Chemokines play important roles in leukocyte trafficking as well as function regulation. In this study, we described the identification and characterization of a novel CXC chemokine from a human dendritic cell (DC) cDNA library, the full-length cDNA of which contains an open reading frame encoding 111 aa with a putative signal peptide of 34 aa. This CXC chemokine shares greatest homology with macrophage inflammatory protein (MIP)-2αβ, hence is designated as MIP-2γ. Mouse MIP-2γ was identified by electrocloning and is highly homologous to human MIP-2γ. Northern blotting revealed that MIP-2γ was constitutively and widely expressed in most normal tissues with the greatest expression in kidney, but undetectable in most tumor cell lines except THP-1 cells. In situ hybridization analysis demonstrated that MIP-2γ was mainly expressed by the epithelium of tubules in the kidney and hepatocytes in the liver. Although no detectable expression was observed in freshly isolated or PMA-treated monocytes, RT-PCR analysis revealed MIP-2γ expression by monocyte-derived DC. Recombinant MIP-2γ from 293 cells is about 9.5 kDa in size and specifically detectable by its polyclonal Ab developed by the immunization with its 6His-tagged fusion protein. The eukaryotically expressed MIP-2γ is a potent chemoattractant for neutrophils, and weaker for DC, but inactive to monocytes, NK cells, and T and B lymphocytes. Receptor binding assays showed that MIP-2γ does not bind to CXCR2. This implies that DC might contribute to the innate immunity through the production of neutrophil-attracting chemokines and extends the knowledge about the regulation of DC migration.

Chemokines are a family of proinflammatory cytokines of low molecular mass (8–11 kDa) characterized by a structurally conserved motif and their ability to mediate leukocyte chemotaxis. Some chemokines are also involved in hematopoieses, angiogenesis, and oncogenesis (1, 2, 3). In addition, several CC chemokines, including RANTES, macrophage inflammatory protein (MIP)4-1α, and MIP-1β, have been found to be capable of inhibiting HIV infection (4). The chemokine family can be divided into four major subfamilies based on the positions of amino-terminal cysteine residues. In the CXC chemokines, the first two cysteines are separated by a nonconserved amino acid, while in the CC chemokine subfamily, these two cysteines are adjacent to each other. The C chemokine subfamily with the only member of lymphotactin lacks the second and fourth cysteines, which are conserved in the CXC and CC chemokines. The CX3C membrane-bound chemokines have 3 aa between the first two cysteines, a long mucin-like stalk, and a short transmembrane domain (5, 6). In general, the CXC chemokines primarily recruit neutrophils, while the CC chemokines primarily attract monocytes and also lymphocytes, basophils, and/or eosinophils with variable selectivity. The C chemokine of lymphotactin seems to act specifically on T lymphocytes and NK cells (7, 8).

Dendritic cells (DC) are the uniquely potent APCs involved in immune responses (9). As adjuvants for Ag delivery, immature DC pick up Ags in the periphery and carry them to the T cell area in lymphoid organs to prime the immune responses, meanwhile undergoing maturation (10). Chemokines play a vital role in DC trafficking, maturation, and function. In this work, we identified and characterized a novel human CXC chemokine from human DC cDNA library, which showed the highest homology to MIP-2αβ (11) and was chemoattractant for neutrophils and DC.

Peripheral mononuclear cells were isolated by Histopaque-1077 (Sigma, St. Louis, MO) density gradient centrifugation of heparinized blood from healthy adult donors and cultured in six-well plates (Nunclon, Naperville, IL) at 37°C for 2 h in RPMI 1640 medium containing 10% (v/v) FBS (Life Technologies, Grand Island, NY), 10 mM glutamine, and penicillin/steptomycin. Nonadherent cells were removed by gentle washing twice with prewarmed HBSS solution, and the resultant CD14+ monocytes accounted for >90% of the remaining adherent cells by FACS analysis. Monocytes were cultured in RPMI 1640 complete medium containing 100 ng/ml recombinant human GM-CSF and 500 U/ml IL-4 (Sigma). Cytokines were replenished on day 3, and cell differentiation was monitored by light microscopy. On day 7, nonadherent cells were harvested as the DC population by gentle aspiration and followed by minimagnetic bead-mediated enrichment. For positive selection, the DC population was labeled with mouse anti-human CD1a mAb (PharMingen, San Diego, CA) for 30 min at 4°C and followed by labeling with minimagnetic bead-conjugated rabbit anti-mouse mAb for 30 min at 15°C. The labeled cell suspension was passed through a separation column placed in a magnetic field (MiniMACS; Milteny1 Biotec, Bergisch, Germany). The resultant positive fractions were over 95% CD1a+CD83+ and used as DC, which were characterized further by phenotype analysis and allogeneic MLR. Total cellular RNA was isolated from the DC using Trizol reagent (Life Technologies), and poly(A)+ RNA was purified with a mRNA isolation kit (Boehringer Mannheim, Mannheim, Germany) for cDNA synthesis or Northern blotting.

cDNA was synthesized and cloned into pSPORT vector at the sites of SalI and NotI using the Superscript plasmid system (Life Technologies) followed by transformation into Escherichia coli DH10B bacteria. Plasmid DNA was prepared from randomly picked individual transformants and was used as a template for large-scale DNA sequencing of the insert 5′ end to create an expressed sequence tag (EST). Sequencing reactions were performed on thermocycler PCR9600 (Perkin-Elmer, Norwalk, CT) by BigDye terminator sequencing Kit (Perkin-Elmer) with SP6 primer. Reaction products were electrophoresed on ABI377 DNA sequencers (Perkin-Elmer), and the raw sequence data were automatically recorded. Approximately 400–600 bp of the 5′ end of plasmid inserts were sequenced and compared with EMBL using BLAST in the Genetics Computer Group program package (Madison, WI). An in-house EST database was generated for human monocyte-derived DC, from which a full-length cDNA clone SBBI25 was identified as the candidate for a human CXC chemokine designated as MIP-2γ. By BLAST analysis against mouse database EST from the National Center for Biotechnology Information, two mouse ESTs (GenBank accession nos. AU035952 and W59562) were found to be highly homologous to human MIP-2γ, from which mouse MIP-2γ full-length cDNA was obtained by contig and confirmed by RT-PCR cloning from mouse kidney. Mouse MIP-2γ full-length cDNA inserted into pGEM-3Zf vector (Promega, Madison, WI) was used as a template (pGEM-mMIP2γ) to synthesize RNA probes for in situ hybridization analysis.

