
Received for publication September 22, 2003. Accepted for publication December 22, 2003.
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s mediate antiviral protection through a distinct class II cytokine receptor complex. Nat. Immunol. 4:69.[Medline]
) is a common chain of both the IL-10 and IL-22 (IL-10-related T cell-derived inducible factor, IL-TIF) receptor complexes. J. Biol. Chem. 276:2725.This article has been cited by other articles:
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R. P. Donnelly, F. Sheikh, S. V. Kotenko, and H. Dickensheets The expanded family of class II cytokines that share the IL-10 receptor-2 (IL-10R2) chain J. Leukoc. Biol., August 1, 2004; 76(2): 314 - 321. [Abstract] [Full Text] [PDF]
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