International Immunology

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International Immunology, Vol. 13, No. 8, 1021-1029, August 2001
© 2001 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 Sakaguchi^1,3, Ryuichi Fujisawa², Sumio Tanase¹, Hiroshi Nishiura⁴, Kenjiro Matsuno⁵, Hiroshi Takamori³, Youichi Tabira³, Tetsuro Yamamoto⁴, Retsu Miura¹ and Osamu Yoshie²

¹ Departments of Biochemistry,
³ Surgery,
⁴ Molecular Pathology and
⁵ Anatomy, Kumamoto University Medical School, Honjo, Kumamoto 860-0811, Japan
² Department of Microbiology, Kinki University School of Medicine, Osaka-Sayama, Osaka 589-8511, Japan

Correspondence to: H. Nomiyama

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
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.

Keywords: chemokine, chemokine receptor, hepatocyte, HIV-1, plasma

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Chemokines are a family of chemotactic cytokines that play important roles in inflammatory responses and lymphocyte homing ( 1 , 2 ). Based on the arrangement of the N-terminal cysteine residues, chemokines are grouped into four subfamilies, the CXC, CC, C and CX ₃ C subfamilies. One amino acid residue separates the first two conserved cysteine residues in CXC chemokines, while the first two cysteine residues are juxtaposed in CC chemokines. The majority of CXC chemokines primarily attract neutrophils and their genes are clustered at chromosome 4q12–13 in humans ( 1 ). The majority of CC chemokines primarily attract monocytes and their genes are clustered at chromosome 17q11.2 in humans ( 1 , 3 ). Therefore, these chemokines are important mediators of inflammatory responses and could be collectively called inflammatory chemokines. Recently, novel CXC and CC chemokines as well as the members of the C and CX ₃ C chemokine subfamilies have been rapidly identified, mostly through application of bioinformatics on Expressed Sequence Tag (EST) databases ( 2 , 4 ). The majority of these novel chemokines have turned out to be directed at lymphocytes, and their genes are mapped at loci different from the classical chemokine gene clusters on chromosomes 4 and 17 ( 4 ). Because of their essential roles in the development, homeostasis and function of the immune system, these chemokines may be collectively called immune (system) chemokines ( 5 ). Chemokines are also known to signal via a group of seven transmembrane G-protein-coupled receptors ( 1 , 6 ). Notably, most inflammatory chemokines have highly promiscuous ligand–receptor relationships, whereas immune chemokines display a more restricted receptor usage ( 1 , 2 ). Chemokine receptors such as CCR5 and CXCR4 are also known to act as entry co-receptors for HIV-1 and -2 ( 7 ).

Liver-expressed chemokine (LEC), which was originally identified from the GenBank EST database and termed novel CC chemokine (NCC)-4 ( 3 ), is a human CC chemokine expressed highly selectively in the liver ( 8 ). LEC has also been reported as human CC chemokine (HCC)-4 ( 9 ), lymphocyte and monocyte chemoattractant (LMC) ( 10 ) and liver-specific CC chemokine (LCC)-1 ( 11 ). In the recently proposed systematic nomenclature of the chemokine ligands, LEC is listed as CCL16 ( 2 ). Previously, we showed that the human LEC gene is located in the major CC chemokine gene cluster on chromosome 17 ( 3 ). The mouse has, however, only a pseudogene for LEC ( 12 ). Mature LEC protein is 97 amino acids long and shows 19–38% identity to other human CC chemokines with the highest identity to HCC-1/CCL14 ( 8 ). LEC was shown to be inducible in monocytes by IL-10 ( 9 ), and chemotactic for monocytes and lymphocytes ( 9 , 10 ). In addition, this chemokine was shown to have potent myelosuppressive activity comparable to that of macrophage inflammatory protein (MIP)-1/CCL3 ( 10 ) and to induce tumor rejection ( 13 ). However, its biological activity has not been studied in detail yet. Here, we report that LEC is a low- affinity functional ligand for CCR1, CCR2 and CCR5. We also show that the LEC protein is constitutively expressed in liver parenchymal cells and present at high levels in normal human plasma.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Chemokines and antibodies
Recombinant human chemokines [LEC/CCL16, MIP-1, MCP-1/CCL2, eotaxin/CCL11, RANTES/CCL5, I-309/CCL1, thymus-expressed chemokine (TECK)/CCL25, BLC/CXCL13 and stromal cell-derived factor (SDF)-1/CXCR12] and cytokines [IL-1, tumor necrosis factor (TNF)-, IL-4 and IFN-] were purchased from PeproTech EC (London, UK). TARC/CCL17, LARC/CCL20, SLC/CCL21, fractalkine/CX ₃ CL1 and single cysteine motif (SCM)-1/lymphotactin/XCL1 were prepared as described previously ( 14 , 15 ). Lipopolysaccharide (LPS) was purchased from Sigma (St Louis, MO). MCP-2/CCL8 was kindly provided by Dr G. Opdenakker (University of Leuven, Belgium). Rabbit polyclonal anti-human LEC was purchased from PeproTech EC. A murine monoclonal anti-human LEC (clone 70218.11) was purchased from R & D Systems (Minneapolis, MN).

