I-309 Michael Steven Krangel* Immunology, Duke University Medical Center, PO Box 3010, Durham, NC 27710, USA *corresponding author tel: 919-684-4985, fax: 919-684-8982, e-mail: [email protected] DOI: 10.1006/rwcy.2000.11003. SUMMARY structure has not been determined. Although the proteinisassumed,onthebasisofsequencehomology, tosharewithother(cid:12)chemokinesthebasicchemokine I-309isaCCchemokinethatisproducedbyactivated fold, I-309 displays several structural features that T lymphocytes, monocytes, and a mast cell leukemia. distinguish it from other chemokines. These features Ithasstructuralfeaturesthatdistinguishitfromother includeanadditionalpairofcysteinesthatformathird chemokines, including a third intrachain disulfide intramolecular disulfide bond, and a propensity to bond and a monomeric association state even at high remain monomeric at concentrations at which many concentrations.I-309bindsselectivelytoCCR8andis otherchemokinesdimerize(Paolinietal.,1994). chemotactic for monocytes and TH2-differentiated T cells in vitro. Main activities and BACKGROUND pathophysiological roles Discovery I-309 was initially shown to be chemotactic for human monocytes (Miller and Krangel, 1992). AnI-309cDNAclonewasinitiallydiscoveredaspart Subsequent studies have attributed additional activ- of a subtractive hybridization strategy designed to ities, most notably the ability to protect the murine identify cDNA clones representing transcripts that hybridoma BW5147 from dexamethasone-induced were expressed in the activated human (cid:13)(cid:14) T cell line growth inhibition and apoptosis (van Snick et al., IDP2 but not expressed in the Epstein–Barr virus 1996),andchemoattractantactivitytowardsactivated (EBV)-transformedBcelllineJY(Milleretal.,1989). TH2-polarized T cells (Zingoni et al., 1998). The cDNA was shown to direct the synthesis of a secreted protein in transiently or stably transfected cell lines (Miller et al., 1989; Miller and Krangel, 1992), and the purified protein was shown to possess GENE AND GENE REGULATION monocyte chemoattractant activity (Miller and Krangel, 1992). Accession numbers Alternative names Gene: M57506 cDNA: M57502 The murine homolog of I-309 is TCA3. Structure Chromosome location I-309isahighlybasic73aminoacidproteinthatbelongs Human chromosome 17q11.2 (Miller et al., 1990; totheCCor(cid:12)chemokinefamily.Athree-dimensional Naruse et al., 1996). 1168 Michael Steven Krangel Relevant linkages cell line migrates on SDS-PAGE as a 15–16kDa doublet due to the presence of a heterogeneous, N-linked oligosaccharide (Miller and Krangel, 1992; I-309mapswithintheCCchemokinegeneclusterthat Selvan et al., 1994; Selvan et al., 1997). I-309 is includes MCP-1, MCP-3, MIP-1(cid:11), MIP-1(cid:12), and presumedtohaveamonomerstructurethatissimilar RANTES (Naruse et al., 1996). to other (cid:12) chemokines; however, a three-dimensional structure has not yet been determined. I-309 includes Regulatory sites and corresponding the four cysteine residues characteristic of all (cid:12) chemokines(C10,C11,C34,andC50).Inaddition,I- transcription factors 309 includes a pair of cysteine residues not found in other (cid:12) chemokines (C26 and C68). These cysteines Over 800bp of sequence is available upstream of the have been demonstrated to be linked in an intra- cap site, and there is substantial homology with the molecular disulfide bond that covalently couples the correspondingregionoftheTCA3gene(Milleretal., predicted C-terminal (cid:11) helix to the first (cid:12) strand 1990).However,therehasbeennoexplicitanalysisof (Paolini et al., 1994). Another distinctive feature of regulatory sites and factors. the I-309 protein is its propensity to remain mono- meric at concentrations at which most other chemo- kines self-associate (Paolini et al., 1994). Cells and tissues that express the gene Important homologies T lymphocytes: CD4(cid:135)CD8(cid:255) (cid:11)(cid:12), CD4(cid:255)CD8(cid:135) (cid:11)(cid:12), I-309 displays homology to other human (cid:12) chemo- and CD4(cid:255)8(cid:255) (cid:13)(cid:14)T cell lines and clones (Miller et al., kines (for example, 37% amino acid identity to 1989),JurkatTcellleukemia(Milleretal.,1989),Mo human MCP-3). Interestingly, homology of I-309 to T cell leukemia (van Snick et al., 1996). Monocytes: