Title: Interleukin (IL)-22, a Novel Human Cytokine That Signals through the Interferon Receptor-related Proteins CRF2–4 and IL-22R
Abstract: We report the identification of a novel human cytokine, distantly related to interleukin (IL)-10, which we term IL-22. IL-22 is produced by activated T cells. IL-22 is a ligand for CRF2–4, a member of the class II cytokine receptor family. No high affinity ligand has yet been reported for this receptor, although it has been reported to serve as a second component in IL-10 signaling. A new member of the interferon receptor family, which we term IL-22R, functions as a second component together with CRF2–4 to enable IL-22 signaling. IL-22 does not bind the IL-10R. Cell lines were identified that respond to IL-22 by activation of STATs 1, 3, and 5, but were unresponsive to IL-10. In contrast to IL-10, IL-22 does not inhibit the production of proinflammatory cytokines by monocytes in response to LPS nor does it impact IL-10 function on monocytes, but it has modest inhibitory effects on IL-4 production from Th2 T cells. We report the identification of a novel human cytokine, distantly related to interleukin (IL)-10, which we term IL-22. IL-22 is produced by activated T cells. IL-22 is a ligand for CRF2–4, a member of the class II cytokine receptor family. No high affinity ligand has yet been reported for this receptor, although it has been reported to serve as a second component in IL-10 signaling. A new member of the interferon receptor family, which we term IL-22R, functions as a second component together with CRF2–4 to enable IL-22 signaling. IL-22 does not bind the IL-10R. Cell lines were identified that respond to IL-22 by activation of STATs 1, 3, and 5, but were unresponsive to IL-10. In contrast to IL-10, IL-22 does not inhibit the production of proinflammatory cytokines by monocytes in response to LPS nor does it impact IL-10 function on monocytes, but it has modest inhibitory effects on IL-4 production from Th2 T cells. interleukin interferon signal transducers and activators of transcription reverse transcriptase-polymerase chain reaction The class II cytokine receptor family, also known as the interferon receptor family, includes the IL-10R,1 tissue factor, the two subunits of the IFNγ receptor, the two subunits of the IFNα/β receptor, and CRF2–4. Additional members of this family also exist in human genomic sequence (1Adams, R. L., Farrah, T. M., Jelmberg, A. C., Lok, S., and Whitemore, T. E. (1999) Patent WO9907848-A1 and U. S. Patent 5,965,704.Google Scholar). 2M-H. Xie, S. Aggarwal, W-H. Ho, J. Foster, Z. Zhang, J. Stinson, W. I. Wook, A. D. Goddard and A. L. Gurney, unpublished observations. The known biological actions mediated by these molecules are diverse and include the antiviral actions of IFNα, IFNβ, and IFNγ, the immunomodulatory effects of IFNγ, a TH1 cytokine that potentiates inflammatory responses and promotes cell-mediated immune responses, the multiple and generally immunosuppressive actions of IL-10, and the role of tissue factor as a high affinity receptor for plasma factor VII/VIIa involved in cellular initiation of the coagulation cascade. The class II cytokine receptors are evolutionarily related and are characterized by the presence of a single transmembrane domain and an extracellular domain that contains several fibronectin-type three repeats. Given the great importance of members of the interferon receptor family, it is of interest to identify proteins that interact with this system as they are likely to perform significant immune functions. In this report we identify a new human cytokine, IL-22, that signals through a receptor complex that contains CRF2–4 and a new member of the class II cytokine receptor family. CRF2–4 has previously been demonstrated to be a functional component of the IL-10 signaling complex. This is the first example within the class II cytokine receptor family of a receptor being utilized as a component of multiple distinct cytokine signaling complexes. Antibodies used for STAT supershift experiments were