Cells
Mouse L1.2 pre-B cells stably expressing a panel of 12 human chemokine receptors (CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CXCR5, XCR1 and CX ₃ CR1) were generated as described previously ( 14 , 15 ). L1.2 cells expressing CCR2 express one of the two splicing variants, CCR2b, which is a major type of CCR2. A human monocytic cell line THP-1 and a human hepatocarcinoma cell line HepG2 were obtained from ATCC (Manassas, VA).

Calcium mobilization assay
Intracellular calcium mobilization was measured as described previously ( 16 ). In brief, cells were suspended at 1x10 ⁶ cells/ml in HBSS containing 1 mg/ml of BSA and 10 mM HEPES, pH 7.4, and incubated with 3 µM Fura 2-AM (Molecular Probes, Eugene, OR) ( Fig. 1 ) or 4 µM Fluo 3-AM (Dojindo, Kumamoto, Japan) ( Fig. 2 ) fluorescence dye at room temperature for 1 h in the dark. After washing twice, cells were resuspended at 5x10 ⁶ cells/ml. Cells in 100 µl were placed in a quartz cuvette on a F-4500 fluorescence spectrometer (Hitachi, Tokyo, Japan) and treated with chemokines at 10 or 100 nM. Emission fluorescence at 510 (Fura 2-AM) or 530 (Fluo 3-AM) nm was measured upon excitation at 340 and 380 nm with a time resolution of 5 points/s to obtain fluorescence intensity ratio of R _340/380 (Fura 2-AM) or at 480 nm (Fluo 3-AM).

View larger version (43K): [in this window] [in a new window]   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-1 were used at 100 nM, while other chemokines were used at 10 nM. Intracellular calcium mobilization was measured on a fluorescence spectrophotometer. All assays were done 3 times and representative results are shown. (A) Calcium mobilization by LEC. (B) Desensitization experiments.

 

View larger version (18K): [in this window] [in a new window]   Fig. 2. Calcium mobilization in THP-1 cells by LEC. A human monocytic cell line THP-1 was loaded with Fluo 3-AM and intracellular calcium mobilization in response to different chemokines was measured as described in Fig. 1 . LEC was used at 100 nM while other chemokines were used at 10 nM.

 
Migration assay
Chemotaxis assays were carried out using Transwell plates with 5-µm pore polycarbonate membrane (Costar, Acton, MA) as described previously ( 15 ). Cells at 1x10 ⁶ /ml in 100 µl of RPMI 1640 containing 0.5% BSA and 10 mM HEPES, pH 7.4, were added to the upper chambers, and 600 µl of the same medium containing each chemokine at various concentrations was added to the lower chambers. After incubation at 37°C for 4 h in 5% CO ₂ air, cells in the lower chambers were counted using a FACScan (Becton Dickinson, Mountain View, CA). Migrated cells were calculated as a percentage of input cells. All assays were done in triplicate.

