IL17

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IL-17 Serge Lebecque*, FrancËois Fossiez and Elizabeth Bates Laboratory for Immunological Research, Schering-Plough, 27 Chemin des Peupliers, Dardilly, 69572, France * corresponding author tel: (33) 4 72 17 27 00, fax: (33) 4 78 35 47 50, e-mail: [email protected] DOI: 10.1006/rwcy.2000.03010.

SUMMARY

GENE AND GENE REGULATION

IL-17 is a cytokine secreted in large amounts exclusively by T cells upon activation, which acts directly on stromal cells and induces their production of several proinflammatory and hematopoietic bioactive molecules. A functional homolog of IL-17 is present in the genome of the T lymphotropic herpesvirus saimiri (HVS). A receptor of low affinity for IL17 has been cloned and is ubiquitously expressed.

Accession numbers

BACKGROUND

Discovery The IL-17 cDNA was first isolated as the murine cytotoxic T lymphocyte antigen 8 (CTLA-8) from a subtracted cDNA library of a PMA ‡ ionomycinactivated hybridoma resulting from the fusion of mouse cytotoxic T cells with a rat T lymphoma cell line (Rouvier et al., 1993). The CTLA-8 sequence presented several features of a cytokine gene: eight AU-rich repeats in the 30 untranslated region, an open reading frame encoding a putative secreted protein of 150 amino acids, a 57% homology with the putative protein encoded by the ORF13 gene of HVS, a T lymphotropic virus well known for its ability to transform human T cells (Biesinger et al., 1992). Evidence for the capture of the CTLA-8 sequence by a virus further supported the hypothesis that this molecule could play some role in the immune response (Spriggs, 1994). Using the rat sequence, two groups independently cloned the human counterpart (Fossiez et al., 1996; Yao et al., 1995b) and partially characterized the biological activity of this novel T cell-secreted molecule. The designation of IL-17 was proposed for the protein product.

Human IL-17 (CTLA serine esterase 8), complete cds: U32659, NM_002190 Mouse IL-17 (CTLA-8) mRNA, complete cds: U43088 Rat (clone 2.6) CTLA-8 mRNA sequence, complete cds: L13839 Herpesvirus saimiri DNA for ORF 13: Y13183 Pig IL-17 gene, partial cds: AF166489 Sheep IL-17 gene, partial cds: AF166488 Bovine IL-17 gene, partial cds: AF166487

Sequence See Figure 1. Four IL-17 full-length cDNAs: the human (hIL-17) (Fossiez et al., 1996; Yao et al., 1995b), mouse (mIL-17), rat IL-17 (rIL-17) as well as the ORF13 of HVS, hereafter named viral IL-17 (vIL17) have been cloned. The two human cDNA nucleotide sequences differ only in the 30 UTR, being 661 nucleotides longer in the report from Yao, a likely result of the use of alternative polyadenylation sites. As suspected by Rouvier et al. (1993) the CTLA-8 clone corresponds to the rat IL-17, as two group have subsequently characterized the mouse IL-17 sequence (Yao et al., 1995b; Kennedy et al., 1996). Partial IL-17 sequences from pig, cow, and sheep are also available in public databases. Comparison between the IL-17 nucleotides sequences reveals the interspecies conservation of several AU-rich repeats in the 30 UTRs which are probably involved in the rapid degradation of mRNA (Shaw and Kamen, 1986).