its murine homolog is relatively poor (37% amino adherence purified peripheral blood monocytes acididentityforthematureproteins).However,I-309 (Selvan et al., 1997). Mast cells: HMC-1 mast cell and TCA3 share the distinctive pair of additional leukemia (Selvan et al., 1994). cysteine residues. PROTEIN Posttranslational modifications Accession numbers I-309 bears a single, N-linked oligosaccaride at residue 29. P22362 A37236 CELLULAR SOURCES AND Sequence TISSUE EXPRESSION Cellular sources that produce See Figure 1. I-309 is inducible in T lymphocytes, including Description of protein CD4(cid:135)CD8(cid:255) (cid:11)(cid:12), CD4(cid:255)CD8(cid:135) (cid:11)(cid:12), and CD4(cid:255) CD8(cid:255) (cid:13)(cid:14) T cell lines and clones (Miller et al., The mature I-309 protein is 73 amino acids in length. 1989),aswellasintheTcellleukemiasJurkat(Miller I-309 secreted from transfected cells, from stimulated et al., 1989) and Mo (van Snick et al., 1996). I-309 is monocytes, and from a stimulated mast cell leukemia also inducible in the HMC-1 mast cell leukemia Figure 1 Amino acid sequence for I-309. KSMQVPFSRC CFSFAEQEIP LRAILCYRNT SSICSNEGLI FKLKRGKEAC ALDTVGWVQR HRKMLRHCPS KRK I-309 1169 (Selvan et al., 1994), and in adherence purified onthetwopopulations(Zingonietal.,1998).I-309is peripheral blood monocytes (Selvan et al., 1997). chemotactic for the cell line K562 in the 1–10nM concentration range (Horuk et al., 1998) and for the cell line THP1 at concentrations of 10nM and above Eliciting and inhibitory stimuli, (van Snick et al., 1996). I-309 has been shown to stimulate an increase in cytoplasmic free calcium in including exogenous and peripheral blood monocytes (Miller and Krangel, endogenous modulators 1992),inactivatedTH2-differentiatedTcells(Zingoni et al., 1998), in HL-60 cells following differentiation In T leukemia cell lines as well as T lymphocyte lines in butyric acid and IL-5 (Tiffany et al., 1997) and in and clones, stimuli such as phorbol myristate acetate CCR8 transfectants (Roos et al., 1997; Tiffany et al., (PMA), phytohemagglutinin (PHA), anti-CD3 plus 1997; Goya et al., 1998). PMA, and PMA plus PHA, will induce I-309 (Miller I-309 has also been shown to protect the murine etal.,1989;Selvanetal.,1994;vanSnicketal.,1996). thymoma BW5147 from dexamethasone-induced In mast cell leukemia HMC-1, PMA induces I-309 apoptosis and growth inhibition, with effects on mRNA and protein secretion (Selvan et al., 1994). In growthdemonstratedatconcentrationsinthe100pM adherence-purified peripheral blood monocytes, sti- range and an antiapoptotic effect explicitly demon- mulationwithimmobilizedIgGorlipopolysaccharide stratedataconcentrationthatisseveralhundred-fold (LPS) alone will induce low levels of I-309 mRNA higher (van Snick et al., 1996). I-309 was also found and secreted protein, whereas the combination of the toinhibitCCR8-dependentHIV-1envelope-mediated two stimuli will induce substantially higher levels cell fusion and virus infection (Horuk et al., 1998). (Selvan et al., 1997). IL-1(cid:11) or IL-1(cid:12) can replace LPS and synergize with immobilized IgG to stimulate I-309 production in stimulated monocytes (Selvan Regulatory molecules: Inhibitors et al., 1997). and enhancers In monocytes stimulated by immobilized IgG plus LPS, IL-1(cid:11) that is produced in response to these stimulatorsisacriticalendogenousmediatorofI-309 Pertussis toxin inhibits the effects of I-309 on induction (Selvan et al., 1997). In T cells stimulated BW5147 (van Snick et al., 1996), HL-60 (Tiffany with anti-CD3 plus PMA, the glucocorticoid methyl- et al., 1997), and CCR8 transfectants (Goya et al., prednisolone inhibits the induction of I-309 tran- 1998). scripts (Selvan et al., 1994). Bioassays used RECEPTOR UTILIZATION Chemotaxis in response to I-309 is measured using a The only identified receptor for I-309 is CCR8 (Roos standard Boyden chamber microchemotaxis assay et al., 1997; Tiffany et al., 1997; Goya et al., 1998). (Miller and Krangel, 1992; van Snick et al., 1996; Tiffanyetal.,1997;Horuketal.,1998;Zingonietal., 1998). Elevation of cytoplasmic free calcium in response IN VITRO ACTIVITIES to I-309 is measured by monitoring cells loaded with calcium-sensitive fluorescent dyes such as Indo-1/ In vitro findings AM, Fura-2/AM, or