Human CC Chemokine Liver-Expressed Chemokine/CCL16 Is a Functional Ligand for CCR1, CCR2 and CCR5, and Constitutively Expressed by Hepatocytes

International Immunology, Vol. 13, No. 8, pp. 1021–1029 © 2001 The Japanese Society for Immunology Human CC chemokine liver-expressed chemokine/CCL16 is a functional ligand for CCR1, CCR2 and CCR5, and constitutively expressed by hepatocytes Hisayuki Nomiyama1, Kunio Hieshima2, Takashi Nakayama2, Tomonori Sakaguchi1,3, Ryuichi Fujisawa2, Sumio Tanase1, Hiroshi Nishiura4, Kenjiro Matsuno5, Hiroshi Takamori3, Youichi Tabira3, Tetsuro Yamamoto4, Retsu Miura1 and Osamu Yoshie2 Departments of 1Biochemistry, 3Surgery, 4Molecular Pathology and 5Anatomy, Kumamoto University Medical School, Honjo, Kumamoto 860-0811, Japan 2Department of Microbiology, Kinki University School of Medicine, Osaka-Sayama, Osaka 589-8511, Japan Keywords: chemokine, chemokine receptor, hepatocyte, HIV-1, plasma Abstract Liver-expressed chemokine (LEC)/CCL16 is a human CC chemokine selectively expressed in the liver. Here, we investigated its receptor usage by calcium mobilization and chemotactic assays using mouse L1.2 pre-B cell lines stably expressing a panel of 12 human chemokine receptors. At relatively high concentrations, LEC induced calcium mobilization and chemotaxis via CCR1 and CCR2. LEC also induced calcium mobilization, but marginal chemotaxis via CCR5. Consistently, LEC was found to bind to CCR1, CCR2 and CCR5 with relatively low affinities. The binding of LEC to CCR8 was much less significant. In spite of its binding to CCR5, LEC was unable to inhibit infection of an R5-type HIV-1 to activated human peripheral blood mononuclear cells even at high concentrations. In human liver sections, hepatocytes were strongly stained by anti-LEC antibody. HepG2, a human hepatocarcinoma cell line, was found to constitutively express LEC. LEC was also present in the plasma samples from healthy adult donors at relatively high concentrations (0.3–4 nM). Taken together, LEC is a new low-affinity functional ligand for CCR1, CCR2 and CCR5, and is constitutively expressed by liver parenchymal cells. The presence of LEC in normal plasma at relatively high concentrations may modulate inflammatory responses. Introduction Chemokines are a family of chemotactic cytokines that play (1,3). Therefore, these chemokines are important mediators important roles in inflammatory responses and lymphocyte of inflammatory responses and could be collectively called homing (1,2). Based on the arrangement of the N-terminal inflammatory chemokines. Recently, novel CXC and CC cysteine residues, chemokines are grouped into four sub- chemokines as well as the members of the C and CX3C families, the CXC, CC, C and CX3C subfamilies. One amino chemokine subfamilies have been rapidly identified, mostly acid residue separates the first two conserved cysteine through application of bioinformatics on Expressed Sequence residues in CXC chemokines, while the first two cysteine Tag (EST) databases (2,4). The majority of these novel chemo- residues are juxtaposed in CC chemokines. The majority of kines have turned out to be directed at lymphocytes, and CXC chemokines primarily attract neutrophils and their genes their genes are mapped at loci different from the classical are clustered at chromosome 4q12–13 in humans (1). The chemokine gene clusters on chromosomes 4 and 17 (4). majority of CC chemokines primarily attract monocytes and Because of their essential roles in the development, their genes are clustered at chromosome 17q11.2 in humans homeostasis and function of the immune system, these Correspondence to: H. Nomiyama Transmitting editor: M. Miyasaka Received 1 March 2001, accepted 7 May 2001 1022 Human chemokine LEC expressed in liver chemokines may be collectively called immune (system) variants, CCR2b, which is a major type of CCR2. A human chemokines (5). Chemokines are also known to signal via a monocytic cell line THP-1 and a human hepatocarcinoma cell group of seven transmembrane G-protein-coupled receptors line HepG2 were obtained from ATCC (Manassas, VA). (1,6). Notably, most inflammatory chemokines have highly promiscuous ligand–receptor relationships, whereas immune Calcium mobilization assay chemokines display a more restricted receptor usage (1,2). Intracellular calcium mobilization was measured as described Chemokine receptors such as CCR5 and CXCR4 are also previously (16). In brief, cells were suspended at 1ϫ106 cells/ known to act as entry co-receptors for HIV-1 and -2 (7). ml in HBSS containing 1 mg/ml of BSA and 10 mM HEPES, Liver-expressed chemokine (LEC), which was originally pH 7.4, and incubated with 3 µM Fura 2-AM (Molecular identified from the GenBank EST database and termed novel Probes, Eugene, OR) (Fig. 1) or 4 µM Fluo 3-AM (Dojindo, CC chemokine (NCC)-4 (3), is a human CC chemokine Kumamoto, Japan) (Fig. 2) fluorescence dye at room expressed highly selectively in the liver (8). LEC has also temperature for 1 h in the dark. After washing