242 Serge Lebecque, FrancËois Fossiez and Elizabeth Bates

Figure 1 Nucleotide sequences for human, mouse, rat, and herpesvirus saimiri IL-17. The start codons are in bold; the stop codons are underlined. Human IL-17 complete CDS GAATTCCGGCAGGCACAAACTCATCCATCCCCAGTTGATTGGAAGAAACAACGATGACTCCTGGGAAGACCTCATTGGTGTCACTGCT ACTGCTGCTGAGCCTGGAGGCCATAGTGAAGGCAGGAATCACAATCCCACGAAATCCAGGATGCCCAAATTCTGAGGACAAGAACTTC CCCCGGACTGTGATGGTCAACCTGAACATCCATAACCGGAATACCAATACCAATCCCAAAAGGTCCTCAGATTACTACAACCGATCCA CCTCACCTTGGAATCTCCACCGCAATGAGGACCCTGAGAGATATCCCTCTGTGATCTGGGAGGCAAAGTGCCGCCACTTGGGCTGCAT CAACGCTGATGGGAACGTGGACTACCACATGAACTCTGTCCCCATCCAGCAAGAGATCCTGGTCCTGCGCAGGGAGCCTCCACACTGC CCCAACTCCTTCCGGCTGGAGAAGATACTGGTGTCCGTGGGCTGCACCTGTGTCACCCCGATTGTCCACCATGTGGCCTAAGAGCTCT GGGGAGCCCACACTCCCCAAAGCAGTTAGACTATGGAGAGCCGACCCAGCCCCTCAGGAACCCTCATCCTTCAAAGACAGCCTCATTT CGGACTAAACTCATTAGAGTTCTTAAGGCAGTTTGTCCAATTAAAGCTTCAGAGGTAACACTTGGCCAAGATATGAGATCTGAATTAC CTTTCCCTCTTTCCAAGAAGGAAGGTTTGACTGAGTACCAATTTGCTTCTTGTTTACTTTTTTAAGGGCTTTAAGTTATTTATGTATT TAATATGCCCTGAGATAACTTTGGGGTATAAGATTCCATTTTAATGAATTACCTACTTTATTTTGTTTGTCTTTTTAAAGAAGATAAG ATTCTGGGCTTGGGAATTTTATTATTTAAAAGGTAAAACCTGTATTTATTTGAGCTATTTAAGGATCTATTTATGTTTAAGTATTTAG AAAAAGGTGAAAAAGCACTATTATCAGTTCTGCCTAGGTAAATGTAAGATAGAATTAAATGGCAGTGCAAAATTTCTGAGTCTTTACA ACATACGGATATAGTATTTCCTCCTCTTTGTTTTTAAAAGTTATAACATGGCTGAAAAGAAAGATTAAACCTACTTTCATATGTATTA ATTTAAATTTTGCAATTTGTTGAGGTTTTACAAGAGATACAGCAAGTCTAACTCTCTGTTCCATTAAACCCTTATAATAAAATCCTTC TGTAATAATAAAGTTTCAAAAGAAAATGTTTATTTGTTCTCATTAAATGTATTTTAGCAAACTCAGCTCTTCCCTATTGGGAAGAGTT ATGCAAATTCTCCTATAAGCAAAACAAAGCATGTCTTTGAGTAACAATGACCTGGAAATACCCAAAATTCCAAGTTCTCGATTTCACA TGCCTTCAAGACTGAACACCGACTAAGGTTTTCATACTATTAGCCAATGCTGTAGACAGAAGCATTTTGATAGGAATAGAGCAAATAA GATAATGGCCCTGAGGAATGGCATGTCATTATTAAAGATCATATGGGGAAAATGAAACCCTCCCCAAAATACAAGAAGTTCTGGGAGG AGACATTGTCTTCAGACTACAATGTCCAGTTTCTCCCCTAGACTCAGGCTTCCTTTGGAGATTAAGGCCCCTCAGAGATCAACAGACC AACATTTTTCTCTTCCTCAAGCAACACTCCTAGGGCCTGGCTTCTGTCTGATCAAGGCACCACACAACCCAGAAAGGAGCTGATGGGG CAGAATGAACTTTAAGTATGAGAAAAGTTCAGCCCAAGTAAAATAAAAACTCAATCACATTCAATTCCAGAGTAGTTTCAAGTTTCAC ATCGTAACCATTTTCGCCCGGAATTC Murine IL-17 mRNA complete CDS: GAGGCTCAAGTGCACCCAGCACCAGCTGATCAGGACGCGCAAACATGAGTCCAGGGAGAGCTTCATCTGTGTCTCTGATGCTGTTGCT GCTGCTGAGCCTGGCGGCTACAGTGAAGGCAGCAGCGATCATCCCTCAAAGCTCAGCGTGTCCAAACACTGAGGCCAAGGACTTCCTC CAGAATGTGAAGGTCAACCTCAAAGTCTTTAACTCCCTTGGCGCAAAAGTGAGCTCCAGAAGGCCCTCAGACTACCTCAACCGTTCCA CGTCACCCTGGACTCTCCACCGCAATGAAGACCCTGATAGATATCCCTCTGTGATCTGGGAAGCTCAGTGCCGCCACCAGCGCTGTGT CAATGCGGAGGGAAAGCTGGACCACCACATGAATTCTGTTCTCATCCAGCAAGAGATCCTGGTCCTGAAGAGGGAGCCTGAGAGCTGC CCCTTCACTTTCAGGGTCGAGAAGATGCTGGTGGGTGTGGGCTGCACCTGCGTGGCCTCGATTGTCCGCCAGGCAGCCTAAACAGAGA CCCGCGGCTGACCCCTAAGAAACCCCCACGTTTCTCAGCAAACTTACTTGCATTTTTAAAACAGTTCGTGCTATTGATTTTCAGCAAG GAATGTGGATTCAGAGGCAGATTCAGAATTGTCTGCCCTCCACAATGAAAAGAAGGTGTAAAGGGGTCCCAAACTGCTTCGTGTTTGT TTTTCTGTGGACTTTAAATTATTTGTGTATTTACAATATCCCAAGATAACTTTGAAGGCGTAACTTATTTAATGAAGTATCTACATTA TTATTATGTTTCTTTCTGAAGAAGACAAAATTCAAGACTCAGAAATTTTATTATTTAAAAGGTAAGCCTATATTTATATGAGCTATTT ATGAATCTATTTATTTTTCTTCAGTATTTGAAGTATTAAGAACATGATTTTCAGATCTACCTAGGGAAGTCCTAAGTAAGATTAAATA TTAATGGAAATTTCAGCTTTACTATTTGGTTGATTTAAGGTTCTCTCCTCTGAATGGGGTGAAAACCAAACTTAGTTTTATGTTTAAT AACTTTTTAAATTATTGAAGATTCAAAAAATTGGATAATTTAGCTCCCTACTCTGTTTT Rat CTLA-8 mRNA sequence: GAATTCCATCCATGTGCCTGATGCTGTTGCTGCTACTGAACCTGGAGGCTACAGTGAAGGCAGCGGTACTCATCCCTCAAAGTTCAGT GTGTCCAAACGCCGAGGCCAATAACTTTCTCCAGAACGTGAAGGTCAACCTGAAAGTCATCAACTCCCTTAGCTCAAAAGCGAGCTCC AGAAGGCCCTCAGACTACCTCAACCGTTCCACTTCACCCTGGACTCTGAGCCGCAATGAGGACCCTGATAGATATCCTTCTGTGATCT GGGAGGCACAGTGCCGCCACCAGCGCTGTGTCAACGCTGAGGGGAAGTTGGACCACCACATGAATTCTGTTCTCATCCAGCAAGAGAT CCTGGTCCTGAAGAGGGAGCCTGAGAAGTGCCCCTTCACTTTCCGGGTGGAGAAGATGCTGGTGGGCGTGGGCTGCACCTGCGTTTCC TCTATTGTCCGCCATGCGTCCTAAACAGAGACCTGAGGCTAGCCCCTAAGAAACCCCTGCGTTTCTCTGCAAACTTCCTTGTCTTTTT AAAACAGTTCACAGTTGAATCTCAGCAAGTGATATGGATTTAAAGGCGGGGTTAGAATTGTCTGCCTTCCACCCTGAAAAGAAGGCGC AGAGGGGATATAAATTGCTTCTTGTTTTTCTGTGGGCTTTAAATTATTTATGTATTTACTCTATCCCGAGATAACTTTGAGGCATAAG TTATTTTAATGAATTATCTACATTATTATTATGTTTCTTAATGCAGAAGACAAAATTCAAGACTAAGAAATTTTATTATTTAAAAGGT AAAACCTATATTTATATGAGCTATTTATGGGTCTATTTATTTTTCTTCAGTGCTAAGATCATGATTATCAGATCTACCTAAGGAAGTC CTAAATAATATTAAATATTAATTGAAATTTCAGTTTTACTATTTGCTTATTTAAGGTTCCCTCCTCTGAATGGTGTGAAATCAAACCT CGTTTTATGTTTTTAAATTATTGAGGCTTCGAAAAATTGGGCAATTTAGCTTCCTACTGTGTGTTTAAAAACCTTGTAACAATATCAC TATAATAAATTTTTGGAAGAAAAT Herpesvirus saimiri DNA for ORF 13: ATGACATTTAGAAAGACTTCACTTGTGTTACTTCTGCTGCTGAGCATAGATTGTATAGTAAAGTCAGAAATAACCAGCGCACAAACCC CAAGATGCTTAGCTGCTAACAATAGCTTCCCACGGTCTGTGATGGTTACTTTGAGCATCCGTAACTGGAATACTAGTTCTAAAAGGGC TTCAGACTACTACAATAGATCTACGTCTCCTTGGACTCTCTATCGCAATGAAGATCAAGATAGATATCCTTCTGTGATTTGGGAAGCA AAGTGTCGCTACTTAGGATGTGTTAATGCTGATGGGAATGTAGACTACCACATGAACTCAGTCCCTATCCAACAAGAGATTCTAGTAG TGCGCAAAGGGCATAACCCTTGCCCTAATTCATTTCGGCTAGAGAAGATGCTAGTGACTGTAGGTTGCACATGCGTTACTCCTATTGT TCACAATGTAGACTAA

