| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
from Normal and Transformed Cells1
Laboratory of Molecular Immunology, Rega Institute for Medical Research, University of Leuven, Leuven, Belgium
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
(MIP-3), Exodus, or CCL20, is a
C-C chemokine that attracts immature dendritic cells and memory T
lymphocytes, both expressing CCR6. Depending on the cell type, this
chemokine was found to be inducible by cytokines (IL-1ß) and by
bacterial, viral, or plant products (including LPS, dsRNA, and PMA) as
measured by a specific ELISA. Although coinduced with monocyte
chemotactic protein-1 (MCP-1) and IL-8 by dsRNA, measles virus, and
IL-1ß in diploid fibroblasts, leukocytes produced LARC/MIP-3 only
in response to LPS. However, in myelomonocytic THP-1 cells
LARC/MIP-3 was better induced by phorbol ester, whereas in HEp-2
epidermal carcinoma cells IL-1ß was the superior inducer. The
production levels of LARC/MIP-3 (1–10 ng/ml) were, on the average,
10- to 100-fold lower than those of IL-8 and MCP-1, but were comparable
to those of other less abundantly secreted chemokines. Natural
LARC/MIP-3 protein isolated from stimulated leukocytes or tumor cell
lines showed molecular diversity, in that NH2- and
COOH-terminally truncated forms were purified and identified by amino
acid sequence analysis and mass spectrometry. In contrast to other
chemokines, including MCP-1 and IL-8, the natural processing did not
affect the calcium-mobilizing capacity of LARC/MIP-3 through its
receptor CCR6. Furthermore, truncated natural LARC/MIP-3 isoforms
were equally chemotactic for lymphocytes as intact
rLARC/MIP-3. It is concluded that in addition to its role in
homeostatic trafficking of leukocytes, LARC/MIP-3 can function as an
inflammatory chemokine during host
defense. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
(MIP-3) (4) or
Exodus (5), are expected to play a crucial role in
trafficking and homing of lymphocytes and dendritic cells into
secondary lymphoid organs and in the maturation of leukocytes
(6, 7, 8). The three different acronyms for this single gene
product are the consequence of the independent identification of
LARC/MIP-3 through bioinformatics on ESTs. In contrast to most human
C-C chemokine genes, which are mapped on chromosome 17, the
LARC/MIP-3 gene is localized on chromosome 2 (3). Based
on its gene SCYA20 (9), LARC/MIP-3 protein
is also designated C-C chemokine ligand 20 (CCL20) following a new
classification system (10).
The spectrum of target cells that chemotactically respond to
LARC/MIP-3 is restricted to memory T lymphocytes (3, 11, 12) and immature dendritic cells (11, 13, 14, 15, 16). In
addition, this chemokine has been reported to inhibit the proliferation
of hemopoietic progenitors in vitro (5, 17). LARC/MIP-3
is exerting its activity through binding to CCR6 (11, 12, 18, 19, 20), which is not shared by any other known chemokine, but
is nevertheless binding a member of the structurally unrelated
ß-defensins (21). Concordant with the target cells for
LARC/MIP-3, CCR6 is found to be expressed on immature dendritic
cells and memory T lymphocytes as well as on B lymphocytes, in various
lymphoid organs, and in pancreas (11, 12, 13, 14, 15, 16, 18, 19, 22, 23, 24).
Up-regulation of CCR6 expression on human neutrophils by cytokines can
explain the slight chemotactic response of these cells to LARC/MIP-3
(3, 25).
LARC/MIP-3 mRNA is reported to be expressed at mucosal sites; in
lymphoid tissues such as liver, lung, peripheral lymph nodes, thymus,
tonsils, and appendix; and in PBMC (3, 4, 5, 11, 13, 26).
However, little is known about the regulation of the LARC/MIP-3
protein production in different cell types. Additionally, many
chemokines are post-translationally modified due to
NH2- and/or COOH-terminal processing. Selective
NH2-terminal cleavage of most C-C chemokines by
enzymes (e.g., CD26/dipeptidyl peptidase IV) results in impaired
receptor recognition and signaling properties and hence loss of
chemotactic activity (27). In contrast,
NH2- or COOH-terminal processing of some
inflammatory C-X-C chemokines causes an increase in chemotactic potency
(28). To better understand the role of natural
LARC/MIP-3 in physiologic and pathologic conditions, we have studied
the gene regulation and molecular diversity of LARC/MIP-3 at the
protein level. Here we report its inducible production in various
normal tissue cells and tumor cell types. In addition, natural
LARC/MIP-3 isoforms, isolated from normal and transformed cell
cultures, were identified and compared biochemically and
biologically.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Human PBMC were isolated from single blood donations (Blood
Transfusion Center, Antwerp, Belgium). Erythrocytes were removed by
sedimentation (30 min) with hydroxyethyl starch (Plasmasteril,
Fresenius Hemotechnology, Bad Homburg, Germany). Mononuclear cells and
granulocytes were separated by density gradient centrifugation
(400 x g, 30 min) on Ficoll-sodium diatrizoate
(Lymphoprep, Life Technologies, Paisley, UK). For induction
experiments, mononuclear cells were seeded at 2 x
106 cells/ml in serum-free Eagle’s MEM with
Earle’s salts (EMEM; Life Technologies) in
25-cm2 flasks (5 ml) or in multiwell dishes (1
ml/well, 24 x 1.9 cm2; Life Technologies).
Different concentrations of the following inducers were added: pure
natural human IL-1ß (29), human rIFN-
(Bioferon,
Laupheim, Germany), the dsRNA polyriboinosinic:polyribocytidylic acid
(poly(rI:rC); P-L Biochemicals, Milwaukee, WI), measles virus
(Attenuvax strain, 106.5 50% tissue culture
infectious doses/ml) (TCID50/ml), PMA (Sigma, St. Louis,
MO), Con A (Calbiochem, La Jolla, CA), or LPS from Escherichia
coli (0111:B4, Difco Laboratories, Detroit, MI). Conditioned
medium was collected after 48 h and was stored at -20°C until
assay.