purchased from Santa Cruz Biotechnology. Antibodies for STAT tyrosine phosphorylation were purchased from Upstate Biotechnology. Antibodies for isolation of T cells were purchased from Pharmingen. Recombinant human IL-4, IFNγ, IL-10, and IL-12 was purchased from R&D Systems. Human TNF, IL-1, IL-4, IL-6, IL-13, and IFNγ enzyme-linked immunosorbent assay were purchased from R&D Systems. A cDNA clone encoding IL-22 was identified in the Lifeseq EST data base (Incyte Pharmaceuticals) and sequenced in its entirety. Coding sequence for IL-22, IL-10, and the interferon family receptors were obtained by PCR amplification. Fc fusion proteins (immunoadhesins) were prepared by fusion of the entire open reading frames of IL-22 and IL-10 in-frame with the Fc region of human IgG1 in the eukaryotic expression vector pRK5tkNEO and the baculovirus vector pHIF, a derivative of pVL1393 purchased from Pharmingen. Fusion proteins were transiently expressed in human 293 cells or Sf9 insect cells and purified over a protein A column. IL-22 was also expressed as a C-terminal 8 × His tag fusion in baculovirus and purified by nickel affinity column. The identity of the purified protein was verified by N-terminal sequence analysis. IL-22 was also expressed with an N-terminal gD epitope tag as described (2Xie M.H. Holcomb I. Deuel B. Dowd P. Huang A. Vagts A. Foster J. Liang J. Brush J. Gu Q. Hillan K. Goddard A. Gurney A.L. Cytokine. 1999; 11: 729-735Crossref PubMed Scopus (232) Google Scholar). The sequences of the DNA constructs were confirmed by DNA sequencing. STAT activation and supershift assays using the SIE sequence and Western blot analysis of STAT tyrosine phosphorylation were performed as described previously (3Gurney A.L. Wong S.C. Henzel W.J. de Sauvage F.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5292-5296Crossref PubMed Scopus (177) Google Scholar) and as recommended by antibody suppliers. Monocytes were enriched from fresh human blood by isolation of leukocytes using Lymphocyte Separation Medium purchased from ICN Biomedicals followed by enrichment of monocytes by adherence to tissue culture flask. Monocytes were then cultured for 24 h in 10% fetal bovine serum RPMI medium plus indicated cytokine treatment. For IL-22 mRNA expression experiments, resting T cells were isolated from leukocytes by negative selection with antibodies to CD14, CD19, CD56, CD11A, and HLA-DR purchased from Pharmingen. Isolated T cells were cultured in 10% fetal bovine serum RPMI medium with anti-CD3 coated plates for 48 h with concanavalin A (2.5 μg/ml). To drive T cell differentiation to Th1 and Th2 T cells, CD4 positive cells were first isolated with anti-CD4 magnetic beads (Pharmingen). Cells were then cultured RPMI with an equal number (∼10 million) of irradiated monocytes in the presence of concanavalin A (2.5 μg/ml) and IL-2 (4 ng/ml) and either IL-12 (8 ng/ml) + anti IL-4 (0.5 μg/ml) or IL-4 (4 ng/ml) + anti-IFNγ (0.6 μg/ml) for Th1 or Th2, respectively. After 3 days, T cells were collected and washed twice and cultured for 24 h in the presence or absence of IL-22-His (8 nm). As part of a larger effort to identify novel secreted proteins, we identified a novel human sequence that bears significant similarity to IL-10 (Fig. 1 A). This protein also strongly resembles a recently identified murine protein termed IL-TIFα, and is likely its human ortholog. Based on its similarity to the cytokine IL-10 and the data presented herein on the identification of its receptor as a member of an established cytokine receptor family and its production within leukocytes and action upon leukocytes, this molecule may appropriately be considered an interleukin. Following convention, we propose terming this molecule human IL-22. The isolated cDNA encodes a protein of 179 amino acids that is 23% similar to IL-10 and 78% identical to IL-TIFα (4Dumoutier L. Louahed J. Renauld J.C. J. Immunol. 