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Chemoattractant for Human Neutrophils and Dendritic Cells1
Department of Immunology and Shanghai Brilliance Biotechnology Institute, Second Military Medical University, Shanghai, People’s Republic of China
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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ß, hence is designated as MIP-2
.
Mouse MIP-2 was identified by electrocloning and is highly
homologous to human MIP-2. Northern blotting revealed that MIP-2
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-2 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-2 expression by monocyte-derived DC.
Recombinant MIP-2 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-2 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-2 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. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and
MIP-1ß, 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-2ß (11) and
was chemoattractant for neutrophils and DC.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
Isolation of a cDNA-encoding MIP-2
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-2. 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-2, from which mouse MIP-2 full-length cDNA was obtained by
contig and confirmed by RT-PCR cloning from mouse kidney. Mouse
MIP-2 full-length cDNA inserted into pGEM-3Zf vector (Promega,
Madison, WI) was used as a template (pGEM-mMIP2) to synthesize RNA
probes for in situ hybridization analysis.
Northern blotting
The cDNA containing the full-length encoding regions of human
MIP-2 or MIP-2 were amplified by PCR, confirmed by DNA
sequencing, and used as templates for synthesis of probes in Northern
blotting. MIP-2 cDNA from nucleotides 41–363 (GenBank accession no.
X53799) was amplified from human placenta cDNA (Clontech Laboratories,
Palo Alto, CA), and MIP-2 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 0.1x SSC and 0.1% SDS, the filters were subjected to
autoradiography. The filters were reprobed with a human ß-actin cDNA
probe (Clontech Laboratories).
RT-PCR analysis for MIP-2 expression
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-2 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-2 is 5'-CTCCCCATGTCCCTGCTC-3', and its downstream primer is
5'-ACCTGCGCTTCTCGTTCC-3', with the predicted product of 328 bp. The
upstream primer of human ß-actin is 5'-GCATCGTGATGGACTCCG-3', and its
downstream primer is 5'-TCGGAAGGTGGACAGCGA-3', with the expected
product of 600 bp.
Preparation of polyclonal Ab against human MIP-2
For expression of 6His-tagged MIP-2 in E. coli, a
BamHI restriction site was introduced by PCR just before the
predicted first codon of mature MIP-2 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-2 cDNA were
confirmed by sequencing. Expression of His-tagged MIP-2 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-2 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-2 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).
Expression of Fc fusion protein of MIP-2
For expression of IgG fusion protein to determine the N terminal
of mature MIP-2, MIP-2 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-2, and a BamHI site was introduced before the stop
codon for in-frame ligation with human IgG1 CH2 and CH3 fragment. The
MIP2-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
MIP2-IgG expression vector into 293 cells with lipofectamine (Life
Technologies), the 48-h culture supernatants were harvested and
subjected to protein A affinity chromatography. MIP2-IgG fusion
protein was blotted onto a polyvinylidene difluoride membrane for amino
acid sequencing of the MIP-2 N terminus.
Eukaryotic expression of recombinant MIP-2 for activity assay
For activity investigation, MIP-2 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-2. EcoRI and
KpnI restriction sites were added before the start codon and
immediately after the stop codon of MIP-2 respectively as
underlined. MIP-2 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-2. MIP-2-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-2
expression was further confirmed by Western blotting with its rabbit
polyclonal Abs.
Western blotting analysis of MIP-2
Forty-eight-hour culture supernatants from MIP-2-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-2 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).
Chemotaxis assay
Polymorphonuclear neutrophils and mononuclear cells were separated heparinized peripheral blood by double gradient centrifugation (30 min, 700 x 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 (2x106cells/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.
Expression of alkaline phosphatase (AP) fusion protein
To express their AP fusion proteins, human IL-8 and MIP-2
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-2 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.
Receptor binding assay
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-2. 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.
In situ hybridization
Sense and antisense digoxigenin-labeled cRNA probes of mouse
MIP-2 were synthesized with a digoxigenin-RNA labeling kit (Roche
Diagnostics, Hong Kong) using linearilzed pGEM-mMIP2 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, 1x 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 4x SSC for 20 min at 37°C, treated with 20
µg/ml RNase in 2x SSC, and followed by rinses with 1x SSC and 0.2x
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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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cDNA from human DC
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-2 (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-2ß
(11), so it was designated as MIP-2. In contrast to
MIP-2ß, MIP-2 does not contain an ELR motif, which is also
found in IL-8 and other CXC chemokines. MIP-2 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-2 cDNA
was in-frame fused with human IgG1 CH2 and CH3 and inserted into
pcDNA3.1 vector to express MIP2-IgG fusion protein in 293 cells.