Receptor binding studies
Radioligand-binding assays were carried out essentially as described previously ( 15 ). In brief, 5x10 ⁵ cells were incubated for 1 h at 16°C with 100 pM of [ ¹²⁵ I]MIP-1, [ ¹²⁵ I] MCP-1 or [ ¹²⁵ I]I-309 (all purchased from Amersham, Little Chalfont, UK) in the presence of increasing concentrations of unlabeled chemokines (10 ^–10 to 10 ^–6 M) in 200 µl of solution containing 50 mM HEPES, pH 7.5, 5 mM MgCl ₂ , 1 mM CaCl ₂ , 0.5% BSA and 0.05% sodium azide. After incubation, cells were washed 5 times and the radioactivity was measured on a -counter (Aloka, Tokyo, Japan). Assays were performed in triplicate and the data were analyzed with the LIGAND program ( 17 ).

Anti-HIV-1 infection assay
This was carried out as described previously ( 18 ). In brief, peripheral blood mononuclear cells (PBMC) obtained from healthy adult donors were stimulated with phytohemagglutinin 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 LEC at 1000 nM, RANTES at 300 nM or SDF-1 at 300 nM. Infected PBMC were further maintained in the continuous presence of each chemokine at the same concentrations and IL-2 at 20 U/ml. Virus growth was monitored by reverse transcriptase activity in the culture supernatants. All assays were done in triplicate.

Immunohistochemical staining
Human liver tissues were obtained from patients with non-hepatic disorders ( n = 2) under informed consent. Cryosections were fixed in 4% paraformaldehyde for 30 min at room temperature and washed in distilled water. Paraffin sections of formaldehyde-fixed normal human liver obtained from Biochain Institute (San Leandro, CA) were also dewaxed and rehydrated. These sections were incubated with rabbit polyclonal anti-human LEC (PeproTech, Rocky Hill, NJ) for 1 h at room temperature and then with an alkaline phosphatase-labeled donkey anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA) for 1 h at room temperature. The sections were developed with New Fuchsin Substrate kit (red) (Dako, Santa Barbara, CA) and lightly counterstained with hematoxylin.

Quantitation of LEC protein
LEC protein was measured by using a specific ELISA. In brief, 96-well microtest plates (Costar) were coated with anti-LEC/CCL16 mAb at a concentration of 2 µg/ml for 2 h at 37°C. After washing with PBS, the plates were blocked with 1% BSA in PBS overnight. Test samples and recombinant human LEC for a standard curve were appropriately diluted in PBS containing 0.1% BSA and 0.05% Tween 20, and were added to the plates. After incubation for 1 h at 37°C, plates were washed with PBS containing 0.05% Tween 20 (PBS/Tween 20) and were incubated for 30 min at 37°C with rabbit polyclonal anti-LEC at a concentration of 100 ng/ml. After washing with PBS/Tween 20, the plates were incubated for 30 min at 37°C with horseradish peroxidase-labeled donkey anti-rabbit IgG (Amersham). After washing with PBS/Tween 20, the plates were developed by tetramethylbenzidine peroxidase substrate and optical density was measured at 450 nm on a microplate reader (Wallac, Turku, Finland). The typical detection range was 500 pg/ml to 10 ng/ml. Each test sample was assayed at two different dilutions to confirm linearity. All assays were done in duplicate.

RT-PCR
This was carried out as described previously ( 16 ). In brief, total RNA was prepared using Trizol reagent (Life Technologies, Rockville, MD) and RNeasy (Qiagen, Hilden, Germany). Reverse transcription of total RNA (1 µg) was carried out using oligo(dT) ₁₈ primer and Superscript II reverse transcriptase (Life Technologies). First-strand DNA (20 ng total RNA equivalent) and original total RNA (20 ng) were amplified in a final volume of 20 µl containing 10 pmol of each primer and 1 U of Ex-Taq polymerase (Takara, Kyoto, Japan). The primers used were: 5'-CTTCTCGCAGCCAGCC- AAAAGTTCCT and 5'-GGAGTTGAGGAGCTGGGGTTGACCAT for LEC, and 5'-GCCAAGGTCATCCATGACAACTTTGG and 5'-GCCTGCTTCACCACCTTCTTGATGTC for G3PDH. Amplification conditions were denaturation at 94°C for 30 s (5 min for the first cycle), annealing at 60°C for 30 s and extension at 72°C for 30 s (5 min for the last cycle) for 29 cycles for LEC and 27 cycles for G3PDH. Amplification products (10 µl each) were analyzed by electrophoresis on 2% agarose and staining with ethidium bromide.