Human diploid fibroblasts (E6SM, a strain of embryonic skin and muscle cells), the human osteosarcoma cell line MG-63, and the human epidermal larynxcarcinoma cell line HEp-2 were grown in EMEM supplemented with 10% FCS (Life Technologies). For induction experiments, confluent monolayers (grown in 25-cm2 flasks) were induced in 5 ml of EMEM containing 0.5% FCS by stimulation with different concentrations of the inducers mentioned above. The human myelomonocytic cell line THP-1 was grown in RPMI 1640 (BioWhittaker Europe, Verviers, Belgium) supplemented with 10% FCS. After centrifugation and resuspension of the THP-1 cells in EMEM containing 2% FCS, cells were seeded at 2 x 106 cells/ml in 25-cm2 flasks, and the above-mentioned inducer substances were added. After 48- to 72-h stimulation at 37°C, cell supernatants were harvested and kept at -20°C until assay.
Chemokine immunoassays
For detection of LARC/MIP-3 protein, a classical sandwich
ELISA was developed. Plates coated with a mouse mAb raised against
human rLARC/MIP-3 (mAb3B7) were provided by Dr. J.-M. Jaspar
(BioSource Europe, Nivelles, Belgium). Recombinant LARC/MIP-3
(PeproTech, Rocky Hill, NJ) was used as a standard. Samples diluted in
blocking buffer (PBS containing 0.05% Tween-20 and 0.1% casein) were
added, as was the polyclonal rabbit anti-human LARC/MIP-3 Ab
(dilution, 1/3000; PeproTech) used for capturing. After 2 h of
incubation, detection was performed with peroxidase-conjugated
F(ab')2 donkey anti-rabbit Ab (Jackson
ImmunoResearch Laboratories, West Grove, PA) and
3,3',5,5'-tetramethylbenzidine dihydrochloride hydrate (Aldrich,
Milwaukee, WI). The detection limit of the LARC/MIP-3 ELISA was
about 0.2 ng/ml, and its specificity was demonstrated by the lack of
cross-reactivity with other chemokines (including granulocyte
chemotactic protein-2, epithelial cell-derived neutrophil attractant-78
(ENA-78), monocyte chemotactic protein-1 (MCP-1), MCP-2, and IL-8 at a
concentration of 100 ng/ml) and other potentially cross-reactive agents
(e.g., IL-1ß, IFN-, poly(rI:rC), PMA, Con A, and LPS, used as
inducers).
Recombinant MCP-1 was a gift from Dr. J. J. Oppenheim (National Cancer Institute, Frederick, MD); natural IL-8 was purified from osteosarcoma cell-conditioned medium (30). MCP-1 and IL-8 were quantified with a classical sandwich ELISA as previously described (28, 31). Polyclonal rabbit anti-human MCP-1 and polyclonal goat anti-human IL-8 (both purified by protein A affinity chromatography) were used for coating in the MCP-1 and IL-8 ELISA, respectively. Monoclonal mouse anti-human MCP-1 and anti-human IL-8 (both from R&D Systems, Abingdon, U.K.) were used as capture Abs. Peroxidase-labeled goat anti-mouse polyclonal Ab (Jackson ImmunoResearch Laboratories) and 3,3',5,5'-tetramethylbenzidine dihydrochloride hydrate were applied for detection. The detection limits for MCP-1 and IL-8 were 0.1 and 0.02 ng/ml, respectively.
Production and purification of natural LARC/MIP-3
Human LARC/MIP-3 protein was purified from conditioned medium
of stimulated MG-63 osteosarcoma cells, myelomonocytic THP-1 cells, and
PBMC. MG-63 cell monolayers (126 flasks, 175 cm2;
Life Technologies) were grown to confluence in EMEM containing 10% FCS
and were stimulated in EMEM plus 2% FCS with a semipurified cytokine
mixture (derived from mitogen-stimulated mononuclear cells). THP-1
cells were grown in suspension in RPMI containing 10% FCS and were
induced with PMA (10 ng/ml) in EMEM containing 2% FCS (42 flasks, 175
cm2, 1.8 x 106
cells/ml). PBMC (50 x 109 cells, isolated
from 116 blood donations; see above) were suspended in Spinner flasks
(5 x 106 cells/ml) in EMEM plus 1% FCS
supplemented with Con A (2 µg/ml) and LPS (2 µg/ml). After 48
h (for MG-63 cells and PBMC) or 72 h (for THP-1 cells) of
incubation at 37°C, conditioned medium was collected and stored at
-20°C until processing.
Natural LARC/MIP-3 was isolated through a four-step concentration
and purification procedure, as previously described (32).
Briefly, the stimulated conditioned medium from THP-1 and MG-63 cells
was concentrated and partially purified by adsorption to controlled
pore glass (CPG) beads (CPG 10-350, Serva, Heidelberg, Germany) and
elution with 0.3 M glycine/HCl, pH 2.0. The acid CPG eluate was
neutralized with NaOH and further purified by Ab affinity
chromatography, using a polyclonal Ab against CPG-purified
fibroblast-derived cytokines. The Ig fraction from 20 ml of the
antiserum was coupled to 6 g of cyanogen bromide-activated
Sepharose 4B (Pharmacia, Uppsala, Sweden). LARC/MIP-3
immunoreactivity was bound to the column (1.5 x 15 cm, 20 ml/h)
at neutral pH and was recovered in 0.5 M NaCl and 0.1 M citrate/HCl, pH
2.0. Alternatively, the PBMC-derived conditioned medium was
concentrated by adsorption to silicic acid (Matrex, Amicon, Beverly,
MA) and elution with 50% ethylene glycol in 1.4 M NaCl/PBS, pH 7.4.
After dialysis against 50 mM NaCl and 50 mM Tris-HCl (pH 7.4), the
silicic acid eluate was further purified by heparin affinity
chromatography. The eluate was loaded onto a heparin-Sepharose column
(Pharmacia), and the proteins were eluted in a NaCl gradient (0.05–2 M
NaCl) in 50 mM Tris-HCl, pH 7.4.