The cDNA containing the full-length encoding regions of human MIP-2α or MIP-2γ were amplified by PCR, confirmed by DNA sequencing, and used as templates for synthesis of probes in Northern blotting. MIP-2α cDNA from nucleotides 41–363 (GenBank accession no. X53799) was amplified from human placenta cDNA (Clontech Laboratories, Palo Alto, CA), and MIP-2γ cDNA for Fc fusion expression was used as a probe template. Ready-to-use blots containing human poly(A)+ RNA from various tissues (2 μg/lane) were purchased from Clontech Laboratories. The filters were hybridized with the 32P-labeled cDNA probes in ExpressHyb hybridization solution (Clontech Laboratories) according to the manufacturer’s instructions. After stringently washing at 50°C for 20 min in 0.1× SSC and 0.1% SDS, the filters were subjected to autoradiography. The filters were reprobed with a human β-actin cDNA probe (Clontech Laboratories).

In addition to human monocytes isolated from peripheral leukocytes and activated by PMA and monocyte-derived DC, the human cell lines used for RT-PCR analysis of MIP-2γ expression included human monocyte THP-1, histiocytic lymphoma U937, acute promyelocytic leukemia cells HL-60, Burkitt’s lymphoma Raji, acute lymphoblastic leukemia Molt-4, acute T cell leukemia Jurkat, cutaneous T lymphoma Hut78, erythroleukemia K562, and lung carcinoma A549. The upstream primer of MIP-2γ is 5′-CTCCCCATGTCCCTGCTC-3′, and its downstream primer is 5′-ACCTGCGCTTCTCGTTCC-3′, with the predicted product of 328 bp. The upstream primer of human β-actin is 5′-GCATCGTGATGGACTCCG-3′, and its downstream primer is 5′-TCGGAAGGTGGACAGCGA-3′, with the expected product of 600 bp.

For expression of 6His-tagged MIP-2γ in E. coli, a BamHI restriction site was introduced by PCR just before the predicted first codon of mature MIP-2γ and also a 6His tag and BamHI restriction site introduced immediately before the termination codon. The 50-μl PCR mixture included a 200-ng template of plasmid SBBI25, 1.5 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl (pH 8.4), 200 μm dNTP, and 0.5 U Taq (Promega). The reactions were incubated in a thermocycler PCR9600 (Perkin-Elmer) for 10 min at 98°C, followed by 25 cycles of denaturation for 15 s at 94°C, annealing for 30 s at 56°C, and extension for 30 s at 72°C. The PCR products were in-frame ligated into pQE60 expression vector (Qiagen, Chatsworth, CA), and MIP-2γ cDNA were confirmed by sequencing. Expression of His-tagged MIP-2γ was induced by adding 1 mM isopropylthiogalactoside to mid-log cultures (A600 = 0.7–0.8). After 4 h of isopropylthiogalactoside induction at 37 °C, the cells were harvested and lysed in buffer B (8 M urea, 0.1 M NaH2PO4, 10 mM Tris-HCl, pH 8.0), and subjected to nitrilotriacetic acid-Ni2+ agarose (Qiagen) for purification under denaturing condition according to the manufacturer’s instructions. The 6His-tagged MIP-2γ eluted by buffer E (8 M urea, 0.1 M NaH2PO4, 10 mM Tris-HCl, pH 4.5) was subsequently purified by HPLC chromatography, which resulted in a purity of >90%. Normal rabbits were immunized three times with 6His-tagged MIP-2γ including two boostings. Two weeks after the last boosting, the antisera were collected and subjected to affinity chromatography using a protein G Hitrap column (Pharmacia Biotech, Piscataway, NJ).

For expression of IgG fusion protein to determine the N terminal of mature MIP-2γ, MIP-2γ cDNA containing the full-length encoding region except stop codon was amplified by PCR using the sense primer of 5′-GGAATTCGCCATGTCCCTGCTCCCACG and the antisense primer of 5′GGGATCCGGTTCTTCGTAGAACCTG. As underlined, an EcoRI restriction site was added before the start codon of MIP-2γ, and a BamHI site was introduced before the stop codon for in-frame ligation with human IgG1 CH2 and CH3 fragment. The MIP2γ-IgG fusion gene was inserted into pcDNA3.1 expression vector at the sites of EcoRI and KpnI, under the control of CMV promoter/enhancer. Seventy-two hours after transfection of the MIP2γ-IgG expression vector into 293 cells with lipofectamine (Life Technologies), the 48-h culture supernatants were harvested and subjected to protein A affinity chromatography. MIP2γ-IgG fusion protein was blotted onto a polyvinylidene difluoride membrane for amino acid sequencing of the MIP-2γ N terminus.

For activity investigation, MIP-2γ cDNA containing the full-length encoding region was amplified by PCR using the primers of 5′-GGAATTCGCCATGTCCCTGCTCC CACG and 5′-GGGTACCTCATTCTTCGTAGAACCTG, with the resultant protein product designated as MIP-2γ. EcoRI and KpnI restriction sites were added before the start codon and immediately after the stop codon of MIP-2γ respectively as underlined. MIP-2γ cDNA was inserted into pcDNA3.1 expression vector, under the control of CMV promoter, followed by transfection into 293 cells for transient expression with lipofectamine (Life Technologies) according to manufacturer’s instructions. Twenty-four hours after transfection, metabolic labeling was performed to monitor the expression and secretion of MIP-2γ. MIP-2γ-transfected or mock-transfected 293 cells in six-well plates were cultured in methioine and cysteine-free DMEM medium (Life Technologies) for 30 min at 37°C and then replaced with the same fresh medium (0.5 ml/well) containing 200 μCi/ml Redivue [35S]methionine and [35S]cysteine (Amersham, Arlington Heights, IL) and 5% dialyzed serum (Life Technologies). After overnight labeling at 37°C, the culture supernatants were harvested and condensed ∼5-fold with Centricon 5K (Millipore, Bedford, MA) before fractionation by 16% SDS-PAGE and subsequent autoradiography. MIP-2γ expression was further confirmed by Western blotting with its rabbit polyclonal Abs.

Forty-eight-hour culture supernatants from MIP-2γ-transfected or mock-transfected 293 cells were condensed with Centricon 5K (Millipore), fractionated by 16% SDS-PAGE, and electrically blotted onto a nitrocellulose membrane (Amersham). The membrane was blocked with 5% nonfat dry milk in TBST buffer (25 mM Tris-HCl pH 8.0, 125 mM NaCl, and 0.1% Tween 20) for 1 h at room temperature before incubation with rabbit polyclonal Abs against MIP-2γ or normal rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at room temperature. After washing three times in TBST, the membrane was incubated for 45 min with HRP-labeled goat anti-rabbit Ab and washed as before. HRP was detected using enhanced chemiluminescence according to the manufacturer’s instructions (Santa Cruz Biotechnology).