	Results

Top Abstract Introduction Methods Results Discussion References

 
LEC induces calcium mobilization via CCR1, CCR2 and CCR5
To determine the receptor usage of LEC, we examined calcium mobilization in a panel of mouse L1.2 pre-B cells each stably expressing one of 12 human chemokine receptors. As shown in Fig. 1(A) , LEC, even though at 100 nM, induced substantial calcium flux in cells expressing CCR1, CCR2 and CCR5. LEC also induced marginal but reproducible calcium flux in cells expressing CCR3, CCR4 and CCR8. Thus, LEC may weakly act on many receptors. Cross-desensitization studies shown in Fig. 1(B) revealed that LEC even at 100 nM was unable to completely desensitize CCR1 to MIP-1, CCR2 to MCP-1 and CCR5 to MIP-1, each at 10 nM. On the other hand, these ligands at 10 nM effectively desensitized CCR1, CCR2 or CCR5 to LEC at 100 nM. Thus, LEC is a low potency agonist for CCR1, CCR2 and CCR5.

Since a human monocytic cell line THP-1 is known to express CCR1 ( 19 ), CCR2 ( 20 ), CCR5 ( 21 ), CCR8 ( 22 ), CX ₃ CR1 ( 23 ) and CXCR4 ( 24 ), we examined the responses of THP-1 cells to LEC ( Fig. 2 ). In contrast to the above results, initial stimulation with RANTES at 10 nM (a ligand for CCR1, CCR3 and CCR5) or MCP-1 at 10 nM (a ligand for CCR2) failed to desensitize THP-1 cells to LEC at 100 nM in calcium mobilization assays. However, combined stimulation with RANTES and MCP-1 each at 10 nM completely abolished subsequent responses to LEC at 100 nM. These results support that LEC signals via CCR1, CCR2 and CCR5 on THP-1 cells.

LEC induces cell migration mainly via CCR1 and CCR2
We next examined chemotactic activity of LEC using the same panel of L1.2 cells stably expressing human chemokine receptors. As shown in Fig. 3 , LEC, even though at relatively high concentrations, induced vigorous cell migration via CCR1 and CCR2. LEC, however, induced only marginal migration in cells expressing CCR5 even at 1000 nM. Consistent with induction of weak calcium flux shown in Fig. 1(A) , LEC also induced marginal migration of cells expressing CCR3, CCR4 and CCR8 at high concentrations. Furthermore, LEC also induced migration of THP-1 cells by a typical bimodal dose–response curve with a peak at 100 nM ( Fig. 4 ).

View larger version (19K): [in this window] [in a new window]   Fig. 3. Induction of cell migration by LEC. Chemotactic activity of LEC was examined using a panel of murine pre-B L1.2 cells stably expressing 12 different human chemokine receptors. Chemotaxis assays were carried out using Transwell plates with 3-µm pore polycarbonate membranes (Costar). All assays were done duplicate. Only the results from transfectants giving positive responses to LEC are shown. Each point represents mean ± SEM from three separate experiments.

 

View larger version (18K): [in this window] [in a new window]   Fig. 4. Chemotactic response of THP-1 cells to LEC. Chemotaxis assays were carried out using Transwell plates with 3-µm pore polycarbonate membranes (Costar). Each point represents mean ± SEM from three separate experiments.