Fractions recovered by Ab or heparin affinity chromatography containing
the LARC/MIP-3 immunoreactivity were prepared for Mono S cation
exchange chromatography (Pharmacia) by dialysis against
equilibration/loading buffer (50 mM formate, pH 4.0). A linear NaCl
gradient (0–1 M) was used to elute (1 ml/min) proteins in 50 mM
formate, pH 4.0. Absorbance was monitored at 280 nm as a measure for
protein concentration. Finally, LARC/MIP-3 was purified to
homogeneity by reverse phase HPLC (RP-HPLC). Samples were injected on a
220 x 2.1-mm C8 Aquapore RP-300 column (PE
Biosystems, Foster City, CA), equilibrated with 0.1% trifluoroacetic
acid in water (pH 2.0), and the proteins were eluted (0.4 ml/min) with
an acetonitrile gradient (0–80%) in 0.1% trifluoroacetic
acid/H2O, pH 2.0. The column effluent was
monitored by a spectrophotometer at 220 nm.
SDS-PAGE, immunoblotting, amino acid sequence analysis, and mass spectrometry
Natural LARC/MIP-3 was analyzed for
Mr and purity by SDS-PAGE under
reducing conditions on Tris/tricine gels (33). Proteins
were stained with silver, and the molecular mass markers used were
carbonic anhydrase (Mr, 29,000),
ß-lactoglobulin (Mr, 18,400),
lysozyme (Mr, 14,400), and bovine
trypsin inhibitor (Mr, 6,200) (Life
Technologies).
Alternatively, the proteins were transferred to a Problot membrane (PE
Biosystems) after SDS-PAGE to enable immunoblotting. The
Mr markers used (Bio-Rad, Hercules,
CA) were the prestained SDS-PAGE standards lysozyme
(Mr, 21,300) and aprotinin
(Mr, 7,600). The Problot membrane was
incubated overnight with specific rabbit polyclonal anti-human
LARC/MIP-3 antiserum (dilution, 1/1000; PeproTech), which was also
used in the LARC/MIP-3 ELISA, and subsequently for 1 h with
alkaline phosphatase-conjugated anti-rabbit IgG (Jackson
ImmunoResearch Laboratories). Immunoreactive proteins were stained by
incubating the membrane in nitro blue
tetrazolium/5-bromo-4-chloro-3-indolyl phosphate color solution
(Sigma).
The identity and the NH2-terminal sequence of purified chemokines were determined by Edman degradation on a pulsed liquid phase protein sequencer (477A/120A, PE Biosystems) with N-methylpiperidine as a coupling base and on-line detection of phenylthiohydantoin amino acids. The presence of Cys residues was obvious from the absence of any detectable signal (32).
The molecular mass of the purified LARC/MIP-3 forms was determined
on an electrospray ion trap mass spectrometer (Drs. A. Schneider and A.
Ingendoh, Esquire, Bruker Daltonik, Bremen, Germany).
C8 RP-HPLC purified proteins were diluted 5-fold
(final LARC/MIP-3 concentration, 0.2–1 µg/ml) in methanol/water
(1/1) including 0.1% acetic acid and applied to the mass spectrometer
by direct infusion at a flow rate of 2 µl/min. Average relative
molecular masses were calculated from the summation of 100 spectra,
with an accuracy of ±1.0.
Chemotaxis and intracellular calcium mobilization
Blood lymphocytes were purified by incubating PBMC (see above) for 15 min at 4°C with paramagnetic microbeads conjugated with mAb against CD3. The cell suspension was passed over a column placed in a magnetic field (VarioMACS, Miltenyi Biotec, Bergisch Gladbach, Germany). After this positive magnetic cell sorting, a cell purity (analyzed by FACS) of >80% was reached for lymphocytes (CD3+).
LARC/MIP-3 was tested for its chemotactic potency on lymphocytes in
the Boyden microchamber (48-well chemotaxis microchamber, Neuro Probe,
Gaithersburg, MD). The lower compartments of the microchamber were
filled with test samples, dilutions of synthetic MCP-3
(34) as a positive control, and the dilution buffer HBSS
(Life Technologies) supplemented with pyrogen-free human serum albumin
(1 mg/ml; Red Cross, Leuven, Belgium) as a negative control (27 µl).
The upper compartments were filled with a lymphocyte suspension (50
µl) at 10 x 106 cells/ml in HBSS with
human serum albumin. The lower compartments were separated from the
upper compartments by a 5-µm pore size, polyvinyl pyrrolidone-free
polycarbonate membrane (Nuclepore, Acton, MA) that was coated with 20
µg/ml fibronectin (Life Technologies) for 24 h at 4°C. After
4-h incubation at 37°C, the cells that migrated through the membrane
were fixed, stained with Hemacolor solutions (Merck, Darmstadt,
Germany), and counted microscopically in 10 oil immersion fields at
x500 magnification. The chemotactic activity of a sample (triplicates
in each chamber) was expressed as a chemotactic index; this was the
number of cells that migrated in response to the test sample divided by
the number of cells that migrated in response to the negative
control.
The intracellular calcium concentration ([Ca2+]i) was measured using the fluorescent indicator fura-2 (fura-2/acetoxymethylester, Molecular Probes Europe, Leiden, The Netherlands). Human CCR6 transfectants were generated using human embryonic kidney cells (HEK 293) (22) and were provided by Dr. G. Márquez (Department of Immunology and Oncology, National Center of Biotechnology, Madrid, Spain). Transfected cells were grown in DMEM with 4.5 g/L glucose and L-Gln (BioWhittaker Europe) supplemented with 10% FCS in the presence of geneticin at 450 µg/ml (G418, Life Technologies). For detection of the intracellular calcium concentration, cells (107 cells/ml) were incubated for 30 min at 37°C in the growth medium containing 2.5 µM fura-2 and 0.01% Pluronic F-127 (Sigma). Cells were washed twice and resuspended at a concentration of 106 cells/ml in HBSS (1 mM Ca2+) supplemented with 0.1% FCS and buffered at pH 7.4 with 10 mM HEPES/NaOH. Cells were allowed to equilibrate at 37°C for 10 min before fura-2 fluorescence was measured in an LS50B luminescence spectrophotometer (PerkinElmer, Norwalk, CT). Upon excitation of the cell suspension at 340 and 380 nm, emission was measured at 510 nm. The [Ca2+]i was calculated according to the Gryn-kiewicz equation (35). To determine the Rmax, cells were lysed by addition of 50 µM digitonin. The Rmin was determined by the addition of 10 mM EGTA to the lysed cells after adjusting the pH to 8.5 with 20 mM Tris. The Kd used for calibration was 224 nM.