2000; 164: 1814-1819Crossref PubMed Scopus (443) Google Scholar). The first 33 amino acids are predicted to function as a signal sequence. N-terminal amino acid analysis of IL-22 expressed and purified using baculovirus confirm the mature sequence begins at amino acid residue 34. Northern expression analysis showed only trace level expression in several peripheral tissues (not shown). RT-PCR analysis showed that IL-22 mRNA is up-regulated in T cells stimulated with anti-CD3 and further induced by exposure to concanavalin A (Fig. 1 B). To establish whether IL-22 was ligand for any of the members of the class II cytokine receptors, each receptor was tested for its ability to bind IL-22. Cells transiently expressing CRF2–4 showed strong binding of IL-22-fc fusion protein (immunoadhesion) (Fig.2 A). In contrast, IL-10-fc bound only to IL-10R. IL-22 also displayed low but detectable binding to a new member of the family of unknown function which we identified by searching sequences present in public sequence databases (1Adams, R. L., Farrah, T. M., Jelmberg, A. C., Lok, S., and Whitemore, T. E. (1999) Patent WO9907848-A1 and U. S. Patent 5,965,704.Google Scholar), which we term IL-22R (Fig. 2 B). IL-22R is a 574-amino acid protein most related to IL-10R and CRF2–4 (Fig. 2 C). To confirm the direct interaction of IL-22 with CRF2–4 and IL-22R, binding studies were conducted with epitope-tagged ligand and soluble receptor immunoadhesins. IL-22 bound to CRF2–4 but not to any of the other members of the interferon receptor family (Fig. 2 D). Direct binding was not observed by Western blot analysis with IL-22R, suggesting the interaction with this component may be of low affinity. Reasoning that IL-22 likely activated the JAK-STAT signaling pathway in a manner analogous to that observed with the interferons and members of the hematopoietic cytokine family, we surveyed a series of cell lines for activation of STAT transcription factors in response to IL-22. Two cell lines were observed to show rapid and robust STAT activation; TK-10, a renal cell carcinoma (Fig.3 A) and SW480, a colon adenocarcinoma (not shown). TK-10 did not induce STAT activity in response to IL-10. Interestingly, in this survey we observed another cell line, MOLT-4, a human lymphoblast cell line that did respond to IL-10 but did not respond to IL-22. The specific STAT proteins activated in response to IL-22 were examined using antibodies to the known STATs. In gel-shift assays, antibodies to STAT1, STAT3, and STAT5 were able to supershift IL-22 induced binding complexes to an SIE sequence (Fig. 3 B) and other related STAT-binding elements (not shown). Furthermore, using antibodies specific for tyrosine-phosphorylated STAT, clear tyrosine phosphorylation of these STATs was observed in response to IL-22 (Fig. 3 C). As IL-22 and IL-10 signal STAT activation in distinct cell lines, we explored the expression pattern of IL-10R, CRF2–4, and IL-22R in these lines. RT-PCR analysis indicated CRF2–4 is expressed in both TK-10 and MOLT-4 (Fig. 4 A). In contrast, expression of IL-10R was detected in MOLT-4 and not in TK-10 and IL-22R expression was detected in TK-10 but not MOLT-4. These results suggested that functional signaling complexes for IL-10 included IL-10R and CRF2–4 as previously reported, and that IL-22 signaled through a complex that included CRF2–4 and IL-22R. To test this hypothesis, these receptors were transfected into COS cells and the ability of IL-10 and IL-22 to mediate STAT activation was determined (Fig.4 B). In agreement with previous reports IL-10 was able to mediate STAT activation in cells transfected with both CRF2–4 and IL-10R. Somewhat weaker activation was also seen in cells transfected with IL-10R alone. In contrast, IL-22 was able to mediate STAT activation in cells transfected with both CRF2–4 and IL-22R but not with either receptor transfected alone. Given the distant homology between IL-22 and IL-10, we examined whether IL-22 had similar biological activities to IL-10. Monocytes from freshly isolated human blood were examined for the ability of IL-22 to effect production of cytokines known to be regulated by IL-10. LPS induces a large increase in TNF production, an effect which