I-309

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 I-309 is a CC chemokine that is produced by activated T lymphocytes, monocytes, and a mast cell leukemia. It has structural features that distinguish it from other chemokines, including a third intrachain disulfide bond and a monomeric association state even at high concentrations. I-309 binds selectively to CCR8 and is chemotactic for monocytes and TH2-differentiated T cells in vitro.

BACKGROUND

Discovery An I-309 cDNA clone was initially discovered as part of a subtractive hybridization strategy designed to identify cDNA clones representing transcripts that were expressed in the activated human  T cell line IDP2 but not expressed in the Epstein±Barr virus (EBV)-transformed B cell line JY (Miller et al., 1989). 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 monocyte chemoattractant activity (Miller and Krangel, 1992).

Alternative names The murine homolog of I-309 is TCA3.

structure has not been determined. Although the protein is assumed, on the basis of sequence homology, to share with other chemokines the basic chemokine fold, I-309 displays several structural features that distinguish it from other chemokines. These features include an additional pair of cysteines that form a third intramolecular disulfide bond, and a propensity to remain monomeric at concentrations at which many other chemokines dimerize (Paolini et al., 1994).

Main activities and pathophysiological roles I-309 was initially shown to be chemotactic for human monocytes (Miller and Krangel, 1992). Subsequent studies have attributed additional activities, most notably the ability to protect the murine hybridoma BW5147 from dexamethasone-induced growth inhibition and apoptosis (van Snick et al., 1996), and chemoattractant activity towards activated TH2-polarized T cells (Zingoni et al., 1998).

GENE AND GENE REGULATION

Accession numbers Gene: M57506 cDNA: M57502

Structure

Chromosome location

I-309 is a highly basic 73 amino acid protein that belongs to the CC or chemokine family. A three-dimensional

Human chromosome 17q11.2 (Miller et al., 1990; Naruse et al., 1996).

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Relevant linkages I-309 maps within the CC chemokine gene cluster that includes MCP-1, MCP-3, MIP-1, MIP-1 , and RANTES (Naruse et al., 1996).

Regulatory sites and corresponding transcription factors Over 800 bp of sequence is available upstream of the cap site, and there is substantial homology with the corresponding region of the TCA3 gene (Miller et al., 1990). However, there has been no explicit analysis of regulatory sites and factors.

Cells and tissues that express the gene T lymphocytes: CD4‡CD8ÿ  , CD4ÿCD8‡  , and CD4ÿ8ÿ T cell lines and clones (Miller et al., 1989), Jurkat T cell leukemia (Miller et al., 1989), Mo T cell leukemia (van Snick et al., 1996). Monocytes: adherence purified peripheral blood monocytes (Selvan et al., 1997). Mast cells: HMC-1 mast cell leukemia (Selvan et al., 1994).

PROTEIN

cell line migrates on SDS-PAGE as a 15±16 kDa doublet due to the presence of a heterogeneous, N-linked oligosaccharide (Miller and Krangel, 1992; Selvan et al., 1994; Selvan et al., 1997). I-309 is presumed to have a monomer structure that is similar to other chemokines; however, a three-dimensional structure has not yet been determined. I-309 includes the four cysteine residues characteristic of all chemokines (C10, C11, C34, and C50). In addition, I309 includes a pair of cysteine residues not found in other chemokines (C26 and C68). These cysteines have been demonstrated to be linked in an intramolecular disulfide bond that covalently couples the predicted C-terminal  helix to the first strand (Paolini et al., 1994). Another distinctive feature of the I-309 protein is its propensity to remain monomeric at concentrations at which most other chemokines self-associate (Paolini et al., 1994).

Important homologies I-309 displays homology to other human chemokines (for example, 37% amino acid identity to human MCP-3). Interestingly, homology of I-309 to its murine homolog is relatively poor (37% amino acid identity for the mature proteins). However, I-309 and TCA3 share the distinctive pair of additional cysteine residues.

Posttranslational modifications

Accession numbers

I-309 bears a single, N-linked oligosaccaride at residue 29.

P22362 A37236

Sequence

CELLULAR SOURCES AND TISSUE EXPRESSION

See Figure 1.