 
Low-affinity binding of LEC to CCR1, CCR2 and CCR5
We next examined LEC binding to CCR1, CCR2, CCR5 and CCR8 through its competition with MIP-1 for CCR1 and CCR5, with MCP-1 for CCR2, and with I-309 for CCR8. As shown in Fig. 5 , cold LEC, even though at relatively high concentrations, fully competed with ¹²⁵ I-labeled MIP-1 for CCR1 (IC ₅₀ ~ 77 nM) and for CCR5 (IC ₅₀ ~ 130 nM), and with ¹²⁵ I-labeled MCP-1 for CCR2 (IC ₅₀ ~ 95 nM). LEC was, however, unable to fully compete with I-309 for CCR8 even at 1000 nM (IC ₅₀ > 1000 nM). Thus, LEC is a low-affinity ligand for CCR1, CCR2 and CCR5 but hardly for CCR8.

View larger version (17K): [in this window] [in a new window]   Fig. 5. Binding of LEC to CCR1, CCR2, CCR5 and CCR8. Binding of 125 I-labeled MIP-1 at 100 pM to CCR1 or CCR5, that of 125 I-labeled MCP-1 at 100 pM to CCR2, or that of 125 I-labeled I-309 at 100 pM to CCR8 was determined in the absence or presence of various concentrations of cold LEC, MIP-1, MCP-1 or I-309. All assays were done in triplicate to obtain mean ± SEM.

 
Effect of LEC on R5 and X4 HIV-1 infections
The finding that LEC was a low-affinity ligand for CCR5 prompted us to examine its effect on HIV-1 infection. As shown in Fig. 6 , LEC even at 1000 nM had little inhibitory effect on infection of R5-type SF-162 or X4-type NL432 HIV-1 strains, while RANTES and SDF-1 at 300 nM significantly suppressed R5 and X4 strains, respectively.

View larger version (31K): [in this window] [in a new window]   Fig. 6. Effect of LEC on HIV-1 infection. Peripheral blood mononuclear cells were stimulated with phytohemagglutinin and infected with HIV-1 SF162 strain (R5) or HIV-1 NL432 strain (X4) in the absence or presence of LEC at 1000 nM, RANTES at 300 nM or SDF-1 at 300 nM. Viral growth was monitored by reverse transcriptase activity in the culture supernatants. All assays were done in triplicate to obtain mean ± SEM.

 
Cellular origin of LEC protein in liver
LEC mRNA is constitutively expressed in the normal liver ( 8 , 11 ). To determine the cells producing LEC protein in the liver, we carried out immunohistochemical staining using rabbit polyclonal anti-LEC. In both cryosections ( Fig. 7A ) and paraffin sections ( Fig. 7B ), most hepatocytes and some bile duct epithelial cells were intensely stained by anti-LEC. There was no positive staining without the primary antibody ( Fig. 7C and D ). In cryosections ( Fig. 7A ), a weak staining was also observed in non-parenchymal cells in the sinusoidal lumen, which may be Kupffer cells.

View larger version (96K): [in this window] [in a new window]   Fig. 7. Expression of LEC by liver parenchymal cells. Human liver cryosections (A and C) and paraffin sections (B and D) were stained with anti-LEC (A and B) or with control antibody (C and D). Hepatocytes are intensely stained (A, red). Sinusoidal cells, probably Kupffer cells, are also weakly stained (A, arrows). Hepatocytes and bile duct epithelial cells (arrow) are stained (B, red). P, portal vein. Bar = 40 µm.

 
To further support LEC production by liver parenchymal cells, we examined a human hepatocarcinoma cell line HepG2 for production of LEC by using a specific sandwich-type ELISA. HepG2 cells indeed produced LEC proteins at a level well over ng/ml. Treatment of HepG2 cells with LPS, IL-1, TNF-, IFN- or IL-4, had, however, no significant positive or negative effects on expression of LEC mRNA ( Fig. 8 ) or secretion of LEC protein (data not shown). These results further support that hepatocytes are the major constitutive producers of LEC.