may be substantially repressed by the presence of IL-10 (Fig.5). We observed that IL-22 had little if any effect on TNF production in the presence or absence of LPS. Furthermore, at 10-fold molar excess IL-10, it did not appear to inhibit the action of IL-10. Similar results were observed measuring production of IL-1 and IL-6 (not shown). The effect of IL-22 on production of cytokines by T cells was studied. Human T cells were polarized in vitro to either Th1 or Th2 differentiation by activation with concanavalin A in the presence of IL-12 and anti-IL-4 antibodies or IL-4 and anti-IFNγ antibodies, respectively. Cells were then washed and cultured in the presence or absence of IL-22 for 24 h. IL-22 treatment had little effect on IFNγ production from Th1 cells, but modestly inhibited production of IL-4 from Th2 cells (TableI).Table IIFNγ and IL-4 production by human T cellsIFNγIL-4Th1Th2Th1Th2−+−+−+−+pg/mlpg/mlExperiment 122507 ± 743422114 ± 114731225 ± 390833 ± 1046.5 ± 0.65.4 ± 0.785.2 ± 1.913.6 ± 2.7Experiment 211356 ± 18038844 ± 5471014 ± 91660 ± 22276 ± 1068 ± 22476 ± 39356 ± 48Experiment 37009 ± 5577714 ± 792247 ± 21138 ± 1027 ± 1.26.6 ± 0.729.6 ± 1.519.6 ± 2.5Experiment 410022 ± 21639241 ± 1232397 ± 39465 ± 19.45.5 ± 0.25.0 ± 0.351 ± 7.510.4 ± 0.7T cells were isolated from fresh human blood and polarized in vitro towards Th1 or Th2 differentiation for 3 days. Cells were then washed and incubated for 24 h in the presence of IL-22-His (8 nm) as indicated for 24 h. Production of IFNγ and IL-4 was measured by enzyme-linked immunosorbent assay. Shown are the results from four independent experiments. Open table in a new tab T cells were isolated from fresh human blood and polarized in vitro towards Th1 or Th2 differentiation for 3 days. Cells were then washed and incubated for 24 h in the presence of IL-22-His (8 nm) as indicated for 24 h. Production of IFNγ and IL-4 was measured by enzyme-linked immunosorbent assay. Shown are the results from four independent experiments. IL-22 signals through a receptor complex that includes CRF2–4 and a new member of the class II cytokine receptor family, IL-22R. Previous reports have demonstrated that CRF2–4 serves as a second component in IL-10 signaling (5Kotenko S.V. Krause C.D. Izotova L.S. Pollack B.P. Wu W. Pestka S. EMBO J. 1997; 16: 5894-5903Crossref PubMed Scopus (333) Google Scholar, 6Spencer S.D. Di Marco F. Hooley J. Pitts-Meek S. Bauer M. Ryan A.M. Sordat B. Gibbs V.C. Aguet M. J. Exp. Med. 1998; 187: 571-578Crossref PubMed Scopus (304) Google Scholar). CRF2–4 was initially discovered and identified as a member of the interferon receptor family on the basis of sequence similarity (7Lutfalla G. Gardiner K. Uze G. Genomics. 1993; 16: 366-373Crossref PubMed Scopus (76) Google Scholar, 8Gibbs V.C. Pennica D. Gene ( Amst. ). 1997; 186: 97-101Crossref PubMed Scopus (29) Google Scholar). The gene is located within a cluster that also includes IFN-αR1, IFN-αR2, and IFN-γR2 on human chromosome 21. A two-component receptor for IL-10 signaling parallels the IFNα/β and IFNγ systems which have each been shown to signal through two component receptors. The data presented here suggest that, in addition, CFR2–4 serves as a binding component of an IL-22 signaling complex. Thus CRF2–4 could serve in combination with IL-10R to transduce IL-10 signaling and also function within a signaling complex for IL-22 that likely includes the new member of this family, IL-22R. Overexpression of CRF2–4 alone or in combination with IL-10R does not appear to be sufficient to enable IL-22-dependent STAT activation, but combined with IL-22R does enable STAT activation. While shared receptor components have not been previously observed among the class II cytokine receptor family, within the large family of hematopoietic cytokine receptors such utilization of receptors in multiple distinct combinations to respond to different cytokines is common. Examples include the common β-chain utilized by IL-3, IL-5, and GM-CSF, the common γ-chain utilized by IL-2, -4, -7, –9, and -15, and gp130 utilized by multiple members of the IL-6 family of cytokines (reviewed in Refs. 9He Y.W. Malek T.R. Crit. Rev. Immunol. 