Cellular sources that produce

Description of protein The mature I-309 protein is 73 amino acids in length. I-309 secreted from transfected cells, from stimulated monocytes, and from a stimulated mast cell leukemia Figure 1

I-309 is inducible in T lymphocytes, including CD4‡CD8ÿ  , CD4ÿCD8‡  , and CD4ÿ CD8ÿ  T cell lines and clones (Miller et al., 1989), as well as in the T cell leukemias Jurkat (Miller et al., 1989) and Mo (van Snick et al., 1996). I-309 is also inducible in the HMC-1 mast cell leukemia

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 peripheral blood monocytes (Selvan et al., 1997).

Eliciting and inhibitory stimuli, including exogenous and endogenous modulators In T leukemia cell lines as well as T lymphocyte lines and clones, stimuli such as phorbol myristate acetate (PMA), phytohemagglutinin (PHA), anti-CD3 plus PMA, and PMA plus PHA, will induce I-309 (Miller et al., 1989; Selvan et al., 1994; van Snick et al., 1996). In mast cell leukemia HMC-1, PMA induces I-309 mRNA and protein secretion (Selvan et al., 1994). In adherence-purified peripheral blood monocytes, stimulation with immobilized IgG or lipopolysaccharide (LPS) alone will induce low levels of I-309 mRNA and secreted protein, whereas the combination of the two stimuli will induce substantially higher levels (Selvan et al., 1997). IL-1 or IL-1 can replace LPS and synergize with immobilized IgG to stimulate I-309 production in stimulated monocytes (Selvan et al., 1997). In monocytes stimulated by immobilized IgG plus LPS, IL-1 that is produced in response to these stimulators is a critical endogenous mediator of I-309 induction (Selvan et al., 1997). In T cells stimulated with anti-CD3 plus PMA, the glucocorticoid methylprednisolone inhibits the induction of I-309 transcripts (Selvan et al., 1994).

RECEPTOR UTILIZATION The only identified receptor for I-309 is CCR8 (Roos et al., 1997; Tiffany et al., 1997; Goya et al., 1998).

IN VITRO ACTIVITIES

In vitro findings At concentrations in the 100 nM range, I-309 has been shown to function as a chemoattractant for peripheral blood monocytes (Miller and Krangel, 1992). However, it has been shown to function as a much more potent chemoattractant for activated TH2-differentiated T cells, with activity in the 10 pM range. It is active on TH1-differentiated T cells at concentrations that are 1000-fold higher, presumably as a consequence of differential expression of CCR8

on the two populations (Zingoni et al., 1998). I-309 is chemotactic for the cell line K562 in the 1±10 nM concentration range (Horuk et al., 1998) and for the cell line THP1 at concentrations of 10 nM and above (van Snick et al., 1996). I-309 has been shown to stimulate an increase in cytoplasmic free calcium in peripheral blood monocytes (Miller and Krangel, 1992), in activated TH2-differentiated T cells (Zingoni et al., 1998), in HL-60 cells following differentiation in butyric acid and IL-5 (Tiffany et al., 1997) and in CCR8 transfectants (Roos et al., 1997; Tiffany et al., 1997; Goya et al., 1998). I-309 has also been shown to protect the murine thymoma BW5147 from dexamethasone-induced apoptosis and growth inhibition, with effects on growth demonstrated at concentrations in the 100 pM range and an antiapoptotic effect explicitly demonstrated at a concentration that is several hundred-fold higher (van Snick et al., 1996). I-309 was also found to inhibit CCR8-dependent HIV-1 envelope-mediated cell fusion and virus infection (Horuk et al., 1998).

Regulatory molecules: Inhibitors and enhancers Pertussis toxin inhibits the effects of I-309 on BW5147 (van Snick et al., 1996), HL-60 (Tiffany et al., 1997), and CCR8 transfectants (Goya et al., 1998).

Bioassays used Chemotaxis in response to I-309 is measured using a standard Boyden chamber microchemotaxis assay (Miller and Krangel, 1992; van Snick et al., 1996; Tiffany et al., 1997; Horuk et al., 1998; Zingoni et al., 1998). Elevation of cytoplasmic free calcium in response to I-309 is measured by monitoring cells loaded with calcium-sensitive fluorescent dyes such as Indo-1/ AM, Fura-2/AM, or Fluo-3 using either a fluorometer or a flow cytometer (Miller and Krangel, 1992; Roos et al., 1997; Tiffany et al., 1997; Goya et al., 1998; Zingoni et al., 1998). Effects of I-309 on growth of BW5147 in the presence of 0.25 mM dexamethasone is measured by assay of [3 H]thymidine incorporation (van Snick et al., 1996). Effects of I-309 on apoptosis of BW5147 in the presence of 0.25 mM dexamethasone is measured using agarose gel electrophoresis to assess DNA fragmentation (van Snick et al., 1996).