Statistical analysis
Statistical analysis was performed using the Mann-Whitney U test. The significance levels of differences with controls are indicated in the figures.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
in human tumor cells
Human osteosarcoma (MG-63) cells are a good source of chemokines
such as IL-8 (30) and MCP-1 (36) when
stimulated with cytokines or after viral infection. Indeed, about 200
ng/ml of IL-8 (Fig. 1
A) and
MCP-1 (36) was induced by 100 U/ml of IL-1ß or by
infection with 105.2 50% tissue culture
infectious doses/ml of measles virus. However, induction of MG-63 cells
for 48 h with IL-1ß yielded 500-fold lower (0.4 ng/ml)
LARC/MIP-3 production as measured by a specific ELISA. A higher
production of LARC/MIP-3 (
2 ng/ml) by MG-63 sarcoma cells was
obtained after infection with measles virus. Although the
tumor-promoting agent PMA did not induce detectable LARC/MIP-3 or
MCP-1 (data not shown) at 100 ng/ml, it clearly stimulated IL-8
production (Fig. 1A). It can be concluded that in MG-63
sarcoma cells, LARC/MIP-3 induction quantitatively and qualitatively
differed from that of other chemokines, such as IL-8 and MCP-1.
|
induction were more similar to those
for IL-8 (Fig. 1
B). IL-1ß, measles virus, and PMA were
capable of inducing both chemokines, whereas IFN-, which induced
MCP-1 in HEp-2 cells (data not shown), failed to stimulate
LARC/MIP-3 and IL-8 production. It should be noted that the maximal
amount of LARC/MIP-3 produced by HEp-2 cells (10 ng/ml) is about
5-fold higher than that produced by MG-63 cells. As a result, HEp-2
cells produced only 10-fold less LARC/MIP-3 compared with IL-8 (Fig. 1B). These data show that LARC/MIP-3 production is
differently regulated in these sarcoma and carcinoma cells. Indeed,
LARC/MIP-3 was best produced by measles virus in MG-63 cells,
whereas in HEp-2 cells IL-1ß was superior.
Further, myelomonocytic THP-1 cells were found to be good producers of
both LARC/MIP-3 and IL-8 after stimulation with PMA or LPS (Fig. 2). In contrast, MCP-1 was
dose-dependently induced in THP-1 cells by both LPS and IFN-,
whereas PMA failed to do so. Measles virus and IL-1ß, which induced
sarcoma cells and carcinoma cells to produce chemokines, did not
stimulate the leukemic cell line to produce LARC/MIP-3, IL-8,
or MCP-1. Finally, these three chemokines were only marginally induced
in THP-1 cells by the mitogen Con A. Collectively, these results
indicate that the production of LARC/MIP-3 varied depending on the
tumor cell line and the inducer used.
|
in diploid fibroblasts and
PBMC
To evaluate LARC/MIP-3 gene regulation and protein secretion by
normal cells, similar induction experiments were performed on cultured
diploid skin-muscle fibroblasts and freshly isolated PBMC. Fig. 3A shows that in diploid
fibroblasts the dsRNA poly(rI:rC), measles virus, and IL-1ß were the
predominant inducers of LARC/MIP-3. This dose-dependent induction
pattern is again similar to that of IL-8 (Fig. 3A). The
finding that normal connective tissue cells responded best to dsRNA to
produce LARC/MIP-3 is in contrast with the fact that the tumor cell
lines (see above) failed to secrete this chemokine after induction with
poly(rI:rC). On a quantitative basis, LARC/MIP-3 production by
normal fibroblasts and sarcoma cells was low compared with that by
carcinoma and myelomonocytic cells, indicating that connective tissue
is not the primary source of LARC/MIP-3.
|
(Fig. 3B). Measles virus also induced LARC/MIP-3 in mononuclear
cells (data not shown), but not in THP-1 cells (Fig. 2). In contrast,
this myelomonocytic cell line was most responsive to PMA, which failed
to induce LARC/MIP-3 in PBMC (Fig. 3B). Although PBMC did
not respond to the other stimuli tested, the capacity of the
mononuclear cell population to respond to other inducers was confirmed
by the significant induction of MCP-1 by poly(rI:rC) and Con A (Fig. 3B). Taken together, the induction experiments demonstrate
that in normal leukocytes and fibroblasts LARC/MIP-3 induction is
differently regulated, and compared with corresponding leukemic and
sarcoma cell lines and to other chemokines, there is only limited
parallelism of inducibility.
Isolation and characterization of natural LARC/MIP-3 isoforms
In view of the existing heterogeneity of chemokines due to
post-translational modification and the subsequent alterations in
biological activities, natural LARC/MIP-3 forms were isolated from
different cellular sources. To that goal, conditioned media from
cytokine-stimulated MG-63 osteosarcoma cells, phorbol ester-induced
monocytic THP-1 cells, and PBMC stimulated by endotoxin and mitogen
were produced on a large scale (3–10 liters). These conditioned media
were processed through a standard procedure for chemokine purification,
including adsorption (to CPG or to silicic acid), affinity
chromatography (Ab or heparin), cation exchange chromatography and
RP-HPLC (32). The LARC/MIP-3 concentration was
quantified by the same ELISA used to measure LARC/MIP-3 in crude
induction samples (see above). After cation exchange chromatography,
LARC/MIP-3 from MG-63 cells eluted in the NaCl gradient as a single
sharp peak of immunoreactivity (data not shown), separate from the
other chemokines present in the crude conditioned medium (IL-8,
granulocyte chemotactic protein-2, GRO, IFN--inducible protein-10,
MCP-1, MCP-2, MCP-3, and RANTES). This confirms the specificity of the
LARC/MIP-3 ELISA. Further purification of this LARC/MIP-3 peak by
RP-HPLC showed that the chemokine again eluted in a single peak of
immunoreactivity corresponding to a 6.0-kDa protein (data not shown).