IL-17 243

Chromosome location Human, mouse, and rat genomes contain a single copy of the IL-17 gene. hIL-17 has been mapped to chromosome 2 (2q31), and mIL-17 to chromosome 1(A1-A4) in a known interspecific syntenic region (Rouvier et al., 1993).

PROTEIN

Accession numbers Human IL-17 protein: Q16552, 1587047, 4504651, AAC50341 Mouse IL-17 protein (Balb/c): Q62386, AAB05222 Rat IL-17 protein: Q61453 Herpesvirus saimiri IL-17 protein: CAA73627 Pig IL-17 partial protein sequence: AAD46378 Sheep IL-17 partial protein sequence: AAD46377 Bovine IL-17 partial protein sequence: AAD46376

Sequence See Figure 2.

Description of protein hIL-17 is secreted, after cleavage of a 23 amino acid signal peptide, as a glycoprotein homodimer of 155 amino acids (Fossiez et al., 1996). F-endoglycosidase digestion shifts the apparent molecular weight of purified recombinant hIL-17 expressed by mammalian cells from 22 kDa to 15 kDa on reducing SDSPAGE, suggesting that the higher molecular weight

species represent N-glycosylated forms. Glycosylation appears not to be essential for the function since all the biological activities tested were retained by recombinant human, mouse, and rat IL-17 produced in E. coli (Kennedy et al., 1996). Table 1 shows the percentage homology between the four IL-17 proteins, and their alignment indicates the conservation of six cysteines, one putative N-glycosylation site, and three consensus phosphorylation sites (two potential sites for protein kinase C and one for tyrosine kinase). The six conserved cysteines share spacing features with the cystine knot motif found in nerve growth factor, TGF , PDGF-BB, human chorionic gonadotropic hormone (McDonald and Hendrickson, 1993), and in the Toll ligand Spatzle (Mizuguchi et al., 1998). Each member of the cystine knot superfamily can form dimers, although the mode of dimerization is different in each case. If indeed its six cysteines participate in the cystine knot superstructure, IL-17 will thus form homodimers noncovalently bound by the cystine knot domains. Disruption of the cystine knot would result in the separation of dimers and would explain the shift of apparent molecular weight observed on reducing gel (Fossiez et al., 1996).