Polymorphonuclear neutrophils and mononuclear cells were separated heparinized peripheral blood by double gradient centrifugation (30 min, 700 × g) on Histopaque-1119 and Histopaque-1077 (Sigma) according to the manufacturer’s instructions. Granulocytes are found at the 1077/1119 interphase, whereas mononuclear cells are found at the plasma/1077 interphase. The total mononuclear cell fraction was used as a source for monocytes and lymphocytes. Monocytes and T lymphocytes were further isolated by magnetic cell sorting (MiniMACS) using positive selection with anti-CD14 or anti-CD3 mAbs, respectively. After positive magnetic cell sorting, monocytes were >85% pure, whereas the purity of I lymphocytes reached >90%. The resultant granulocytes, monocytes, T lymphocytes, and monocyte-derived DC were washed and resuspended in pyrogen-free HBSS containing human plasma protein (1 mg/ml albumin). The monocytic THP-1 cells, grown in RPMI 1640 with 10% FCS (Life Technologies), were used for chemotaxis assay as an alternative to fresh monocytes. The microchemotaxis assay was conducted in duplicate using the Boyden chamber migration assay. Then, 200-μl culture supernatants of appropriate dilutions were added into the lower chambers of the assay assembly (NeuroProbe, Cabin John, MD), and the upper chambers were filled with 200 μl of the appropriate cell suspension (2×106cells/ml). The wells were separated by a 5-μm (neutrophils, lymphocytes, monocytes) or 8-μm (DC, THP-1) pore-size polycarbonate filters (Poretics, Livermore, CA) with a diameter of 13 mm. The chambers were incubated for 1 h (neutrophils), 2 h (monocytes, DC), or 4 h (lymphocytes) at 37°C. After incubation, the filters were removed, fixed, and stained. Data were obtained by counting five nonoverlapping high-power microscope fields from each well. Cells were considered to be chemoattracted if the chemotactic index (number of cells migrating in experimental well per number of cells migrating in media only) was >2.

To express their AP fusion proteins, human IL-8 and MIP-2γ cDNA fragments encoding their full mature proteins were amplified by PCR. Restriction sites at the ends of the amplification primers were cut with BamHI and in-frame inserted into the expression vector pAPtag-4 (GenHunter Corporation, Nashville, TN) at the BglII site, so that both chemokines were fused at the N terminal through a 4-aa linker (Gly-Ser-Gly-Gly) to secreted human placental AP. By transfection of the expression vectors into 293T cells with lipofectamine (Life Technologies) according to manufacturer’s instructions, the AP-IL-8 and AP-MIP-2γ fusion proteins detected up to 500 mU/ml of AP activity (1 unit of enzyme hydrolyzes 1 μmol/min of p-nitropheny1 phosphate at 37°C) in their 72-h culture supernatants of transient expression. The unfused AP with the activity of 560 mU/ml was also produced as mock control by transfection of plasmid pAPtag-4 into 293T cells. The AP-tagged fusion proteins were stable for several months when stored in tissue culture supernatant at 4°C.

A receptor binding assay was performed using AP-tagged ligand proteins according to the manufacturer’s instructions (GenHunter Corporation). Briefly, 106 cells were washed with HBHA buffer (HBSS with 0.5 mg/ml BSA, 0.1% NaN3, 20 mM HEPES, pH 7.0), and incubated with 2 ml of culture medium containing AP fusion proteins. After incubation at room temperature for 90 min, the cells were washed five times with HBHA over a 10-min period, lysed in 500 μl of 1% Triton X-100, 10 mM Tris-HCl (pH 8.0), and vortexed vigorously for 10 s. The nuclei were spun down in a microfuge tube for 2 min, and the supernatants were incubated at 65°C for 10 min to inactivate endogenous AP before AP assay using GenHunter AP assay reagent A as instructed. After incubation of samples in the presence of AP assay reagent A for 20 min at 37°C, the AP activity was determined by OD405 nm in a spectrophotometer. A 293 cell clone stably expressing CXCR2 was established to evaluate the CXCR2 binding capacity to MIP-2γ. The cDNA-encoding full-length CXCR2 protein was amplified from THP-1 cells by RT-PCR, using the primers of 5′-GGAATTCCGCCATGTCAAATATTACAGATCCAC-3′ and 5′-GGGGTACCTCGAGTCAGAGG TTGGAAGAGACATT-3′. After double digestion with EcoRI and KpnI, the PCR products were cloned into pcDNA3.1 vector (Invitrogen, San Diego, CA) and confirmed by DNA sequencing. The resultant CXCR2 expression vector was transfected into 293 cells by lipofectamine (Life Technologies). After 2 wk screening with 800 μg/ml of G418, a positive clone designated 293CXCR2 was obtained, the CXCR2 expression of which was confirmed by FACS analysis using PE-conjugated anti-human CXCR2 mAb (PharMingen). The mock-transfected 293 cell clone was also established by transfection of pcDNA3.1 plasmid into 293 cells.

Sense and antisense digoxigenin-labeled cRNA probes of mouse MIP-2γ were synthesized with a digoxigenin-RNA labeling kit (Roche Diagnostics, Hong Kong) using linearilzed pGEM-mMIP2γ as the template. In situ hybridization was performed according to the method modified from Hoefler et al. (12). The livers and kidneys from 6-wk-old male BALB/c mice were rapidly frozen in −70°C isopentane for 2 min, cut into 10-μm sections in a cryostat, thaw-mounted on poly-l-lycine-coated slides, and air-dried. The sections were fixed in 4% formaldehyde and 0.03% picric acid in 0.1 M phosphate buffer (pH 7.4) for 10 min. After three rinses of PBS and one rinse of 0.1 M glycine/PBS and 0.4% Triton X-100/PBS, the sections were digested with 1 μg/ml of protease K in PBS at 37°C for 30 min, fixed in 4% paraformaldehyde for 5 min, and followed by two rinses of PBS to remove the fixative. The sections were then incubated in 0.25% acetic anhydride with 0.1 M triethanolamine (pH 8.0) for 10 min at room temperature, followed by two rinses of 0.6 M sodium chloride, 0.06 M SSC for 10 min. Digoxigenin-labeled cRNA (0.1–0.5 μg/ml) of either antisense or sense probes was added to the hybridization solution containing 50% formamide, 10% dextran sulfate, 0.05 M Tris-HCl (pH 8.0), 1 mM EDTA, 0.3 M NaCl, 1× Denhardt’s solution, and 250 μg/ml E. coli transfer RNA (RNase-free). After overnight hybridization at 64°C in a hybridization oven, the sections were rinsed with 4× SSC for 20 min at 37°C, treated with 20 μg/ml RNase in 2× SSC, and followed by rinses with 1× SSC and 0.2× SSC at 37°C for 20 min, respectively. After incubation in PBS blocking buffer containing 5% BSA and 0.4% Triton X-100 at room temperature for 30 min, the sections were incubated with AP-conjugated anti-digoxigenin Ab (Roche Diagnostics) in the blocking buffer for 3 h at room temperature. The sections were rinsed four times with PBS before color development with 400 μg/ml nitroblue tetrazolium, 200 μg/ml 5-bromo-4-chloro-3-indolyl phosphate and 100 μg/ml levamisole in 0.1 M Tris-HCl buffer (pH 9.5) at room temperature. The sections were rinsed for 10 min in 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA to stop color development, then mounted with 50% glycerol in the Tris-HCl/EDTA buffer and stored at 4°C in the dark.