View larger version (25K): [in this window] [in a new window]   Fig. 8. Expression of LEC by HepG2 cells. Total RNA was prepared from HepG2 cells treated without or with LPS (30 ng/ml), IL-1 (1 ng/ml), TNF- (50 ng/ml), IFN- (100 ng/ml) or IL-4 (10 ng/ml) for 24 h. RT-PCR analysis was carried out. Representative results from two separate experiments are shown.

 
LEC is present at high concentrations in plasma
The constitutive production of LEC by liver parenchymal cells prompted us to measure LEC in normal plasma by ELISA. LEC was detected in plasma samples of 12 healthy volunteers with a wide range of 3.5–28 ng/ml (mean 11 ng/ml). Thus, LEC is present in the normal blood at relatively high concentrations. Unexpectedly, we did not detect LEC in serum samples from the same donors (<500 pg/ml). This may be due to adsorption of LEC to fibrin during clotting.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
LEC, also reported as HCC-4 ( 9 ), LMC ( 10 ) and LCC-1 ( 11 ), is a human CC chemokine selectively expressed in the liver and has 19–38% identity to other human CC chemokines ( 8 ). Previously, LEC was shown to induce migration of monocytes and lymphocytes ( 9 , 10 ). Here we examined the receptor usage of LEC, and have found that LEC is a low-affinity functional ligand for CCR1, CCR2 and CCR5 ( Figs 1 , 3 and 5 ). However, LEC has no significant inhibitory activity on the entry of R5 HIV-1 via CCR5 ( Fig. 6 ). During the progression of this work, Howard et al. ( 25 ) also reported the receptor usage of LEC. They found that LEC was capable of inducing human embryonal kidney HEK293 cells stably expressing CCR1 or CCR8 to migrate at high concentrations and to adhere to type I collagen or fibronectin at much lower concentrations. These authors, thus, concluded that LEC was a potent agonist for CCR1 and CCR8 especially in the cell adhesion assays ( 25 ). However, we found that the activity of LEC on CCR8-transfected L1.2 cells was marginal if any ( Fig. 3 ) and the binding affinity of LEC to CCR8 was very low ( Fig. 5 ). At present, we have no solution to these discrepancies, which may be due in part to the use of different types of cells for CCR8 expression or to different types of assays. However, there have been other controversies on the functional ligands of CCR8. Previously, Bernardini et al ., by using CCR8-transfected human Jurkat T cells, claimed that, besides I-309, TARC and MIP-1ß/CCL4 were also functional ligands for CCR8 ( 26 ). Howard et al. also found a high-affinity binding of TARC to CCR8 expressed on HEK239 ( 25 ). On the other hand, Garlisi et al. concluded that TARC and MIP-1ß were not the functional ligands for CCR8 by using CCR8-transfected rat Y3 cells and human T _h 2-polarized T cells ( 27 ). We also repeatedly confirmed that TARC did not act on or bind to CCR8 expressed on mouse L1.2 cells (our unpublished results). Future studies using cells naturally expressing CCR8 may be required to solve these issues.

Recently, we have shown that the CC chemokine cluster on human chromosome 17 consists of two subregions, i.e. the MIP subregion and MCP subregion ( 28 ). The chemokines from the MIP subregion (the MIP group) mainly recognize CCR1 and CCR5. On the other hand, the chemokines from the MCP subregion (the MCP group) mainly use CCR2 and CCR3. LEC maps to the MIP subregion ( 3 ). Thus, it may not be surprising that LEC interacts with CCR1 and CCR5. The use of CCR2 by LEC is, however, unique for a MIP group chemokine because CCR2 is the major receptor for the MCP group chemokines. The exceptional usage of CCR2 by LEC may be in part due to its N-terminal resemblance to those of the MCP group chemokines. The N-terminal sequence of LEC is Gln–Pro ( 8 ). This N-terminal sequence is identical to MCP-1, MCP-2, MCP-3/CCL7 and MCP-4/CCL13, all of which signal through CCR2 ( 29 ). Furthermore, the N-terminal residues of MCP chemokines are pyroglutamate and resistant to Edman degradation ( 30 ). The LEC produced in mammalian cells was also found to resist Edman degradation (data not shown), suggesting the presence of N-terminal pyroglutamic acid residue.