CELLULAR SOURCES AND TISSUE EXPRESSION

Cellular sources that produce hIL-17 mRNA appears to be mostly expressed by activated CD4‡ T cells, in particular by CD4‡ CD45RO `memory' T cells (Fossiez et al., 1996). Every T cell activation signal tested so far (including activation with Con A, PHA, anti-CD3 mAB, anti-CD28 mAb, or PI) upregulates IL-17 gene transcription.

Figure 2 Amino acid sequences for human, mouse, rat, and herpesvirus saimiri IL-17. Human IL-17: MTPGKTSLVSLLLLLSLEAIVKAGITIPRNPGCPNSEDKNFPRTVMVNLNIHNRNTNTNPKRSSDYYNRSTSPWNLHRNEDPERYPSV IWEAKCRHLGCINADGNVDYHMNSVPIQQEILVLRREPPHCPNSFRLEKILVSVGCTCVTPIVHHVA Murine IL-17: MSPGRASSVSLMLLLLLSLAATVKAAAIIPQSSACPNTEAKDFLQNVKVNLKVFNSLGAKVSSRRPSDYLNRSTSPWTLHRNEDPDRY PSVIWEAQCRHQRCVNAEGKLDHHMNSVLIQQEILVLKREPESCPFTFRVEKMLVGVGCTCVASIVRQAA Rat IL-17: MCLMLLLLLNLEATVKAAVLIPQSSVCPNAEANNFLQNVKVNLKVINSLSSKASSRRPSDYLNRSTSPWTLSRNEDPDRYPSVIWEAQ CRHQRCVNAEGKLDHHMNSVLIQQEILVLKREPEKCPFTFRVEKMLVGVGCTCVSSIVRHAS Herpesvirus saimiri IL-17: MTFRKTSLVLLLLLSIDCIVKSEITSAQTPRCLAANNSFPRSVMVTLSIRNWNTSSKRASDYYNRSTSPWTLYRNEDQDRYPSVIWEA KCRYLGCVNADGNVDYHMNSVPIQQEILVVRKGHNPCPNSFRLEKMLVTVGCTCVTPIVHNVD

244 Serge Lebecque, FrancËois Fossiez and Elizabeth Bates

Table 1 Summary of data in the literature on the activities of IL-17 in vitro Biological activities

References

T cells

Increase of thymidine incorporation

Yao et al., 1995b

Macrophages

Stimulation of TNF, IL-1 , IL-6, PGE2, IL-10, IL-12, IL-1Ra, and stromelysin secretion

Jovanovic et al., 1998

Mouse macrophages

Absence of stimulation of G-CSF

Cai et al., 1998

Dendritic cells

Induction of maturation

Antonysamy et al., 1999

Induction of IL-6 and IL-8 secretion

Yao et al., 1995a, 1995b; Fossiez et al., 1996; Kennedy et al., 1996

Hematopoietic cells

Stromal cells Fibroblasts

Upregulation of ICAM-1 expression

Yao et al., 1995b

Stimulation of PGE2 secretion

Fossiez et al., 1996

Stimulation of G-CSF secretion

Fossiez et al., 1996; Cai et al., 1998

Stimulation of GM-CSF secretion in combination with TNF

Fossiez et al., 1996

Synoviocytes

Stimulation of LIF secretion

Chabaud et al., 1998

Rat intestinal epithelial cells

Stimulation of CINC and CMP-1 gene expression

Awane et al., 1999

Bronchial epithelial cells

Stimulation of IL-8 and IP-2 secretion

Laan et al., 1999

Endothelial cells

Stimulation of IL-6 and IL-8 secretion

Fossiez et al., 1996; Laan et al., 1999

Chondrocytes

Stimulation of NO production

Attur et al., 1997

Tubular epithelial cells

IL-6, IL-8, MCP-1

Van Kooten et al., 1998

Osteoblasts

Stimulation of PGE2 production, upregulation of OCD

Kotake et al., 1999

Keratinocytes

Stimulation of IL-8 secretion

Teunissen et al., 1998; Albanesi et al., 1999

Stimulation of GM-CSF and IL-6 secretion; upregulation of ICAM-1 and HLA-DR

Teunissen et al., 1998

Melanoma cell lines

Stimulation of IL-6 and IL-8 secretion

Tartour et al., 1999

Cervical carcinoma cell lines

Stimulation of IL-6 and IL-8 secretion

Tartour et al., 1999

Analysis of T cell clones suggests that both TH1 and TH2 CD4= subsets can express IL-17 mRNA at comparable levels (Hans Yssel, unpublished data; Teunissen et al., 1998; Albanesi et al., 1999). However, the production of IL-17 by TH2 cells derived from either synovial fluid or synovial membranes from rheumatoid arthritis patients remains controversial (Aarvak et al., 1999). While activated CD4‡ T cells clearly produce most of the IL-17, CD8‡ T cells might still be activated to secrete some hIL-17. A weak hIL-17 signal was detected in RNA from CD8‡-enriched T cells, but no protein was

detected in the supernatant of CD8‡-enriched T cells cultured for 72 hours in the presence of PMA and ionomycin (Fossiez et al., 1996). Along the same lines, Tartour and coworkers found that the presence of CD4‡, but not of CD8‡ T cells, correlates with the detection by RT-PCR of IL-17 mRNA in human cervical carcinoma biopsies (Tartour et al., 1999). In contrast, Yao et al. (1995b) reported that the supernatants of purified CD8‡ T cells stimulated for 40 hours with PMA and ionomycin or by the combination of anti-CD3 and anti-CD28 mAbs contained low but detectable levels of hIL-17.