By randomly large-scale sequencing of human DC cDNA library, we identified a full-length cDNA clone encoding a novel CXC chemokine designated as MIP-2γ (Fig. 1). It contains an open reading frame of 111 aa with a conserved four-cysteine motif. The first two cysteine residues of the amino terminus are separated by a nonconserved lysine, which is characteristic of CXC chemokines (Fig. 2). The protein product shares 30% identity and 50% similarity with MIP-2αβ (11), so it was designated as MIP-2γ. In contrast to MIP-2αβ, MIP-2γ does not contain an ELR motif, which is also found in IL-8 and other CXC chemokines. MIP-2γ contains a putative signal peptide of 34 aa based on peptide hydrophilicity analysis, so the mature protein consists of 77 aa with a predicted relative molecular mass of 9.5 kDa. To confirm the predicted NH2 terminus of the mature peptide, MIP-2γ cDNA was in-frame fused with human IgG1 CH2 and CH3 and inserted into pcDNA3.1 vector to express MIP2γ-IgG fusion protein in 293 cells. Amino-terminal sequencing of protein A-purified MIP2γ-IgG yields the sequence SKCKCSRKGP, confirming the predicted NH2 terminus of mature MIP-2γ. Mouse MIP-2γ shared 95% and 98% identity with human MIP-2γ at nucleotide and protein levels, respectively (Fig. 2, GenBank accession no. AF252873), suggesting that MIP-2γ was highly conserved.

FIGURE 1.

Sequence and deduced translation of human MIP-2γ cDNA. The predicted signal sequence is underlined. The mature NH2 terminus was confirmed by amino acid sequence of recombinant MIP-2γ produced by 293 cells. The four conserved cysterine residues are indicated by asterisks underneath them. The cDNA sequence of human MIP-2γ has been deposited in GenBank under the accession no. AF106911.

FIGURE 1.

Sequence and deduced translation of human MIP-2γ cDNA. The predicted signal sequence is underlined. The mature NH2 terminus was confirmed by amino acid sequence of recombinant MIP-2γ produced by 293 cells. The four conserved cysterine residues are indicated by asterisks underneath them. The cDNA sequence of human MIP-2γ has been deposited in GenBank under the accession no. AF106911.

Close modal
FIGURE 2.

Alignment of amino acid sequences between MIP-2γ and other CXC chemokines. The cDNA sequence of mouse MIP-2γ has been deposited in GenBank under the accession no. AF252873.

FIGURE 2.

Alignment of amino acid sequences between MIP-2γ and other CXC chemokines. The cDNA sequence of mouse MIP-2γ has been deposited in GenBank under the accession no. AF252873.

Close modal

Northern blotting revealed constitutive expression of MIP-2γ in most normal tissues. The greatest expression of MIP-2γ was observed in the kidney, with weaker expression in small intestine, brain, placenta, skeletal muscle, liver, spleen, thymus, and pancreas (Fig. 3). Very faint expression of MIP-2γ was detected in testis, ovary, heart, and lung, and no expression was seen in PBL. Two different transcripts of human MIP-2γ were detected in normal tissues. The dominant one is ∼2 kb, and another is about 0.5 kb. Constitutive expression of MIP-2α was also detectable in most normal tissues, with the greatest expression in liver and abundant expression in lung, brain, heart, and spleen, but no expression in kidney and PBL was observed (Fig. 3). MIP-2α also has two transcripts with the sizes of 2.5 kb and 1.5kb, respectively, and the larger one seemed to be dominant. These suggested that the expression pattern of MIP-2γ in normal tissues was similar to that of its homologue MIP-2α to some extent.

FIGURE 3.

Northern blotting analysis for human MIP-2γ and MIP-2α in human tissues. Multiple tissue Northern blots were probed with 32P-labeled human MIP-2γ or MIP-2α cDNA and washed with high stringency (0.2× SSC, 50°C). The RNA markers are indicated in kilobases.

FIGURE 3.

Northern blotting analysis for human MIP-2γ and MIP-2α in human tissues. Multiple tissue Northern blots were probed with 32P-labeled human MIP-2γ or MIP-2α cDNA and washed with high stringency (0.2× SSC, 50°C). The RNA markers are indicated in kilobases.

Close modal

In contrast to normal tissues, MIP-2γ expression was undetectable in cancer cell lines by Northern blotting, including HL-60, HeLa, K562, Molt-4, Raji, colorectal adenocarcinoma SW480 cells, lung carcinoma A549 cells, and melanoma G361 cells (data not shown). RT-PCR analysis also demonstrated no expression of MIP-2γ in cancer cell lines detected, including U937, HL-60, K562, Molt-4, Jurkat, Hut78, Raji, and A549, with the exception of THP-1 cell line (Fig. 4,A). Meanwhile, MIP-2α was detectable by Northern blotting in A549, melanoma G361 cells, and HeLa cells (Fig. 3). Although MIP-2γ mRNA expression was not observed in freshly isolated or PMA-treated peripheral monocytes, monocyte-derived DC cultured with GM-CSF/IL-4 did express detectable MIP-2γ by RT-PCR (Fig. 4,B). In situ hybridization demonstrated that MIP-2γ was mainly expressed by parenchyma cells in kidney and liver, including epithelium of uriniferous tubule and liver cells (Fig. 5). This implied that MIP-2γ might possess growth regulatory functions under physiological conditions.

FIGURE 4.

RT-PCR analysis for human MIP-2γ expression. Human tumor cell lines (A) or freshly isolated human peripheral monocytes (Mono) or PMA-treated peripheral monocytes (Mono/PMA) or human monocyte-derived dendritic cells (Mono-DC) (B) were subjected to RT-PCR analysis for human MIP-2γ. Human β-actin was also amplified by RT-PCR as a positive control.