twice, cells been reported as human CC chemokine (HCC)-4 (9), were resuspended at 5ϫ106 cells/ml. Cells in 100 µl were lymphocyte and monocyte chemoattractant (LMC) (10) and placed in a quartz cuvette on a F-4500 fluorescence spectro- liver-specific CC chemokine (LCC)-1 (11). In the recently meter (Hitachi, Tokyo, Japan) and treated with chemokines proposed systematic nomenclature of the chemokine at 10 or 100 nM. Emission fluorescence at 510 (Fura 2-AM) ligands, LEC is listed as CCL16 (2). Previously, we showed or 530 (Fluo 3-AM) nm was measured upon excitation at 340 that the human LEC gene is located in the major CC chemokine and 380 nm with a time resolution of 5 points/s to obtain gene cluster on chromosome 17 (3). The mouse has, however, fluorescence intensity ratio of R340/380 (Fura 2-AM) or at only a pseudogene for LEC (12). Mature LEC protein is 97 480 nm (Fluo 3-AM). amino acids long and shows 19–38% identity to other human CC chemokines with the highest identity to HCC-1/CCL14 (8). Migration assay LEC was shown to be inducible in monocytes by IL-10 (9), Chemotaxis assays were carried out using Transwell plates and chemotactic for monocytes and lymphocytes (9,10). In with 5-µm pore polycarbonate membrane (Costar, Acton, addition, this chemokine was shown to have potent myelo- MA) as described previously (15). Cells at 1ϫ106/ml in 100 µl suppressive activity comparable to that of macrophage of RPMI 1640 containing 0.5% BSA and 10 mM HEPES, inflammatory protein (MIP)-1α/CCL3 (10) and to induce tumor pH 7.4, were added to the upper chambers, and 600 µlof rejection (13). However, its biological activity has not been the same medium containing each chemokine at various studied in detail yet. Here, we report that LEC is a low- concentrations was added to the lower chambers. After affinity functional ligand for CCR1, CCR2 and CCR5. We also incubation at 37°C for 4 h in 5% CO2 air, cells in the lower show that the LEC protein is constitutively expressed in liver chambers were counted using a FACScan (Becton Dickinson, parenchymal cells and present at high levels in normal Mountain View, CA). Migrated cells were calculated as a human plasma. percentage of input cells. All assays were done in triplicate. Receptor binding studies Methods Radioligand-binding assays were carried out essentially as described previously (15). In brief, 5ϫ105 cells were incu- Chemokines and antibodies bated for 1 h at 16°C with 100 pM of [125I]MIP-1α,[125I] Recombinant human chemokines [LEC/CCL16, MIP-1α, MCP-1 or [125I]I-309 (all purchased from Amersham, Little MCP-1/CCL2, eotaxin/CCL11, RANTES/CCL5, I-309/CCL1, Chalfont, UK) in the presence of increasing concentrations of thymus-expressed chemokine (TECK)/CCL25, BLC/CXCL13 unlabeled chemokines (10–10 to 10–6 M) in 200 µl of solution and stromal cell-derived factor (SDF)-1/CXCR12] and cyto- containing 50 mM HEPES, pH 7.5, 5 mM MgCl2, 1 mM CaCl2, kines [IL-1α, tumor necrosis factor (TNF)-α, IL-4 and IFN-γ] 0.5% BSA and 0.05% sodium azide. After incubation, cells were purchased from PeproTech EC (London, UK). TARC/ were washed 5 times and the radioactivity was measured on CCL17, LARC/CCL20, SLC/CCL21, fractalkine/CX3CL1 and a γ-counter (Aloka, Tokyo, Japan). Assays were performed in single cysteine motif (SCM)-1α/lymphotactin/XCL1 were pre- triplicate and the data were analyzed with the LIGAND pared as described previously (14,15). Lipopolysaccharide program (17). (LPS) was purchased from Sigma (St Louis, MO). MCP-2/ CCL8 was kindly provided by Dr G. Opdenakker (University Anti-HIV-1 infection assay of Leuven, Belgium). Rabbit polyclonal anti-human LEC was This was carried out as described previously (18). In brief, purchased from PeproTech EC. A murine monoclonal anti- peripheral blood mononuclear cells (PBMC) obtained from human LEC (clone 70218.11) was purchased from R & D healthy adult donors were stimulated with phytohemagglutinin Systems (Minneapolis, MN). for 2 days and infected with HIV-1 NL432 (an X4 strain) or HIV-1 SF162 (an R5 strain) in the absence or presence of Cells LEC at 1000 nM, RANTES at 300 nM or SDF-1 at 300 nM. Mouse L1.2 pre-B cells stably expressing a panel of 12 Infected PBMC were further maintained in the continuous human chemokine receptors (CCR1, CCR2, CCR3, CCR4, presence of each chemokine at the same concentrations and CCR5, CCR6, CCR7, CCR8, CCR9, CXCR5, XCR1 and IL-2 at 20 U/ml. Virus growth was monitored by reverse CX3CR1) were generated as described previously (14,15). transcriptase activity in the culture supernatants. All assays L1.2 cells expressing CCR2 express one of the two splicing were done in triplicate. Human chemokine LEC expressed in liver 1023 Fig. 1. Calcium mobilization by LEC. A panel of murine pre-B L1.2 cells stably expressing 12 different human chemokine receptors was loaded with Fura 2-AM and stimulated with the indicated chemokines. LEC and SCM-1α were used at 100 nM, while other chemokines were used at 10 nM.

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