IL-17 245 Recently, hIL-17 mRNA was also detected by PCR amplification of cDNA from CD8‡ T cell clones derived from psoriatic skin lesions after anti-CD3 and CD28 (Teunissen et al., 1998) or from PBMC-derived and PMA/ionomycin-activated CD8‡ T cells purified after, but not before, activation (Shin et al., 1998). Indeed, IL-17 expression in memory CD8(‡) T cells may require accessory signals provided by other cells, since culture of ionomycin/PMA-activated CD8(‡) 45RO‡ T cells alone did not result in IL-17 mRNA expression (Shin et al., 1999). However, in neither case was secretion of the hIL-17 protein measured. While hIL-17 mRNA has been recently PCR-amplified from the human myeloma B cell line AF10 (Zhou et al., 1998), the physiological relevance of this observation remains unclear, as no IL-17 transcript could be detected in B cells isolated from the peripheral blood or from tonsil, neither before nor after activation (Fossiez et al., 1996). mRNA was also absent in several fetal tissues (heart, brain, lung, liver, and kidney) as well as adult tissues (heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary, small intestine, and colon) (Fossiez et al., 1996; Yao et al., 1995b). HIL-17 protein secretion was detected by ELISA in the supernatant of PI-activated PBMCs after 16 hours of culture, and reached a plateau of up to 22.7 ng/mL of hIL-17 after 48 hours. In agreement with the induction of a strong IL-17 mRNA signal on northern blot, only purified and activated CD4‡ T cells secreted detectable amounts of hIL-17 (Fossiez et al., 1996). The expression of IL-17 mRNA in mice may differ from that in humans inasmuch as no mIL-17 mRNA was detected by northern blot in purified and activated CD4‡ and CD8‡ T cells or thymocytes. The messenger was exclusively detected in  TCR‡ CD4-CD8- double negative (DN) T cells (Kennedy et al., 1996). As expression was assayed only after activation with anti-CD3 mAb, it is not known whether mIL-17 is constitutively expressed by this T cell subpopulation in vivo. The exact role of  TCR‡ DN T cells, which are found in thymus, spleen, lymph nodes, and bone marrow and which are among the first cells to be activated during immune response, remains to be established. The related  TCR‡DN T cells that express NK1.1 do not produce mIL-17 before or after stimulation (Kennedy et al., 1996). Expression of mIL-17 was also detected by RT-PCR in activated CD4‡ and CD8‡ thymocytes, and at low levels in activated CD8‡ splenic T cells and in the EL-4 T lymphoma cell line (Kennedy et al., 1996; Yao et al., 1995a,b). As described for humans, no IL-17 mRNA was present in a variety of normal mouse

tissues such as heart, brain, spleen, lung, liver, skeletal muscles, kidney, and testis.

RECEPTOR UTILIZATION A ubiquitously expressed, large (864 aa) mouse membrane glycoprotein has been cloned after its binding to soluble vIL-17-Fc fusion protein. This receptor, which also binds mIL-17 is unrelated to previously identified cytokine receptor families. The cDNA encoding a human homolog of the mIL-17R has been isolated by cross-hybridization. Like its mouse counterpart, the human IL-17R (866 aa) is also ubiquitously expressed. Binding studies, doseresponse, and cellular restriction of IL-17 activities suggest the existence of an as yet unidentified highaffinity converting subunit on IL-17-responsive cells.

IN VITRO ACTIVITIES

In vitro findings Activities of IL-17 on Cells of Hematopoietic Origin Initially, a variety of human hematopoietic cell lines were screened for IL-17 biological activity without success (Fossiez et al., 1996). In contrast, Yao and coworkers reported that vIL-17 and mIL-17 can enhance 3- to 4-fold the tritiated thymidine incorporation of purified mouse splenic T cells cultured in presence of suboptimal concentration (1%) of PHA (Yao et al., 1995a). Moreover, high concentrations of a soluble form of the mIL-17R (composed of the extracellular domain of the receptor fused to the Fc portion of human IgG1) inhibited both murine T cell proliferation and IL-2 secretion induced by PHA, Con A, and anti-TCR mAb, suggesting an early autocrine function for IL-17 in the growth control of activated T cells. This might reveal an important difference between human and mouse. Recently, IL-17 was found to exert biological effects on other cells of hematopoietic origin, using two biological readouts. First, Jovanovic and coworkers found that rhIL-17 increased the production of the proinflammatory factors TNF, IL-1 , IL-6, prostaglandin E2 (PGE2), as well as IL-10, IL-12, IL1Ra, and stromelysin by ex vivo enriched human macrophages (Jovanovic et al., 1998). Second, mIL17 was shown to modestly promote the maturation of dendritic cell progenitors, as judged by upregulation of CD40, CD11c, MHC-II, CD80, and CD86 expression (Antonysamy et al., 1999).