FIGURE 4.

RT-PCR analysis for human MIP-2γ expression. Human tumor cell lines (A) or freshly isolated human peripheral monocytes (Mono) or PMA-treated peripheral monocytes (Mono/PMA) or human monocyte-derived dendritic cells (Mono-DC) (B) were subjected to RT-PCR analysis for human MIP-2γ. Human β-actin was also amplified by RT-PCR as a positive control.

Close modal
FIGURE 5.

In situ hybridization analysis for mouse MIP-2γ in normal kidney and liver. The sections from mouse kidney and liver were subjected to hybridization overnight at 64°C with digoxigenin-labeled sense or antisense cRNA probes, incubated with AP-conjugated anti-digoxigenin, and then developed by AP staining with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate. A, Kidney section probed with mouse MIP-2γ sense cRNA probe (magnification, ×100). B, Kidney section probed with mouse MIP-2γ antisense cRNA, and the positive uriniferous tubules were marked with → (magnification, ×200). C, Liver section probed with mouse MIP-2γ sense cRNA (magnification, ×100). D, Liver section probed with mouse MIP-2γ antisense cRNA. The central vein was marked with ➱, the sinus hepaticus was marked with ➡, and positive liver cells were marked with → (magnification, ×200).

FIGURE 5.

In situ hybridization analysis for mouse MIP-2γ in normal kidney and liver. The sections from mouse kidney and liver were subjected to hybridization overnight at 64°C with digoxigenin-labeled sense or antisense cRNA probes, incubated with AP-conjugated anti-digoxigenin, and then developed by AP staining with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate. A, Kidney section probed with mouse MIP-2γ sense cRNA probe (magnification, ×100). B, Kidney section probed with mouse MIP-2γ antisense cRNA, and the positive uriniferous tubules were marked with → (magnification, ×200). C, Liver section probed with mouse MIP-2γ sense cRNA (magnification, ×100). D, Liver section probed with mouse MIP-2γ antisense cRNA. The central vein was marked with ➱, the sinus hepaticus was marked with ➡, and positive liver cells were marked with → (magnification, ×200).

Close modal

To detect MIP-2γ protein expression, we developed rabbit polyclonal Abs against human MIP-2γ by immunizing with 6His-tagged MIP-2γ, which was expressed in bacteria and purified by nickel-nitrilotriacetic acid-mediated affinity chromatography. For bioactivity analysis, human MIP-2γ cDNA with a full-length encoding region was inserted into pcDNA3.1 expression vector and expressed transiently in human embryonic kidney 293 cell line. By 35S metabolic labeling and autoradiography, the protein product of ∼9.5 kDa could be detected in the supernatants from MIP-2γ-transfected 293 cells (Fig. 6,A), which was confirmed to be human MIP-2γ by Western blotting with rabbit polyclonal Abs against human MIP-2γ (Fig. 6 B), whereas untransfected or mock-transfected 293 cells didn’t express any detectable human MIP-2γ by Western blotting.

FIGURE 6.

Recombinant expression and Western blotting analysis of human MIP-2γ. A, Metabolic labeling of recombinant human MIP-2γ. MIP-2γ gene-transfected or mock-control 293 cells were labeled with [35S]methionine and [35S]cysteine overnight, and the culture supernatants were condensed ∼5-fold with Centricon 5K before fractionated by 16% Tris-glycine SDS-polyacrylamide electrophoresis. B, Western blotting analysis for the culture supernatants using rabbit anti-MIP-2γ polyclonal Abs. The supernatants from human MIP-2γ gene-transfected (lanes 2 and 3) or mock-control 293 cells (lane 1) were fractionated on 16% Tris-glycine SDS-PAGE gel, electrically blotted onto a nitrocellulose membrane, and followed by detection with anti-MIP-2γ polyclonal Abs (lanes 1 and 2) or control rabbit IgG. The protein markers are indicated in kilodaltons.

FIGURE 6.

Recombinant expression and Western blotting analysis of human MIP-2γ. A, Metabolic labeling of recombinant human MIP-2γ. MIP-2γ gene-transfected or mock-control 293 cells were labeled with [35S]methionine and [35S]cysteine overnight, and the culture supernatants were condensed ∼5-fold with Centricon 5K before fractionated by 16% Tris-glycine SDS-polyacrylamide electrophoresis. B, Western blotting analysis for the culture supernatants using rabbit anti-MIP-2γ polyclonal Abs. The supernatants from human MIP-2γ gene-transfected (lanes 2 and 3) or mock-control 293 cells (lane 1) were fractionated on 16% Tris-glycine SDS-PAGE gel, electrically blotted onto a nitrocellulose membrane, and followed by detection with anti-MIP-2γ polyclonal Abs (lanes 1 and 2) or control rabbit IgG. The protein markers are indicated in kilodaltons.

Close modal

For microchemotactic assay, the 24-h serum-free culture supernatants from MIP-2γ-transfected or mock-transfected 293 cells were harvested 48 h after transfection. The culture supernatants from MIP-2γ-transfected 293 cells could attract neutrophils markedly even at the 100-fold dilution, but not T lymphocytes or monocytes (Fig. 7), which is consistent with most of CXC chemokines. To a lesser extent, it was also chemoattractive to human monocyte-derived DC, but inactive on B lymphocytes or NK cells, whereas the mock control supernatants had no obvious chemoattractant activity on neutrophils, T lymphocytes, monocytes, or DC.

FIGURE 7.

Chemotactic activity of human MIP-2γ. The culture supernatants from MIP-2γ gene-transfected (□) or mock-control 293 cells (○) direct the migration of freshly isolated neutrophils (A), T lymphocytes (B), monocytes (C) from human peripheral blood, or monocyte-derived DC (D). Migration is presented with chemotactic index. The data are representative of three individual experiments.

FIGURE 7.

Chemotactic activity of human MIP-2γ. The culture supernatants from MIP-2γ gene-transfected (□) or mock-control 293 cells (○) direct the migration of freshly isolated neutrophils (A), T lymphocytes (B), monocytes (C) from human peripheral blood, or monocyte-derived DC (D). Migration is presented with chemotactic index. The data are representative of three individual experiments.