Amino-terminal sequencing of protein A-purified MIP2-IgG yields the
sequence SKCKCSRKGP, confirming the predicted NH2
terminus of mature MIP-2. Mouse MIP-2 shared 95% and 98%
identity with human MIP-2 at nucleotide and protein levels,
respectively (Fig. 2, GenBank accession no. AF252873), suggesting that
MIP-2 was highly conserved.
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Northern blotting revealed constitutive expression of MIP-2 in
most normal tissues. The greatest expression of MIP-2 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-2 was
detected in testis, ovary, heart, and lung, and no expression was seen
in PBL. Two different transcripts of human MIP-2 were detected in
normal tissues. The dominant one is 2 kb, and another is about 0.5
kb. Constitutive expression of MIP-2 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-2 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-2 in normal tissues was similar to that of its homologue
MIP-2 to some extent.
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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-2 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. 4A). Meanwhile, MIP-2 was
detectable by Northern blotting in A549, melanoma G361 cells, and HeLa
cells (Fig. 3
). Although MIP-2 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-2 by RT-PCR
(Fig. 4B). In situ hybridization demonstrated that MIP-2
was mainly expressed by parenchyma cells in kidney and liver, including
epithelium of uriniferous tubule and liver cells (Fig. 5). This implied that MIP-2 might
possess growth regulatory functions under physiological conditions.
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protein
To detect MIP-2 protein expression, we developed rabbit
polyclonal Abs against human MIP-2 by immunizing with 6His-tagged
MIP-2, which was expressed in bacteria and purified by
nickel-nitrilotriacetic acid-mediated affinity chromatography. For
bioactivity analysis, human MIP-2 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-2-transfected 293 cells (Fig. 6A), which was confirmed to be
human MIP-2 by Western blotting with rabbit polyclonal Abs against
human MIP-2 (Fig. 6B), whereas untransfected or
mock-transfected 293 cells didn’t express any detectable human
MIP-2 by Western blotting.
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For microchemotactic assay, the 24-h serum-free culture
supernatants from MIP-2-transfected or mock-transfected 293 cells
were harvested 48 h after transfection. The culture supernatants
from MIP-2-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.
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Putatively, ELR+ CXC chemokine mediates the
chemotaxis of neutrophils via CXCR2. Although MIP-2 was absent of
the ELR motif, it did attract neutrophils. To evaluate whether CXCR2
mediates the chemotactic capacity of MIP-2, 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-2 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-2 didn’t bind to receptor CXCR2 and suggested that other
chemokine receptors might mediate the biological functions of
MIP-2.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß (11),
and can attract neutrophils markedly, so it’s designated as MIP-2.
In contrast to MIP-2ß, MIP-2 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-2 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-2. Interestingly, MIP-2 is also a chemoattractant of
monocyte-derived DC. The identification of a MIP-2 receptor may
facilitate understanding the chemotactic features of MIP-2.
Although it was isolated from human monocyte-derived DC, no detectable
expression of human MIP-2 was observed in PBL by Northern blotting,
or in freshly isolated or PMA-treated monocytes by RT-PCR, suggesting
MIP-2 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-1, MIP-1ß, MIP-1, 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-2 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-1, MCP-3, and RANTES can direct the migration of immature DC
located in the periphery, whereas MIP-3ß 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--inducible protein-10, and growth-related oncogene-ß, 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-2
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-2 binding and function needs to be identified
and characterized for better understanding the functional role of
MIP-2 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-2 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-2. MIP-2 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-2
expression in the loci. Moreover, the constitutive expression in normal
tissues was also observed for chemokine other than MIP-2, e.g.,
MCP-2 and MIP-1 (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-2 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-2ß 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-2 as a potential hepatic
regenerative factor is consistent with our finding of abundant MIP-2
expression in the liver. So, the growth regulatory functions of
MIP-2 may be predictable. Presently, we did not find any obviously
stimulatory or inhibitory activity of MIP-2 in
GM-CSF/IL-3/erythyropoietin-stimulating colony formation assay using
CD34+ hemopoietic progenitors (data not shown).
The expression of MIP-2 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-2 isolated from
monocyte-derived DC that exhibited potent chemotaxis on neutrophils,
indicating its potential roles in innate immunity. Most interestingly,
MIP-2 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.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint request to Dr. Xuetao Cao, Department of Immunology, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, People’s Republic of China.
3 X.C. and W.Z. contributed equally to this work.
4 Abbreviations used in this paper: MIP, macrophage inflammatory protein; DC, dendritic cells; MCP, monocyte chemoattractant protein; AP, alkaline phosphatase; EST, expressed sequence tag.
Received for publication May 28, 1999. Accepted for publication June 20, 2000.
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(magnification, x200). C, Liver
section probed with mouse MIP-2
, the sinus hepaticus was
marked with 
, and positive liver cells were marked with 
) 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.