Although only a minute amount of purified LARC/MIP-3 protein was
recovered, NH2-terminal sequence analysis of this
6.0-kDa protein allowed us to identify it as LARC/MIP-3. The natural
product showed heterogeneity, in that
NH2-terminally intact LARC/MIP-3 and an
isoform missing the NH2-terminal Ala were
detected. Thus, on the basis of the ELISA only a single chemokine,
i.e., LARC/MIP-3, was purified from a crude chemokine mixture.
Because THP-1 cells stimulated with PMA were quantitatively a better
source of LARC/MIP-3 than MG-63 cells (Figs. 1A and 2),
an identical isolation procedure was applied for THP-1 cell-conditioned
medium. LARC/MIP-3 from THP-1 cells eluted at a similar position
from the cation exchange column and yielded again a single 6.0-kDa
protein peak on RP-HPLC (data not shown).
NH2-terminal sequence analysis and mass
spectrometry revealed the presence of the two isoforms, intact
LARC/MIP-31–70 and truncated
LARC/MIP-32–70, that differ in length by only
one residue at the NH2 terminus. However, both
isoforms were COOH-terminally intact. The amounts of purified
LARC/MIP-3 protein were insufficient to continue further
fractionation.
To determine whether LARC/MIP-3 heterogeneity also occurred in
normal cells, LARC/MIP-3 present in the conditioned medium from
stimulated PBMC was purified to homogeneity (Fig. 4). All LARC/MIP-3 immunoreactivity
specifically bound to the heparin-Sepharose column, but was not
separated from other chemokines present, such as MIP-1, MIP-1ß,
ENA-78, MCP-1, and IL-8 (data not shown). After subsequent cation
exchange chromatography, LARC/MIP-3 eluted over a broad range
(0.55–0.90 M NaCl), indicating additional heterogeneity of
leukocyte-derived LARC/MIP-3 (Fig. 4A). The LARC/MIP-3
immunoreactivity was not due to cross-reactivity with other abundantly
present chemokines such as MCP-1, as can be seen by its distinct
elution pattern (Fig. 4A). Final purification of
leukocyte-derived LARC/MIP-3 by RP-HPLC revealed two separate peaks
of LARC/MIP-3 protein (Fig. 4B). The first peak (fraction
70) eluting at 34% acetonitrile corresponded to a 6.0-kDa protein upon
SDS-PAGE (Fig. 5A). Its
LARC/MIP-3 authenticity was confirmed by immunoblotting in parallel
with rLARC/MIP-3 (Fig. 5B). However, this LARC/MIP-3
form appeared to be underestimated in the ELISA, indicating a
biochemical difference from the rLARC/MIP-3 used as a standard. The
second, major LARC/MIP-3 peak (fractions 73 and 74) eluting on
RP-HPLC at 35% acetonitrile (Fig. 4B) clearly contained a
5- to 5.5-kDa protein doublet (Fig. 5A), which was confirmed
to be LARC/MIP-3 by immunoblotting (Fig. 5B). Sufficient
LARC/MIP-3 protein was available from this second LARC/MIP-3 peak
to demonstrate again by amino acid sequence analysis the presence of
NH2-terminal heterogeneity identical with that
for LARC/MIP-3 forms recovered from THP-1 cells. However, this minor
difference (one amino acid) in length at the NH2
terminus could not explain the LARC/MIP-3 protein doublets (5 and
5.5 kDa, fractions 73–74) isolated from PBMC.
|
|
, RP-HPLC fractions were subjected to ion trap mass
spectrometry. It can be seen from the deconvoluted spectra (Fig. 6A) that 6.0-kDa LARC/MIP-3
(fraction 70) from leukocytes is composed of proteins for which the
average relative molecular mass corresponds to intact
LARC/MIP-31–70 and the truncated
LARC/MIP-32–70 and
LARC/MIP-31–69 forms, missing one residue at the
NH2 and COOH terminus, respectively. Indeed, the
average relative molecular mass (7895.1 ± 1.0) of
LARC/MIP-31–69 fully corresponded to the
theoretical value (Mr, 7894.3) of
LARC/MIP-3 missing the COOH-terminal Met. Because the experimentally
obtained average relative molecular masses for
LARC/MIP-31–70 and
LARC/MIP-32–70 were about 16 ± 1 Da
higher than the theoretical masses, in all probability the
COOH-terminal Met residue is oxidized. The composition of the 5- to
5.5-kDa LARC/MIP-3 doublet (fraction 73) was found to be even more
complex in COOH-terminal processing (Fig. 6B).
LARC/MIP-31–67 and
LARC/MIP-31–64, missing three and six
COOH-terminal residues, respectively, were the predominant
LARC/MIP-3 forms in this fraction. In addition, their corresponding
forms lacking the NH2-terminal Ala residue, i.e.,
LARC/MIP-32–67 and
LARC/MIP-32–64 were present. Finally, two
additional LARC/MIP-3 forms were discovered, of which the obtained
average relative molecular masses (7713.8 ± 1.0 and 7360.2
± 1.0, respectively) did not correspond to the theoretical value of
any intact or truncated LARC/MIP-3. These minor forms showed,
respectively, 132.2 ± 1.0 and 134.7 ± 1.0 Da of
difference in molecular mass from
LARC/MIP-32–67 and
LARC/MIP-32–64, which may be explained by
O-glycosylation, e.g., with a single ribose. It should be
noted that intact LARC/MIP-31–70, without any
minor modification such as an oxidized COOH-terminal Met and with a
molecular mass corresponding to the theoretical one, was not recovered
from any of the PBMC- or THP-1 cell-derived preparations tested. In
view of their physical separation (RP-HPLC elution pattern) and their
different compositions (molecular mass), the first and second RP-HPLC
peaks of leukocyte-derived LARC/MIP-3 were further compared at the
biological level.