Close modal

Putatively, ELR+ CXC chemokine mediates the chemotaxis of neutrophils via CXCR2. Although MIP-2γ was absent of the ELR motif, it did attract neutrophils. To evaluate whether CXCR2 mediates the chemotactic capacity of MIP-2γ, we carry out CXCR2 binding assay. CXCR2-transfected 293 cells (293CXCR2) were established, CXCR2 expression of which was confirmed by FACS analysis (data not shown). AP-tagged MIP-2γ fusion protein could bind efficiently to neutrophils, but bind poorly to 293CXCR2 cells (Fig. 8). As a positive control, IL-8 AP fusion protein could bind efficiently to 293CXCR2 cells. These showed that MIP-2γ didn’t bind to receptor CXCR2 and suggested that other chemokine receptors might mediate the biological functions of MIP-2γ.

FIGURE 8.

CXCR2 binding assay of MIP-2γ. Neutrophils or CXCR2-transfected 293 cells were incubated with culture medium containing AP-tagged IL-8 or MIP-2γ fusion proteins for 90 min at 37°C, washed with HBHA, and lysed with 1% Triton X-100/10 mM Tris-HCl. After the nuclei were spun down, the supernatants were incubated for 10 min at 65°C to inactivate endogenous AP and were subjected to AP assay by incubation with AP assay reagent A for 20 min at 37°C. The AP activities were determined by OD405 nm read in a spectrophotometer and represented receptor binding capacity. ∗, p < 0.01 in comparison with AP control. The data are representative of three individual experiments.

FIGURE 8.

CXCR2 binding assay of MIP-2γ. Neutrophils or CXCR2-transfected 293 cells were incubated with culture medium containing AP-tagged IL-8 or MIP-2γ fusion proteins for 90 min at 37°C, washed with HBHA, and lysed with 1% Triton X-100/10 mM Tris-HCl. After the nuclei were spun down, the supernatants were incubated for 10 min at 65°C to inactivate endogenous AP and were subjected to AP assay by incubation with AP assay reagent A for 20 min at 37°C. The AP activities were determined by OD405 nm read in a spectrophotometer and represented receptor binding capacity. ∗, p < 0.01 in comparison with AP control. The data are representative of three individual experiments.

Close modal

A new chemokine cDNA was isolated from a human DC cDNA library, which encodes 111 aa with a putative signal peptide of 34 aa. It contains a CXC motif characteristic of CXC chemokine, shares high homology with the previously identified MIP-2αβ (11), and can attract neutrophils markedly, so it’s designated as MIP-2γ. In contrast to MIP-2αβ, MIP-2γ doesn’t contain the ELR motif, which is found in the CXC chemokines attracting neutrophils via CXCR1 or CXCR2. We have no evidence that MIP-2γ could bind to CXCR1 (data not shown) or CXCR2, which implies that a novel CXC chemokine receptor other than CXCR1 or CXCR2 may mediate neutrophil trafficking by MIP-2γ. Interestingly, MIP-2γ is also a chemoattractant of monocyte-derived DC. The identification of a MIP-2γ receptor may facilitate understanding the chemotactic features of MIP-2γ.

Although it was isolated from human monocyte-derived DC, no detectable expression of human MIP-2γ was observed in PBL by Northern blotting, or in freshly isolated or PMA-treated monocytes by RT-PCR, suggesting MIP-2γ expression is tightly regulated under physiological conditions. DC are regarded as the most powerful APCs in vivo, and chemokines expressed by DC may partially account for the potential roles of DC in immune responses. Up to now, several CC chemokines including MIP-1α, MIP-1β, MIP-1γ, RANTES, monocyte chemoattractant protein (MCP)-1, and DC-CK1 have been found to be expressed in DC (13, 14, 15, 16), and MCP-3 was also found in our EST database from the DC cDNA library (data not shown). Recently, it was reported that monocyte-derived chemokine (MDC) expression was up-regulated on DC maturation (17). CC chemokines expressed by DC may facilitate DC actively attracting T cells and subsequently priming T cell-mediated immunity. This notion is supported by our previous studies that augmenting DC’s preferential chemotaxis on T cells could enhance the induction of T cell immune responses (18, 19). Besides CC chemokines, DC has been shown to express CXC chemokine, e.g., IL-8, which is the potent chemoattractant for neutrophils (13). To our knowledge, MIP-2γ is the second CXC chemokine reported to be expressed by DC, which supports the hypothesis that DC could contribute to innate immunity through the production of inflammatory cytokines.

The in vivo trafficking of DC is highly regulated by chemokines under resting or stimulated conditions. DC has been found to express appreciable levels of the CCR1, CCR2, CCR3, CCR5, and CCR7 receptors for the CC chemokines and CXCR1, CXCR2, and CXCR4 for CXC chemokines (20, 21), which are vital for DC trafficking, in vivo localization, and Ag presentation (22, 23, 24). Chemokine receptor expression was observed to be strictly regulated on DC differentiation and maturation. The CC chemokine receptors CCR3 and CCR5 were found to be down-regulated, while CCR7 and CXC chemokine receptor CXCR4 were enhanced on DC maturation (25, 26, 27). So, it was postulated that different chemokines and chemokine receptors may be involved in DC migration in vivo, depending on the functional and maturation status of DC (27). It seems likely that MIP-1α, MCP-3, and RANTES can direct the migration of immature DC located in the periphery, whereas MIP-3β can mediate the trafficking of Ag-carrying DC from peripheral inflammatory sites, where DC are stimulated to up-regulate the expression of CCR7, to lymphoid organs (20, 21, 27, 28). The CXC chemokines seem to be less important in regulating DC trafficking. Although DC express CXC chemokine receptors, most CXC chemokines, including IL-8, IFN-γ-inducible protein-10, and growth-related oncogene-β, are inactive on DC (29), and stromal cell-derived factor-1 was the only CXC chemokine found to be chemoattractive on DC (26). Our finding that another CXC chemokine MIP-2γ other than stromal cell-derived factor-1 can mediate DC migration will extend the knowledge about the regulation of DC trafficking. The receptor mediating MIP-2γ binding and function needs to be identified and characterized for better understanding the functional role of MIP-2γ in DC migration and maturation. Recently, the concept of DC subpopulation has been postulated on the basis of the cytokine profile (30); it will be interesting to characterize the chemokine profile and the chemokine responsiveness of different DC subpopulations.