1998; 18: 503-524Crossref PubMed Google Scholar, 10de Groot R.P. Coffer P.J. Koenderman L. Cell. Signal. 1998; 10: 619-628Crossref PubMed Scopus (187) Google Scholar, 11Heinrich P.C. Behrmann I. Muller-Newen G. Schaper F. Graeve L. Biochem. J. 1998; 334: 297-314Crossref PubMed Scopus (1749) Google Scholar). The LIF receptor serves both as a ligand binding subunit for LIF and CT-1 and also as a second component in signaling complexes for oncostatin M and ciliary neurotrophic factor (12Pennica D. Wood W.I. Chien K.R. Cytokine Growth Factor Rev. 1996; 7: 81-91Crossref PubMed Scopus (91) Google Scholar,13Grotzinger J. Kurapkat G. Wollmer A. Kalai M. Rose-John S. Proteins. 1997; 27: 96-109Crossref PubMed Scopus (96) Google Scholar). In each of these cases the ligands, as in the case of IL-10 and IL-22, are evolutionarily related. Activation of STATs 1, 3, and 5 has also been reported for both IL-10 and murine IL-TIFα (4Dumoutier L. Louahed J. Renauld J.C. J. Immunol. 2000; 164: 1814-1819Crossref PubMed Scopus (443) Google Scholar, 14Finbloom D.S. Winestock K.D. J. Immunol. 1995; 155: 1079-1090PubMed Google Scholar, 15Wehinger J. Gouilleux F. Groner B. Finke J. Mertelsmann R. Weber-Nordt R.M. FEBS Lett. 1996; 394: 365-370Crossref PubMed Scopus (141) Google Scholar). These same STATs have also been shown to be activated by a great number of hematopoietic cytokines, suggesting that other considerations such as the pattern of receptor expression influence the biological responses (reviewed in Ref.16Chatterjee-Kishore M. van den Akker F. Stark G.R. Trends Cell Biol. 2000; 10: 106-111Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Mice with targeted disruption of either CRF2–4 and IL-10 have been reported (6Spencer S.D. Di Marco F. Hooley J. Pitts-Meek S. Bauer M. Ryan A.M. Sordat B. Gibbs V.C. Aguet M. J. Exp. Med. 1998; 187: 571-578Crossref PubMed Scopus (304) Google Scholar, 17Kuhn R. Lohler J. Rennick D. Rajewsky K. Muller W. Cell. 1993; 75: 263-274Abstract Full Text PDF PubMed Scopus (3668) Google Scholar, 18Rennick D.M. Fort M.M. Davidson N.J. J. Leukocyte Biol. 1997; 61: 389-396Crossref PubMed Scopus (260) Google Scholar). The phenotypes bear some similarity but also significant differences. Mice deficient in IL-10 developed chronic enterocolitis, were anemic, and were growth retarded when raised in a conventional environment. In comparison only 60% of the CRF2–4 mice raised under similar conditions had colitus but this was limited to the large intestine and did not involve the small intestine. The mice did not develop anemia and unlike the IL-10-deficient mice the CRF2–4 mice displayed a 4-fold increase in spleen to body weight with extramedullary hematopoiesis in the spleen of erythroid, myeloid, and megakaryocytic lineages. These differences may be due in part to contributions of environment or genetic background, however, in light of the existence of a specific ligand for CRF2–4, the function of IL-10 in the CRF2–4-deficient mice should be re-evaluated. The broad distribution of CRF2–4 expression is reminiscent of the expression patterns seen for the receptors for IFNα/β and IFNγ whereas the expression of the IL-10R is restricted mainly to hematopoietic cells. Preliminary data suggests IL-22R is also restricted in its expression, but is detectable by RT-PCR in CD3 positive T cells and at low levels in several peripheral tissues (not shown). The broad expression of CRF2–4 may reflect the existence of additional ligands yet to be discovered which also share CRF2–4 as a common subunit. Interestingly, IL-10 and IL-22 appear to have somewhat mirror actions acting to promote Th2 or Th1 type responses, respectively. The ability of IL-22 to suppress IL-4 production may have therapeutic potential, particularly in the treatment of asthma. We thank Diane Pennica, Sherman Fong, and Paul Godowski for helpful discussions and Amy Carlow, Jeffrey Hooly, Peter Ng, and Mark Vasser for technical assistance.