|
isoforms
Natural LARC/MIP-3 was tested for its capacity to mobilize
intracellular calcium in HEK 293 cells transfected with CCR6, the
single receptor for LARC/MIP-3. Fig. 7A indicates that both natural
6-kDa LARC/MIP-3 (RP-HPLC fraction 70) and 5- to 5.5-kDa
LARC/MIP-3 (RP-HPLC fraction 73) could dose-dependently induce
increases in [Ca2+]i;
1–3 ng/ml was the minimal effective concentration. As a control,
rLARC/MIP-3 was already active at 3–10 ng/ml. It must therefore be
concluded that the natural LARC/MIP-3 isoforms that differ in
COOH-terminal truncation (fraction 70 vs fraction 73) were equally
potent in exerting calcium mobilization via CCR6.
|
has been reported to chemoattract lymphocytes,
natural LARC/MIP-3 was tested in the microchamber migration assay
using CD3+ lymphocytes freshly purified from
peripheral blood. Fig. 7B shows that the 5- to 5.5-kDa
LARC/MIP-3 isoforms were already chemotactic for lymphocytes at
10 ng/ml, whereas rLARC/MIP-3 was still active at 30 ng/ml. In
view of the fact that the natural LARC/MIP-3 preparations are still
mixtures of various isoforms, minor differences in chemotactic potency
cannot be excluded. |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
) and to bacterial
and viral products (1, 37). These inducible chemokines are
mainly responsible for the recruitment of phagocytes to sites of
inflammation or infection. Other chemokines, such as stromal
cell-derived factor-1, are secreted constitutively and are probably
more implicated in trafficking of leukocytes under physiologic
conditions. However, many chemokines can be classified in both
categories (37). LARC/MIP-3, also designated Exodus and
CCL20 (following a new classification system), is such a recently
discovered C-C chemokine (3, 4, 5, 10) that stimulates
migration of monocyte-derived and CD34+-derived
immature dendritic cells as well as of memory T cells through its
receptor CCR6 (3, 11, 12, 13, 14, 15, 16). LARC/MIP-3 mRNA is expressed
in various tissues, including lymphoid organs (3, 4, 5, 11),
and occurs in different transcripts (3, 4). However, the
regulated production of LARC/MIP-3 protein in various cell types as
well as the biochemical and biological characteristics of natural
LARC/MIP-3 protein isoforms have not yet been evaluated.
In this study we report the induction of LARC/MIP-3 protein in
normal and transformed cell types as measured by a specific and
sensitive ELISA. It was found that the induction of LARC/MIP-3 is
differently regulated in skin fibroblasts and PBMC, which responded
best to dsRNA and LPS, respectively. In fibroblasts, LARC/MIP-3 was
coinduced with the proinflammatory chemokines IL-8 and MCP-1
(36) by dsRNA, IL-1ß, and measles virus. In leukocytes,
MCP-1 was best induced by plant (Con A) and viral (dsRNA) products,
whereas LARC/MIP-3 was predominantly induced by bacterial endotoxin.
This corresponds with the reported detection of LARC/MIP-3 mRNA in
PBMC after induction with LPS (5). The production levels
of LARC/MIP-3 protein in normal fibroblasts and leukocytes were
about 100-fold lower than those of IL-8 or MCP-1. However a 3- to
10-fold increase in LARC/MIP-3 protein expression was readily
reached after appropriate induction of these cells.
In IL-1ß-stimulated HEp-2 epidermal carcinoma cells and PMA-treated
THP-1 monocytic cells, 3-fold higher levels of LARC/MIP-3 were
obtained than in normal cells, indicative of a potential role for
LARC/MIP-3 in tumor biology. In this respect, monocytic (U937 and
THP-1) cells and Bowes melanoma cells were reported to contain
increased mRNA levels of LARC/MIP-3 after PMA treatment (3, 5). The finding that epithelial cells are good producers of
LARC/MIP-3 in response to inflammatory stimuli such as IL-1ß fits
with the reported detection of LARC/MIP-3 mRNA in epithelial cells
of the appendix and in pancreatic adenocarcinoma cells (24, 26). In addition, in situ hybridization demonstrated the
presence of LARC/MIP-3 mRNA in epithelial crypts of inflamed tonsils
(13). This indicates that immature dendritic cells are
possibly chemoattracted by LARC/MIP-3 produced by the epithelium to
infiltrate the site of inflammation for Ag uptake. Indeed, immature
dendritic cells derived from CD34+ hemopoietic
progenitor cells or from monocytes, but not mature dendritic cells and
monocytes, are chemotactically responsive to LARC/MIP-3 (11, 13, 14, 15, 16). Expression of LARC/MIP-3 mRNA has also been observed
in lymphoid tissues such as peripheral lymph nodes and thymus, in
addition to liver, lung, PBMC, fetal lung, and fetal liver (3, 4, 5, 11). Finally, LARC/MIP-3 mRNA expression can be
down-regulated in activated monocytes by the anti-inflammatory
cytokine IL-10 (4).