Our data showed that MIP-2γ mRNA was widely and constitutively expressed in normal tissues, including kidney, small intestine, brain, placenta, skeletal muscle, liver, spleen, thymus, and pancreas, with the highest expression observed in kidney. In situ hybridization analysis demonstrated that the parenchymal cells in kidney and liver were the predominant cells to express MIP-2γ. MIP-2γ protein expression in normal kidney was also detectable (data not shown). The phenomenon is interesting in that no neutrophil infiltration occurs in kidney and liver under normal condition despite abundant MIP-2γ expression in the loci. Moreover, the constitutive expression in normal tissues was also observed for chemokine other than MIP-2γ, e.g., MCP-2 and MIP-1γ (31, 32). Therefore, we predicted that there might exist certain mechanisms under normal physiological condition to suppress or reverse the chemokine-mediated accumulation of inflammatory cells and the potential for self-perpetuation of inflammation. Our hypothesis is that migratory responses of target cells to a certain chemokine may be dependent of the physiological status of themselves as well as the chemokine concentration gradient in the loci. This is supported by the recent report that high concentrations of stromal cell-derived factor-1 could drive T cells to move away from it and inhibit T cell accumulation in inflammatory loci (33). Whether MIP-2γ could bidirectionally regulate the migration of neutrophils is under investigation in our laboratory. In contrast the abundant expression of chemokines in normal tissues also indicates that they may possess other important biological activities in vivo than chemotaxis. It is evident that some chemokines could regulate cell proliferation and differentiation, participate in hemopoiesis, immunoregulation, angiogenesis, and oncogenesis (1, 2, 34). MIP-2αβ could augment GM-CSF-mediated hemopoiesis and stimulate the proliferation of alveolar epithelial cells (1, 35) and enhance liver regeneration after acute liver injury (36). The contribution of MIP-2α as a potential hepatic regenerative factor is consistent with our finding of abundant MIP-2α expression in the liver. So, the growth regulatory functions of MIP-2γ may be predictable. Presently, we did not find any obviously stimulatory or inhibitory activity of MIP-2γ in GM-CSF/IL-3/erythyropoietin-stimulating colony formation assay using CD34+ hemopoietic progenitors (data not shown). The expression of MIP-2γ by parenchyma cells in kidney and liver may imply its potential involvement in the tissue regeneration, and its abundant expression by normal tissues but poor expression by cancer cells suggest its potential roles in oncogenesis. These need to be characterized by further investigation.

In this report, we described a new CXC chemokine MIP-2γ isolated from monocyte-derived DC that exhibited potent chemotaxis on neutrophils, indicating its potential roles in innate immunity. Most interestingly, MIP-2γ can drive the migration of DC. This will extend the knowledge about the controlling of DC migration and the contribution of DC to innate immunity.

We acknowledge Dr. Zhenghua Xiang, Mei Jin, Xuebing Wu, and Dongming Zhang for their excellent technical assistance.

1

This work was supported in part by Natural Science Foundation of Shanghai, China.

4

Abbreviations used in this paper: MIP, macrophage inflammatory protein; DC, dendritic cells; MCP, monocyte chemoattractant protein; AP, alkaline phosphatase; EST, expressed sequence tag.