246 Serge Lebecque, FrancËois Fossiez and Elizabeth Bates

Activities of IL-17 on Stromal Cells hIL-17 induces the secretion of IL-6 by skin and lung fibroblasts and by normal and rheumatoid synoviocytes. It also stimulates the production of IL-8, PGE2, G-CSF, and LIF by rheumatoid synoviocytes (Fossiez et al., 1996; Chabaud et al., 1998), and upregulates the expression of ICAM-1 by foreskin fibroblasts (Yao et al., 1995b). The increase of G-CSF production by fibroblasts in the presence of IL-17 does not require de novo protein synthesis, but appears to result at least in part from posttranscriptional mechanisms (Cai et al., 1998). Human IL-17 stimulates the production of nitric oxide by normal and osteoarthritic human articular chondrocytes (Attur et al., 1997; Shalom-Barak et al., 1998) and increases their production of IL-1 , IL-6, stromelysin, the inducible nitric oxide synthase (iNOS), and cyclooxygenase 2 (COX-2) (ShalomBarak et al., 1998). When cultured in the presence of IL-17, kidney carcinoma cell lines and keratinocytes also express increased amounts of IL-6 and IL-8 mRNA and/or protein, and they slightly upregulate their expression of ICAM-1 and HLA-DR (Teunissen et al., 1998). IL-17 induces primary human proximal tubular epithelial cells, a type of cell regulating local interstitial inflammatory responses, to secrete higher levels of IL-6, IL-8, and complement component C3 (Van Kooten et al., 1998). IL-17 stimulates the production by endothelial and epithelial cells of several chemokines known to contain NFB-recognition sites in their promoters, including the CXC chemokines IL-8 (Fossiez et al., 1996) and the rat CINC (a homolog of the human GRO), but also of the CC chemokines MIP-2 (Laan et al., 1999), and MCP-1 (Awane et al., 1999; Van Kooten et al., 1998). IL-17 increases the secretion of G-CSF by human synoviocytes (Fossiez et al., 1996), and by murine fibroblasts (Cai et al., 1998) leading fibroblasts to support the growth and differentiation of CD34‡ hematopoietic progenitors into mature neutrophils in coculture experiments (Fossiez et al., 1996). IL-17 synergizes with several proinflammatory factors in doing so. For example, IL-17 cooperates with TNF, IFN , and IL-1 to induce the secretion of IL-6 by rheumatoid synoviocytes (Fossiez et al., 1996; Chabaud et al., 1998), and with IFN to increase the production of IL-6 and IL-8 and to upregulate the expression of ICAM-1 and HLA-DR by human keratinocytes (Teunissen et al., 1998). Likewise, IL-17 and TNF cooperate in causing CXC chemokine release from airway epithelial cells (Laan et al., 1999). Moreover, whereas neither hIL-17 nor

TNF alone had any effect on the secretion of GMCSF, the combination of these two cytokines induced synovial fibroblasts to produce GM-CSF (Fossiez et al., 1996). IL-17 does not appear to have any direct effect on the growth of either normal stromal cells (Fossiez et al., 1996) or tumor cell lines (Tartour et al., 1999). IL-17 decreases the proliferation of intestinal epithelial cells, but this effect was masked by the presence of growth factors in the serum as it could only be detected in culture without serum (Awane et al., 1999). Table 1 summarizes the data gathered in the literature regarding the activities of IL-17 in vitro on human cells (or mouse cells when indicated). Regarding the dose-response curves, with the single and notable exception of the mouse T cell proliferative response (which required almost 1 mg/mL of IL-17; Yao et al., 1995a), all the biological activities reported so far were half-maximal within a range of 2±50 ng/mL of IL-17. As human, mouse, rat, and viral IL-17 proteins induce IL-6 secretion by mouse stromal cells, they all appear to recognize the mouse receptor (Yao et al., 1995a; Kennedy et al., 1996). The Role of vIL-17 vIL-17, which is expressed only during lytic virus replication, has been disrupted in the viral genome, and appears to be neither required for T cell transformation in vitro, nor for pathogenicity in New World primates (Knappe et al., 1998). However, as vIL-17 induces IL-8 secretion by fibroblasts and epithelial cells (Fossiez et al., 1996), it may still exert a positive feedback regulation on infected T cells via the lytically expressed vIL-8R (Ahuja and Murphy, 1993).

IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS

Normal physiological roles Data available in vivo have supported both the proinflammatory and the hematopoietic activities of IL-17. In vivo, intratracheal instillation of hIL-17 increased levels of rat macrophage inflammatory protein 2 (rMIP-2) in bronchoalveolar lavage (BAL) fluid which selectively recruited neutrophils into rat airways (Hoshino et al., 1999; Laan et al., 1999). This recruitment of neutrophils into the airways was