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References Goya, I., Gutierrez, J., Varona, R., Kremer, L., Zaballos, A., and Marquez, G. (1998). Identification of CCR8 as the specific receptor for human -chemokine I-309: cloning and molecular characterization of murine CCR8 as the receptor for TCA-3. J. Immunol. 160, 1975±1981. Horuk, R., Hesselgesser, J., Zhou, Y., Faulds, D., Halks-Miller, M., Harvey, S., Taub, D., Samson, M., Parmentier, M., Rucker, J., Doranz, B., and Doms, R. W. (1998). The CC chemokine I-309 inhibits CCR8-dependent infection by diverse HIV-1 strains. J. Biol. Chem. 273, 386±391. Miller, M. D., and Krangel, M. S. (1992). The human cytokine I-309 is a monocyte chemoattractant. Proc. Natl Acad. Sci. USA 89, 2950±2954. Miller, M. D., Hata, S., de Waal Malefyt, R., and Krangel, M. S. (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., O'Brien, S. J., and Krangel, M. S. (1990). Sequence chromosomal location of the I-309 gene: relationship to genes encoding a family of inflammatory cytokines. J. Immunol. 145, 2737± 2744. Naruse, K., Ueno, M., Satoh, T., Nomiyama, H., Tei, H., Takeda, M., Ledbetter, D. H., van Coillie, E., Opdenakker, G., Gunge, N., Sakaki, Y., Iio, M., and Miura, R. (1996). A YAC contig of the human CC chemokine genes clustered on chromosome 17q11.2. Genomics 34, 236±240. Paolini, J. F., Willard, D., Consler, T., Luther, M., and Krangel, M. S. (1994). The chemokines IL-8, monocyte chemoattractant protein-1, I-309 are monomers at physiologically relevant concentrations. J. Immunol. 153, 2704±2717. Roos, R. S., Loetscher, M., Legler, D. F., Clark-Lewis, I., Baggiolini, M., and Moser, B. (1997). Identification of CCR8, the receptor for the human CC chemokine I-309. J. Biol. Chem. 272, 17251±17254. Selvan, R. S., Butterfield, J. H., and Krangel, M. S. (1994). Expression of multiple chemokine genes by a human mast cell leukemia. J. Biol. Chem. 269, 13893±13898.

Selvan, R. S., Zhou, L.-J., and Krangel, M. S. (1997). Regulation of I-309 gene expression in human monocytes by endogenous interleukin-1. Eur. J. Immunol. 27, 687±694. Tiffany, H. L., Lautens, L. L., Gao, J.-L., Pease, J., Locati, M., Combadiere, C., Modi, W., Bonner, T. I., and Murphy, P. M. (1997). Identification of a human monocyte thymus receptor for the CC chemokine I-309. J. Exp. Med. 186, 165±171. van Snick, J., Houssiau, F., Proost, P., van Damme, J., and Renauld, J.-C. (1996). I-309/T cell activation gene-3 chemokine protects murine T cell lymphomas against dexamethasoneinduced apoptosis. J. Immunol. 157, 2570±2576. Zingoni, A., Soto, H., Hedrick, J. A., Stoppacciaro, A., Storlazzi, C. T., Sinigaglia, F., D'Ambrosio, D., O'Garra, A., Robinson, D., Rocchi, M., Santoni, A., Zlotnik, A., and Napolitano, M. (1998). The chemokine receptor CCR8 is preferentially expressed in TH2 but not in TH1 cells. J. Immunol. 161, 547±551.

LICENSED PRODUCTS New England Nuclear (Boston, MA, USA): 125 Irecombinant human I-309, Bolton-Hunter labeled (catalog no. NEX-364) Peprotech (Rocky Hill, NJ, USA): Human I-309 (catalog no. 300-37) Research Diagnostics (Flanders, NJ, USA): Human I-309 (catalog no. RDI-3037) R&D Systems (Minneapolis, MN, USA): Human I-309 (catalog no. 272-1-010) R&D Systems (Minneapolis, MN, USA): Goat antihuman I-309, IgG fraction (catalog no. AB-272-PB) Sigma (St Louis, MO, USA): Goat anti-human I-309, IgG fraction (catalog no. I7391) Toyobo (Japan): Human I-309 (catalog no. PT 300-37)