Until the present, little has been known about the molecular
heterogeneity of LARC/MIP-3 protein. Although messages of different
sizes have been demonstrated for LARC/MIP-3 (3, 4), it
is not clear how this is reflected at the protein level. This study
demonstrates that the bulk of LARC/MIP-3, derived from either normal
leukocytes or tumor cells, is secreted as two protein isoforms that
differ in one additional amino acid at the NH2
terminus. This NH2-terminal truncation at the
protein level is in agreement with published data showing that some of
the ESTs containing the coding region for human or mouse LARC/MIP-3
lack the codon for the Ala27 residue (3, 5, 11, 26, 38). Inspection of the genomic sequence of murine
LARC/MIP-3 revealed two potential splice acceptor sites in the
boundary of the first intron and the second exon. As the resulting two
species of transcripts corresponded exactly to those for human
LARC/MIP-3 with and without the codon for the
Ala27 residue, and both transcripts appeared to
be expressed in various mouse tissues, it was concluded that such
LARC/MIP-3 heterogeneity at the transcriptional level is due to
alternative splicing in both humans and mice (26). In
addition, post-translational modification of LARC/MIP-3 protein is
implicated. Indeed, mass spectrometry on LARC/MIP-3 from stimulated
PBMC and THP-1 cells has revealed the existence of COOH-terminal
processing, generating LARC/MIP-3 with an oxidized Met or truncated
isoforms missing one, three, or six residues. Although it was
impossible to further fractionate these isoforms chromatographically
and hence to test these as individual molecules, it can indirectly be
deduced that neither NH2-terminal nor
COOH-terminal processing has significant consequences for CCR6 receptor
recognition. Indeed, it was demonstrated that separated PBMC-derived
LARC/MIP-3 isoforms that differ in COOH-terminal truncation (RP-HPLC
fraction 70 vs fraction 73) were equally potent in mobilizing
intracellular calcium (Fig. 7A). In addition, intact
recombinant LARC/MIP-3 has been shown to be only 2-fold more active
than the truncated isoform missing the
NH2-terminal Ala in chemoattracting T cells
(11). Furthermore, in chemotaxis assays using mononuclear
cells it was demonstrated that the two synthetic
NH2-terminal isoforms of LARC/MIP-3 were
nearly equipotent (5). This is in contrast with most
chemokines, which are rather sensitive to
NH2-terminal truncation, e.g., by CD26/dipeptidyl
peptidase IV, resulting in partial or complete loss of receptor
binding, signaling, and chemotactic capacity (27). Because
LARC/MIP-3 has no Pro or Ala at the penultimate position, it cannot
be a substrate for CD26. Nevertheless, CD26 has been reported to be
coexpressed with CCR6 on memory T lymphocytes (12).
Because the ELISA for LARC/MIP-3, like other chemokine ELISAs, does
not discriminate between intact and NH2- or
COOH-terminally truncated forms, it is difficult to say whether a
particular isoform is produced in response to a particular stimulus or
under specific pathologic conditions. Such evidence can only be
provided by production and purification of chemokines for each
induction condition tested, followed by identification of the purified
isoforms through mass spectrometry and sequencing. Complete
purification of LARC/MIP-3 isoforms derived from monocytic THP-1
cells (stimulated with PMA) and from PBMC (induced by LPS and Con A)
demonstrated the presence of equal amounts of the two
NH2-terminally processed forms of LARC/MIP-3.
Although based on rather limited experience, it was also noticed that
the COOH-terminally truncated forms were only recovered from PBMC and
not from THP-1 cells. It is not clear, however, whether this reflects a
difference between normal vs tumor cells in producing certain isoforms,
because PBMC contain a mixture of cell types and possibly a higher
content of proteolytic activity. Similarly, relatively more truncated
isoforms of the chemokines GRO and ENA-78 were recovered when isolated
from PBMC compared with tissue or tumor cells (28).
It can be concluded that LARC/MIP-3 not only acts as a
constitutively expressed chemokine involved in the trafficking of
dendritic cells and lymphocytes. Indeed, this study clearly indicates
that the production of LARC/MIP-3 is positively regulated by
inflammatory signals such as cytokines and micro-organisms.
Furthermore, neutrophils can be induced by cytokines to express CCR6
and to migrate in response to LARC/MIP-3 (25). This
latter is in agreement with the initial observation that LARC/MIP-3
was weakly active on human neutrophils (3). Like many
inflammatory chemokines (e.g., MCP-1, IL-8), LARC/MIP-3 was
inducible in most cell types, including epithelial cells, fibroblasts,
leukocytes, and tumor cells, indicative of its possible role in various
disease states. However, only a limited parallelism in inducibility
with other chemokines was obtained at several levels. Epithelial cells
were the best cell sources for LARC/MIP-3, whereas fibroblasts and
leukocytes were superior for MCP-1 and IL-8. Secondly, the amount of
LARC/MIP-3 produced was much lower than that of IL-8 and MCP-1, but
was comparable to those of MCP-2 (31) and MCP-3
(39). Finally, the optimal inducers for LARC/MIP-3
production were different from those of other chemokines. This further
illustrates that the chemokine network is only apparently redundant and
that LARC/MIP-3 has at least in part a specific role in the immune
response. This study confirms that natural LARC/MIP-3 exerts
chemotactic activity on lymphocytes and mobilizes intracellular calcium
via CCR6. Additional regulation of activity by post-translational
processing, as described for many inflammatory chemokines, seems not to
apply for LARC/MIP-3, because the various natural molecular forms of
this chemokine behave similarly in signaling through their receptor
CCR6. However, this does not exclude minor biological differences at
the level of cell migration, as post-translational modifications can
differently alter the chemotactic and calcium-mobilizing capacities of
chemokines. The finding that ß-defensins can compete for CCR6 binding
sites and exert chemotactic activity on the same target cells as
LARC/MIP-3 (21) indicates an additional but unique
mechanism of intervention via CCR6 during host defense against
microbial infection.
|
Acknowledgments |
|---|
ELISA. |
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Jo Van Damme, Laboratory of Molecular Immunology, Rega Institute for Medical Research, University of Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium.
3 Abbreviations used in this paper: ESTs, expressed sequence tags; [Ca2+]i, intracellular calcium concentration; CPG, controlled pore glass; EMEM, Eagle’s MEM with Earle’s salts; ENA-78, epithelial cell-derived neutrophil attractant-78; HEK, human embryonic kidney; LARC, liver and activation-regulated chemokine; MCP, monocyte chemotactic protein; MIP, macrophage inflammatory protein; poly(rI:rC), polyriboinosinic:polyribocytidylic acid; RP, reverse phase.