IL-17 247 inhibited by either an anti-hIL-17 antibody or an anti-rMIP-2 antibody. Combined with the observation that conditioned medium from airway epithelial cells treated with IL-17 causes IL-8-dependent neutrophil chemotaxis, these data demonstrate that IL-17 can indirectly recruit neutrophils into the airways via the release of CXC chemokines by bronchial epithelial cells (Laan et al., 1999). The striking, though indirect, effect (via the induction of G-CSF and IL-6 production) of IL-17 on the generation of human neutrophils in vitro (Fossiez et al., 1996) led to the testing of its role in acute neutrophilia. Injection of 10 mg of highly purified and endotoxin-free mouse recombinant IL17 into C57BL6/N mice resulted within 2 hours in a small increase in total white blood count and a 3- to 4-fold increase exclusively of neutrophils. After daily injections, neutrophils numbers increased up to 5-fold at the third day and were maintained during 7 days of treatment. The neutrophilia was likely to be caused by demargination of neutrophils as no mobilization of myeloid progenitors from the bone marrow could be observed after IL-17 treatment, indicating no or insufficient G-CSF induction in vivo (Metcalf, 1991). Using adenovirus-mediated gene transfer of the murine IL-17 cDNA targeted to liver, Schwarzenberger and coworkers observed a similar increase (5-fold) in the peripheral white blood count, including a 10-fold rise in the absolute neutrophil count. This was associated with profound stimulation of splenic hematopoiesis with a doubling in the spleen size over 7±14 days after gene transfer, which returned to near baseline by day 21, although the white blood cell count remained elevated (Schwarzenberger et al., 1998). The acute IL-17-induced neutrophilia could protect mice against gram-negative bacteria. Seven out of 10 mice injected daily with 10 mg of IL-17/ 100 mL for 3 days and who received a single LD90 dose (2  107 cfu) of E. coli 2 hours after the last injection survived, compared with only one surviving mouse from the control group injected with PBS. IL17 protection was mediated by IL-6, as IL-6 knockout mice were not protected from a lethal dose of E. coli. (Krishna et al., manuscript in preparation). In conclusion, through the induction of G-CSF, IL-6 and IL-8 release by stromal cells, IL-17 triggers acute neutrophilia that permits prompt nonspecific immune response against infectious agents. Two human cervical tumor cell lines transfected with a cDNA encoding hIL-17 secreted increased amounts of IL-6 in vitro, but their rate of proliferation in vitro was unchanged. When transplanted into nude mice, the growth of these tumor cells was accelerated as compared to the parent tumor, and was associated with increased expression of mIL-6 and macrophage

recruitment at the tumor site (Tartour et al., 1999). IL-17 therefore behaved like a T cell-specific cytokine with paradoxical tumor-promoting activity.

Knockout mouse phenotypes There have been no reports on IL-17 gene disruption in mice. However, no obvious phenotype was observed after disruption of the IL-17R in mice, although experiments addressing functional alterations have not yet been reported (Spriggs, 1997).

PATHOPHYSIOLOGICAL ROLES IN NORMAL HUMANS AND DISEASE STATES AND DIAGNOSTIC UTILITY

Normal levels and effects No IL-17 has been detected in biological fluids under normal conditions. The physiological functions of IL17 remain to be established, in vitro and mouse in vivo data indicate that IL-17 might play a central role in several inflammatory diseases known to involve activated CD4‡ T cells. The finding that IL-17 can specifically and selectively recruit neutrophils into the airways via the release of IL-8 and MIP-2 from bronchial epithelial cells suggests a novel mechanism linking the activation of T lymphocytes to recruitment of neutrophils in inflammatory airway diseases (Hoshino et al., 1999; Laan et al., 1999). Regarding inflammatory skin diseases, the IL-17induced stimulation of proinflammatory cytokines secretion by keratinocytes in vitro (Teunissen et al., 1998) combined with the finding that the majority of the CD4‡ and CD8‡ T cell clones derived from lesional psoriatic skin express IL-17 mRNA, and that IL-17 mRNA can be detected in biopsies from lesional skin from psoriasis and atopic dermatitis patients, and but not in nonlesional control biopsies (Teunissen et al., 1998 and F. Fossiez, unpublished observations) suggest that IL-17 could amplify and support the maintenance of chronic dermatoses, through stimulation of keratinocytes (Teunissen et al., 1998). IL-17 has been detected by RT-PCR (Van Kooten et al., 1998) and shown by immunofluorescence to be produced by infiltrating lymphocytes in kidney biopsies from patients suffering from graft rejection, whereas pretransplant biopsies and normal kidneys

248 Serge Lebecque, FrancËois Fossiez and Elizabeth Bates were negative. IL-17 expression could also be found in in vitro cultured and activated graft-infiltrating T cells (Van Kooten et al., 1998). Indeed, the use of IL17 detection as an early marker for kidney rejection was further suggested in a recent study from Strehlau and coworkers who showed that the presence of IL-17 transcripts in renal biopsies was both sensitive and specific (Strehlau et al., 1997). IL-17R:Fc administration i.p. for different durations posttransplant to murine recipients of MHC-mismatched cardiac allografts, significantly prolonged nonvascularized or vascularized cardiac allograft median survival time (Antonysamy et al., 1999). Taken together, these results indicate that IL-17 may be important in the regulation of local inflammatory responses during allograft rejection. Biologically active IL-17 is spontaneously present in the supernatant from rheumatoid arthritis synovium pieces but not from osteoarthritis synovium, which contains a reduced T cell infiltrate. Immunostaining of the synovial tissues of RA patients has confirmed the presence of IL-17 in a subset of CD4‡CD45RO‡ T cells (Chabaud et al., 1998, 1999; Kotake et al., 1999). Furthermore, CD4‡ T cells associated with a TH1/TH0 phenotype recovered from rheumatoid arthritis synovial tissue secrete IL17 in vitro upon activation. Given its capacity to activate synoviocytes, chondrocytes or osteoblasts to express genes such as IL-1 , IL-6, stromelysin, iNOS and COX-2 (Shalom-Barak et al., 1998) and to produce PGE2 (Fossiez et al., 1996; Kotake et al., 1999) and NO (Attur et al., 1997; Shalom-Barak et al., 1998) that are bioactive molecules implicated in cartilage degradation and joint inflammation, IL-17 production appears of importance in the inflammatory reaction in RA. Indeed, blocking IL-17 in RA synovial cultures with a neutralizing antibody resulted in a 20±50% decrease in the production of IL-6 (Chabaud et al., 1999). Moreover, in cocultures of bone marrow cells and osteoblasts, IL-17 first acts on osteoblasts and stimulates both COX-2-dependent PGE2 synthesis and osteoclast differentiation factor (ODF, which is identical to TRANCE/RANKL/ OPGL) gene expression, which in turn induces differentiation of osteoclast progenitors into mature osteoclasts (Kotake et al., 1999). Anti-IL-17 antibody significantly inhibited osteoclast formation induced by culture media of RA synovial tissues (Kotake et al., 1999), further suggesting that IL-17 is a crucial, although not exclusive cytokine (Rifas and Avioli, 1999) for osteoclastic bone resorption in RA patients. Multiple sclerosis (MS) is a CNS inflammatory disease whose pathogenesis involves myelin-directed autoimmunity. Matusevicius and coworkers have observed higher numbers of IL-17 mRNA-expressing