1
Broxmeyer, H. E., B. Sherry, L. Lu, S. Cooper, C. Carow, S. D. Wolpe, A. Cerami.
1989
. Myelopoietic enhancing effects of murine macrophage inflammatory proteins 1 and 2 on colony formation in vitro by murine and human bone marrow granulocyte/macrophage progenitor cells.
J. Exp. Med.
170
:
1583
2
Cao, Y., C. Chen, J. A. Weatherbee, M. Tsang, J. Folkman.
1995
. Gro-β, a C-X-C- chemokine, is an angiogenesis inhibitor that suppresses the growth of Lewis lung carcinoma in mice.
J. Exp. Med.
182
:
2069
3
Strieter, R. M., P. J. Polverini, S. L. Kunkel, D. A. Arenberg, M. D. Burdick, J. Kasper, J. Dzuiba, J. Van Damme, A. Walz, D. Marriott.
1995
. The functional role of the ELR motif in CXC chemokine-mediated angiogenesis.
J. Biol. Chem.
270
:
27348
4
Cocchi, F., A. L. DeVico, A. Garzino-Demo, S. K. Arya, R. C. Gallo, P. Lusso.
1995
. Identification of RANTES, MIP-1α, and MIP-1β as the major HIV-suppressive factors produced by CD8+ T cells.
Science
270
:
1811
5
Bazan, J. F., K. B. Bacon, G. Hardiman, W. Wang, K. Soo, D. Rossi, D. R. Greaves, A. Zlotnik, T. J. Schall.
1997
. A new class of membrane-bound chemokine with a CX3C motif.
Nature
385
:
640
6
Pan, Y., C. Lloyd, H. Zhou, S. Dolich, J. Deeds, J. A. Gonzalo, J. Vath, M. Gosselin, J. Ma, B. Dussault, et al
1997
. Neurotactin, a membrane-anchored chemokine upregulated in brain inflammation.
Nature
387
:
611
7
Kelner, G. S., J. Kennedy, K. B. Bacon, S. Kleyensteuber, D. A. Largaespada, N. A. Jenkins, N. G. Copeland, J. F. Bazan, K. W. Moore, T. J. Schall, A. Zlotnik.
1994
. Lymphotactin: a cytokine that represents a new class of chemokine.
Science
266
:
1395
8
Kennedy, J., G. S. Kelner, S. Kleyensteuber, T. J. Schall, M. C. Weiss, H. Yssel, P. V. Schneider, B. G. Cocks, K. B. Bacon, A. Zlotnik.
1995
. Molecular cloning and functional characterization of human lymphotactin.
J. Immunol.
155
:
203
9
Banchereau, J., R. M. Steinman.
1998
. Dendritic cells and the control of immunity.
Nature
392
:
245
10
Steinman, R. M., M. Pack, K. Inaba.
1997
. Dendritic cells in the T-cell areas of lymphoid organs.
Immunol. Rev.
156
:
25
11
Tekamp-Olson, P., C. Gallegos, D. Bauer, J. McClain, B. Sherry, M. Fabre, S. van Deventer, A. Cerami.
1990
. Cloning and characterization of cDNAs for murine macrophage inflammatory protein 2 and its human homologues.
J. Exp. Med.
172
:
911
12
Hoefler, H., H. Childers, M. R. Montminy, R. M. Lechan, R. H. Goodman, H. J. Wolfe.
1986
. In situ hybridization methods for the detection of somatostatin mRNA in tissue sections using antisense RNA probes.
Histochem. J.
18
:
597
13
Caux, C., C. Massacrier, B. Vanbervliet, B. Dubois, C. Van Kooten, I. Durand, J. Banchereau.
1994
. Activation of human dendritic cells through CD40 cross-linking.
J. Exp. Med.
180
:
1263
14
Zhou, L. J., T. F. Tedder.
1995
. A distinct pattern of cytokine gene expression by human CD83+ blood dendritic cells.
Blood
86
:
3295
15
Mohamadzadeh, M., A. N. Poltorak, P. R. Bergstressor, B. Beutler, A. Takashima.
1996
. Dendritic cells produce macrophage inflammatory protein-1 gamma, a new member of the CC chemokine family.
J. Immunol.
156
:
3102
16
Adema, G. J., F. Hartgers, R. Verstraten, E. de Vries, G. Marland, S. Menon, J. Foster, Y. Xu, P. Nooyen, T. McClanahan, K. B. Bacon, C. G. Figdor.
1997
. A dendritic-cell-derived C-C chemokine that preferentially attracts naive T cells.
Nature
387
:
713
17
Tang, H. L., J. G. Cyster.
1999
. Chemokine up-regulation and activated T cell attraction by maturing dendritic cells.
Science
284
:
819
18
Cao, X., W. Zhang, L. He, Z. Xie, S. Ma, Q. Tao, Y. Yu, H. Hamada, J. Wang.
1998
. Lymphotactin gene-modified bone marrow dendritic cells act as more potent adjuvants for peptide delivery to induce specific antitumor immunity.
J. Immunol.
161
:
6238
19
Zhang, W., L. He, Z. Yuan, Z. Xie, J. Wang, H. Hamada, X. Cao.
1999
. Enhanced therpeutic efficacy of tumor RNA-pulsed dendritic cells after genetic modification with lymphotactin.
Hum. Gene Ther.
10
:
1151
20
Sozzani, S., W. Luini, A. Borsatti, N. Polentarutti, D. Zhou, L. Piemonti, G. D’Amico, C. A. Power, T. N. Wells, M. Gobbi, P. Allavena, A. Mantovani.
1997
. Receptor expression and responsiveness of human dendritic cells to a defined set of CC and CXC chemokines.
J. Immunol.
159
:
1993
21
Dieu, M. C., B. Vanbervliet, A. Vicari, J. M. Bridon, E. Oldham, S. Ait-Yahia, F. Briere, A. Zlotnik, S. Lebecque, C. Caux.
1998
. Selective recruitment of immature and mature dendritic cells by distinct chemokines expressed in different anatomic sites.
J. Exp. Med.
188
:
373
22
Gunn, M. D., S. Kyuwa, C. Tam, T. Kakiuchi, A. Matsuzawa, L. T. Williams, H. Nakano.
1999
. Mice lacking expression of secondary lymphoid organ chemokine have defects in lymphocyte homing and dendritic cell localization.
J. Exp. Med.
189
:
451
23
Saeki, H., A. M. Moore, M. J. Brown, S. T. Hwang.
1999
. Cutting edge: secondary lymphoid-tissue chemokine (SLC) and CC chemokine receptor 7 (CCR7) participate in the emigration pathway of mature dendritic cells from the skin to regional lymph nodes.
J. Immunol.
162
:
2472
24
Sato, K., H. Kawasaki, H. Nagayama, R. Serizawa, J. Ikeda, C. Morimoto, K. Yasunaga, N. Yamaji, K. Tadokoro, T. Juji, T. A. Takahashi.
1999
. CC chemokine receptors, CCR-1 and CCR-3, are potentially involved in antigen-presenting cell function of human peripheral blood monocyte-derived dendritic cells.
Blood
93
:
34
25
Delgado, E., V. Finkel, M. Baggiolini, C. R. Mackay, R. M. Steinman, A. Granelli-Piperno.
1998
. Mature dendritic cells respond to SDF-1, but not to several β-chemokines.
Immunobiology
198
:
490
26
Lin, C. L., R. M. Suri, R. A. Rahdon, J. M. Austyn, J. A. Roake.
1998
. Dendritic cell chemotaxis and transendothelial migration are induced by distinct chemokines and are regulated on maturation.
Eur. J. Immunol.
28
:
4114
27
Yanagihara, S., E. Komura, J. Nagafune, H. Watarai, Y. Yamaguchi.
1998
. EBI1/CCR7 is a new member of dendritic cell chemokine receptor that is up-regulated upon maturation.
J. Immunol.
161
:
3096
28
Sozzani, S., P. Allavena, G. D’Amico, W. Luini, G. Bianchi, M. Kataura, T. Imai, O. Yoshie, R. Bonecchi, A. Mantovani.
1998
. Differential regulation of chemokine receptors during dendritic cell maturation: a model for their trafficking properties.
J. Immunol.
161
:
1083
29
Sozzani, S., F. Sallusto, W. Luini, D. Zhou, L. Piemonti, P. Allavena, J. Van Damme, S. Valitutti, A. Lanzavecchia, A. Mantovani.
1995
. Migration of dendritic cells in response to formyl peptides, C5a, and a distinct set of chemokines.
J. Immunol.
155
:
3292
30
Rissoan, M. C., V. Soumelis, N. Kadowaki, G. Grouard, F. Briere, R. de Waal Malefyt, Y. J. Liu.
1999
. Reciprocal control of T helper cell and dendritic cell differentiation.
Science
283
:
1183
31
Van Coillie, E., P. Fiten, H. Nomiyama, Y. Sakaki, R. Miura, O. Yoshie, J. Van Damme, G. Opdenakker.
1997
. The human MCP-2 gene (SCYA8): cloning, sequence analysis, tissue expression, and assignment to the CC chemokine gene contig on chromosome 17q11.2.
Genomics
40
:
323
32
Poltorak, A. N., F. Bazzoni, Smirnova, II, E. Alejos, P. Thompson, G. Luheshi, N. Rothwell, and B. Beutler. 1995. MIP-1γ: molecular cloning, expression, and biological activities of a novel CC chemokine that is constitutively secreted in vivo. J. Inflamm. 45:207.
33
Poznansky, M. C., I. T. Olszak, R. Foxall, R. H. Evans, A. D. Luster, D. T. Scadden.
2000
. Active movement of T cells away from a chemokine.
Nat. Med.
6
:
543
34
Ward, S. G., K. Bacon, J. Westwick.
1998
. Chemokines and T lymphocytes: more than an attraction.
Immunity
9
:
1
35
Driscoll, K. E., D. G. Hassenbein, B. W. Howard, R. J. Isfort, D. Cody, M. H. Tindal, M. Suchanek, J. M. Carter.
1995
. Cloning, expression, and functional characterization of rat MIP-2: a neutrophil chemoattractant and epithelial cell mitogen.
J. Leukocyte Biol.
58
:
359
36
Hogaboam, C. M., C. L. Bone-Larson, M. L. Steinhauser, N. W. Lukacs, L. M. Colletti, K. J. Simpson, R. M. Strieter, S. L. Kunkel.
1999
. Novel CXCR2-dependent liver regenerative qualities of ELR-containing CXC chemokines.
FASEB J.
13
:
1565