Fluo-3 using either a fluoro- meter or a flow cytometer (Miller and Krangel, 1992; At concentrations in the 100nM range, I-309 has Roos et al., 1997; Tiffany et al., 1997; Goya et al., been shown to function as a chemoattractant for 1998;Zingonietal.,1998).EffectsofI-309ongrowth peripheral blood monocytes (Miller and Krangel, of BW5147 in the presence of 0.25mM dexametha- 1992). However, it has been shown to function as a sone is measured by assay of [3H]thymidine incor- much more potent chemoattractant for activated poration (van Snick et al., 1996). Effects of I-309 on TH2-differentiated T cells, with activity in the 10pM apoptosis of BW5147 in the presence of 0.25mM range. It is active on TH1-differentiated T cells at dexamethasone is measured using agarose gel elec- concentrations that are 1000-fold higher, presumably trophoresis to assess DNA fragmentation (van Snick as a consequence of differential expression of CCR8 et al., 1996). 1170 Michael Steven Krangel References Selvan,R.S.,Zhou,L.-J.,andKrangel,M.S.(1997).Regulation of I-309 gene expression in human monocytes by endogenous interleukin-1.Eur.J.Immunol.27,687–694. Goya,I.,Gutierrez,J.,Varona,R.,Kremer,L.,Zaballos,A.,and Tiffany, H. L., Lautens, L. L., Gao, J.-L., Pease, J., Locati, M., Marquez, G. (1998). Identification of CCR8 as the specific Combadiere,C.,Modi,W.,Bonner,T.I.,andMurphy,P.M. receptorforhuman(cid:12)-chemokineI-309:cloningandmolecular (1997).Identificationofahumanmonocytethymusreceptorfor characterization of murine CCR8 as the receptor for TCA-3. theCCchemokineI-309.J.Exp.Med.186,165–171. J.Immunol.160,1975–1981. van Snick, J., Houssiau, F., Proost, P., van Damme, J., and Horuk,R.,Hesselgesser,J.,Zhou,Y.,Faulds,D.,Halks-Miller,M., Renauld,J.-C.(1996).I-309/Tcellactivationgene-3chemokine Harvey,S.,Taub,D.,Samson,M.,Parmentier,M.,Rucker,J., protects murine T cell lymphomas against dexamethasone- Doranz, B., and Doms, R. W. (1998). The CC chemokine inducedapoptosis.J.Immunol.157,2570–2576. I-309inhibitsCCR8-dependentinfectionbydiverseHIV-1strains. Zingoni, A., Soto, H., Hedrick, J. A., Stoppacciaro, A., J.Biol.Chem.273,386–391. Storlazzi, C. T., Sinigaglia, F., D’Ambrosio, D., O’Garra, A., Miller, M. D., and Krangel, M. S. (1992). The human cytokine Robinson, D., Rocchi, M., Santoni, A., Zlotnik, A., and I-309isamonocytechemoattractant.Proc.NatlAcad.Sci.USA Napolitano,M.(1998).ThechemokinereceptorCCR8isprefer- 89,2950–2954. entiallyexpressedinTH2butnotinTH1cells.J.Immunol.161, Miller,M.D.,Hata,S.,deWaalMalefyt,R.,andKrangel,M.S. 547–551. (1989). A novel polypeptide secreted by activated human T lymphocytes.J.Immunol.143,2907–2916. Miller, M. D., Wilson, S. D., Dorf, M. E., Seuanez, H. N., LICENSED PRODUCTS O’Brien, S. J., and Krangel, M. S. (1990). Sequence chromo- somallocationoftheI-309gene:relationshiptogenesencoding a family of inflammatory cytokines. J. Immunol. 145, 2737– New England Nuclear (Boston, MA, USA): 125I- 2744. recombinant human I-309, Bolton-Hunter labeled Naruse, K., Ueno, M., Satoh, T., Nomiyama, H., Tei, H., (catalog no. NEX-364) Takeda, M., Ledbetter, D. H., van Coillie, E., Opdenakker, G., Gunge, N., Sakaki, Y., Iio, M., and Peprotech (Rocky Hill, NJ, USA): Human I-309 Miura,R.(1996).AYACcontigofthehumanCCchemokine (catalog no. 300-37) genesclusteredonchromosome17q11.2.Genomics34,236–240. Research Diagnostics (Flanders, NJ, USA): Human Paolini, J. F., Willard, D., Consler, T., Luther, M., and I-309 (catalog no. RDI-3037) Krangel, M. S. (1994). The chemokines IL-8, monocyte che- R&D Systems (Minneapolis, MN, USA): Human moattractant protein-1, I-309 are monomers at physiologically relevantconcentrations.J.Immunol.153,2704–2717. I-309 (catalog no. 272-1-010) Roos, R. S., Loetscher, M., Legler, D. F., Clark-Lewis, I., R&D Systems (Minneapolis, MN, USA): Goat anti- Baggiolini, M., and Moser, B. (1997). Identification of CCR8, human I-309, IgG fraction (catalog no. AB-272-PB) thereceptorforthehumanCCchemokineI-309.J.Biol.Chem. Sigma(StLouis,MO,USA):Goatanti-humanI-309, 272,17251–17254. IgG fraction (catalog no. I7391) Selvan, R. S., Butterfield, J. H., and Krangel, M. S. (1994). Expression of multiple chemokine genes bya human mast cell Toyobo (Japan): Human I-309 (catalog no. PT leukemia.J.Biol.Chem.269,13893–13898. 300-37)