cells in cerebrospinal fluid (CSF) versus blood from patients with MS compared to healthy individuals, with the highest numbers in blood during clinical exacerbation's (Matusevicius et al., 1999), again suggesting a pathogenic role for this cytokine. Of potential clinical importance, while glucocorticoids downregulate some proinflammatory functions of IL-17, such as the increase of CXC chemokine production by bronchial epithelial and endothelial cells (Laan et al., 1999), they could not block the IL17-dependent NO production in human osteoarthritis cartilage explants (Attur et al., 1997). The detection of IL-17 mRNA in cervical tumor biopsies from patients with CD4 infiltration combined with the growth-promoting effect on cervical tumors in nude mice supports the idea that IL-17 may have a deleterious tumor-promoting activity in human cervical cancer, and perhaps also in melanomas (Tartour et al., 1999).

IN THERAPY

Preclinical ± How does it affect disease models in animals? While preliminary data advocate the involvement of IL-17 in inflammatory conditions as well as in acute reactive neutrophilia, it is fair to anticipate that the functional importance of IL-17 has not yet been fully appraised. This novel cytokine produced by activated memory T cells appears to play an upstream role in T cell-triggered inflammation and hematopoiesis, by stimulating stromal cells to secrete other cytokines and growth factors. The hematopoietic effect of IL-17, and in particular its ability to trigger indirectly an acute neutrophilia might have therapeutic applications in the context of immunosuppression, i.e. after bone marrow transplantation. In contrast, IL-17 might represent a target for therapeutic inhibition in T cell-dependent autoimmune diseases (like rheumatoid arthritis or multiple sclerosis), in chronic inflammatory conditions of the lung (chronic pulmonary obstructive disease or asthma) of the skin (psoriasis, atopic dermatitis) and of the intestinal tract (inflammatory bowel diseases), in organ graft rejection, and in some cancers.

Pharmacokinetics There are currently no data available regarding the pharmacokinetics of IL-17.

IL-17 249

Toxicity There are currently no data available regarding the toxicity of IL-17.

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Polyclonal anti-mIL-17 goat antiserum (catalog no.: AF-421-NA) Monoclonal anti-mIL-17 mouse IgG2a antibody, selected to neutralize the bioactivity of mIL-17 (catalog no.: MAB421) Monoclonal anti-mIL-17 mouse IgG2a antibody, selected as a capture antibody in mouse IL-17 sandwich ELISAs (catalog no.: MAB721) Quantitative mIL-17 colorimetric sandwich ELISA (catalog no.: D1700) Biosource Quantitative hIL-17 colorimetric solid phase ELISA (catalog no.: KAC1591, KAC1592) Pharmingen Monoclonal anti-mIL-17 mouse IgG1 rat antibody, selected as a capture antibody in mouse IL-17 sandwich ELISAs (catalog no.: 23290D, purified 23291A/D, PE-conjugated 23295A) Monoclonal anti-mIL-17 mouse IgG1 rat antibody, biotinylated and selected as a detection antibody in mouse IL-17 sandwich ELISAs (catalog no.: 23282D)

ACKNOWLEDGEMENTS The authors wish to thank first P. Golstein and E. Rouvier for sharing the early CTLA-8 data. The members of LIR and of DNAX are aknowledged for their contribution to the identification and the determination of IL-17 functions: J. Abrams, S. AitYahia, J. Banchereau, E. Bates, F. Bazan, J. C. Bories, F. BrieÁre, C. Caux, P. Chomarat, B. Das Mahapatra, O. Djossou, L. Flores-Romo, C. Gaillard, E. Garcia, P. Garrone, D. Gorman, C. H. Hannum, R. Kastelein, J. Kennedy, P. Krishna, C. Maat, K. Moore, R. Murray, C. Perone, J. J. Pin, S. Saeland, A. Zlotnik, G. Zurawsky, and S. Zurawsky. They also want to thank J. Chiller, D. de Groote, J. F. Nicolas, P. Miossec, S. Narula, M. Spriggs, E. Tartour, and C. Von Kooten for exchange of unpublished informations and discussions. The editorial assistance of S. Bourdarel has been greatly appreciated.