Received for publication March 15, 2000. Accepted for publication July 25, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and MIP-3ß. J. Immunol. 158:1033.[Abstract]
from lung dendritic cells. J. Exp. Med. 186:825.. J. Immunol. 162:186. and IL-4. J. Leukocyte Biol. 66:837.[Abstract]
is involved in the constitutive trafficking of epidermal Langerhans cells. J. Exp. Med. 190:1755. and is highly expressed in human dendritic cells. J. Exp. Med. 186:837.. Biochem. Biophys. Res. Commun. 236:212.[Medline]
/LARC/Exodus, a macrophage proinflammatory chemokine, and its CCR6 receptor in human pancreatic cancer. Int. J. Cancer 81:650.[Medline]
and GRO from tumor cells and leukocytes reveals NH2-terminal heterogeneity: functional comparison of different natural isoforms. Eur. J. Biochem. 260:421.[Medline]
. FEBS Lett. 440:188.[Medline]
/ß and interferon- reveals MCP-3 heterogeneity. Eur. J. Immunol. 29:678.[Medline]
This article has been cited by other articles:
|
|
 |
|
  M. Gouwy, S. Struyf, H. Verbeke, W. Put, P. Proost, G. Opdenakker, and J. Van Damme CC chemokine ligand-2 synergizes with the nonchemokine G protein-coupled receptor ligand fMLP in monocyte chemotaxis, and it cooperates with the TLR ligand LPS via induction of CXCL8 J. Leukoc. Biol., September 1, 2009; 86(3): 671 - 680. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Liston, R. E. Kohler, S. Townley, S. Haylock-Jacobs, I. Comerford, A. C. Caon, J. Webster, J. M. Harrison, J. Swann, I. Clark-Lewis, et al. Inhibition of CCR6 Function Reduces the Severity of Experimental Autoimmune Encephalomyelitis via Effects on the Priming Phase of the Immune Response J. Immunol., March 1, 2009; 182(5): 3121 - 3130. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Battaglia, S. Delfino, E. Merello, M. Puppo, R. Piva, L. Varesio, and M. C. Bosco Hypoxia transcriptionally induces macrophage-inflammatory protein-3{alpha}/CCL-20 in primary human mononuclear phagocytes through nuclear factor (NF)-{kappa}B J. Leukoc. Biol., March 1, 2008; 83(3): 648 - 662. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Hasan, L. Mazzucchelli, M. Liebi, M. Lis, R. E. Hunger, A. Tester, C. M. Overall, and M. Wolf Function of Liver Activation-Regulated Chemokine/CC Chemokine Ligand 20 Is Differently Affected by Cathepsin B and Cathepsin D Processing. J. Immunol., June 1, 2006; 176(11): 6512 - 6522. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Hausmann, F. Bataille, T. Spoettl, K. Schreiter, W. Falk, J. Schoelmerich, H. Herfarth, and G. Rogler Physiological Role of Macrophage Inflammatory Protein-3{alpha} Induction during Maturation of Intestinal Macrophages J. Immunol., August 1, 2005; 175(3): 1389 - 1398. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Bonecchi, M. Locati, E. Galliera, M. Vulcano, M. Sironi, A. M. Fra, M. Gobbi, A. Vecchi, S. Sozzani, B. Haribabu, et al. Differential Recognition and Scavenging of Native and Truncated Macrophage-Derived Chemokine (Macrophage-Derived Chemokine/CC Chemokine Ligand 22) by the D6 Decoy Receptor J. Immunol., April 15, 2004; 172(8): 4972 - 4976. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. J. Hensbergen, D. Verzijl, C. I. A. Balog, R. Dijkman, R. C. van der Schors, E. M. H. van der Raaij-Helmer, M. J. A. van der Plas, R. Leurs, A. M. Deelder, M. J. Smit, et al. Furin Is a Chemokine-modifying Enzyme: IN VITRO AND IN VIVO PROCESSING OF CXCL10 GENERATES A C-TERMINALLY TRUNCATED CHEMOKINE RETAINING FULL ACTIVITY J. Biol. Chem., April 2, 2004; 279(14): 13402 - 13411. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Sun, K. Nasu, J. Fukuda, S. Mine, M. Nishida, and I. Miyakawa Expression of macrophage inflammatory protein-3{alpha} in an endometrial epithelial cell line, HHUA, and cultured human endometrial stromal cells Mol. Hum. Reprod., October 1, 2002; 8(10): 930 - 933. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. M. Hoover, C. Boulegue, D. Yang, J. J. Oppenheim, K. Tucker, W. Lu, and J. Lubkowski The Structure of Human Macrophage Inflammatory Protein-3alpha /CCL20. LINKING ANTIMICROBIAL AND CC CHEMOKINE RECEPTOR-6-BINDING ACTIVITIES WITH HUMAN beta -DEFENSINS J. Biol. Chem., September 27, 2002; 277(40): 37647 - 37654. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Schutyser, S. Struyf, P. Proost, G. Opdenakker, G. Laureys, B. Verhasselt, L. Peperstraete, I. Van de Putte, A. Saccani, P. Allavena, et al. Identification of Biologically Active Chemokine Isoforms from Ascitic Fluid and Elevated Levels of CCL18/Pulmonary and Activation-regulated Chemokine in Ovarian Carcinoma J. Biol. Chem., June 28, 2002; 277(27): 24584 - 24593. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Chabaud, G. Page, and P. Miossec Enhancing Effect of IL-1, IL-17, and TNF-{alpha} on Macrophage Inflammatory Protein-3{alpha} Production in Rheumatoid Arthritis: Regulation by Soluble Receptors and Th2 Cytokines J. Immunol., November 15, 2001; 167(10): 6015 - 6020. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. M. Perez-Canadillas, A. Zaballos, J. Gutierrez, R. Varona, F. Roncal, J. P. Albar, G. Marquez, and M. Bruix NMR Solution Structure of Murine CCL20/MIP-3alpha , a Chemokine That Specifically Chemoattracts Immature Dendritic Cells and Lymphocytes through Its Highly Specific Interaction with the beta -Chemokine Receptor CCR6 J. Biol. Chem., July 20, 2001; 276(30): 28372 - 28379. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
)
and MCP-1 (
) immunoreactivities were measured by ELISA.