The present study has demonstrated that LEC is constitutively expressed by hepatocytes both in vivo and in vitro ( Figs 7 and 8 ). Therefore, LEC may have a role in homeostatic cell migration in the liver. Furthermore, we found that LEC is present at relatively high concentrations (~1 nM) in plasma from healthy adults. Thus, LEC produced in the liver may be released into the circulation of healthy individuals. Human HCC-1, whose gene exists side by side with the LEC gene ( 28 ), has also been shown to be expressed in various tissues including liver and present in the plasma of healthy individuals at high concentrations (1–10 nM) ( 31 ). HCC-1 is also a low-affinity ligand for CCR1 (IC ₅₀ ~ 93 nM in competition with radiolabeled MIP-1) ( 32 ) as LEC (IC ₅₀ ~ 77 nM in competition with radiolabeled MIP-1) ( Fig. 5 ). The biological reason why these chemokines circulate in the blood at relatively high concentrations is unclear. However, their presence in the plasma may keep CCR1-expressing leukocytes less sensitive to low background levels of chemokines such as MIP-1 and RANTES, which are potent high-affinity agonists for CCR1. Thus, a certain amount of CCR1 receptors on leukocytes in the bloodstream may be occupied by chemokines such as LEC and HCC-1 under normal conditions. Leukocytes could still migrate to inflamed tissues since chemokines such as MIP-1 and RANTES would be produced in large quantities in such cases.

Besides LEC and HCC-1, mouse MIP-1/MRP-2/CCF18 has been shown to circulate in the blood of healthy mice at high concentrations (~90 nM) and constitutively expressed in various organs including liver ( 33 ). The human counterpart of MIP-1 appears to be MPIF-1/CCL23 or leukotactin-1/CCL15 ( 28 ). Their genes are both expressed in the liver, and closely clustered with the HCC-1 and LEC genes ( 28 ). This suggests that MPIF-1 and/or leukotactin-1 may also be present at high concentrations in the plasma. Although the concentrations of LEC and HCC-1 in the plasma vary between individuals, the total concentrations of these constitutive chemokines, all of which bind to CCR1, may be >10 nM in most individuals. Thus, these constitutive chemokines as a total could have significant `quenching' effects on various chemokine receptors. Recently, however, Detheux et al. have demonstrated that a natural proteolytic processing of HCC-1 generates HCC-1 (9–74) which is a potent agonist for CCR1, CCR3 and CCR5 ( 34 ). Thus, these constitutive chemokines in the plasma may also be precursors of more potent chemokines. Such a possibility is especially high for leukotactin-1 and MPIF-1, both of which have unusually long N-terminal regions like HCC-1 ( 35 , 36 ). On the other hand, LEC, having the N-terminal sequence of Gln–Pro, could be a potential target of CD26/dipeptidyl peptidase IV ( 29 ). It thus remains to be seen whether such a processing occurs in LEC to alter its biological functions.

	Acknowledgments

 
We are very grateful to Dr A. Sato at Shionogi Institute for Medical Science for valuable help in HIV-1 infection assays and Dr T. Imai at Kan Institute, Kyoto for valuable suggestion on receptor binding assay respectively. This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

	Abbreviations

 

BLC B lymphocyte chemoattractant

HCC human CC chemokine

LCC liver-specific chemokine

LEC liver-expressed chemokine

LMC lymphocyte and monocyte chemoatrractant

LPS lipopolysaccharide

MCP monocyte chemotactic protein

MIP macrophage inflammatory protein

MPIF myeloid progenitor inhibitory factor

MRP macrophage inflammatory protein-related protein

NCC novel CC chemokine

PBMC peripheral blood mononuclear cell

SCM single cysteine motif

SDF stromal cell-derived factor

TECK thymus-expressed chemokine

TNF tumor necrosis factor

	Notes

 
Transmitting editor: M. Miyasaka

Received 1 March 2001, accepted 7 May 2001.

	References

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