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Departments of
*
Internal Medicine and
Periodontology, Osaka Dental University, Hanazono-cho, Hirakata-shi, Osaka, Japan;
Department of Microbiology, Kinki University School of Medicine, Ohno-Higashi, Osaka-Sayama, Osaka, Japan;
§
Department of Hematology and Oncology, Kyoto University Graduate School of Medicine, Shogoinn-Kawara-cho, Sakyo-ku, Kyoto, Japan; and
¶
Division of Cellular and Gene Therapies, Center for Biologics Evaluation Research, Food and Drug Administration, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-activated HUVECs.
Moreover, soluble-fractalkine enhanced adhesion of THP-1 cells to
fibronectin and ICAM-1 in a dose-dependent manner. Pertussis toxin, an
inhibitor of Gi, inhibited the fractalkine-mediated
enhancement of THP-1 cell adhesion to fibronectin and ICAM-1. Finally,
we found that soluble-fractalkine also enhanced adhesion of freshly
separated monocytes to fibronectin and ICAM-1. These results indicate
that fractalkine may induce firm adhesion between monocytes and
endothelial cells not only through an intrinsic adhesion function
itself, but also through activation of integrin avidity for their
ligands. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Chemokines were first described as chemoattractant cytokines synthesized at sites of inflammation and are the major regulatory proteins for leukocyte recruitment and trafficking (10, 11, 12, 13, 14, 15). Chemokines are subdivided into four subfamilies, C, CC, CXC, and CX3C chemokine, based on the number and spacing of the first two cysteines in a conserved cysteine structural motif. Different chemokine classes tend to exhibit different ranges of leukocyte specificity. The CXC chemokines seem biased in targeting neutrophils and to a lesser extent lymphocytes, whereas the CC chemokines mainly target monocytes, lymphocytes, basophils, and eosinophils with varying selectivity. The C chemokine is reported to act preferentially on lymphocytes (11, 15, 16).
Fractalkine, a recently identified chemokine, has a unique
architecture, a Cys-X-X-X-Cys chemokine domain presented on top of an
extended mucin-like stalk as a part of transmembrane protein, and is
expressed in a membrane-bound form on TNF-- and IL-1-activated
endothelial cells
(ECs)3 (17, 18). We have previously reported that an "orphan" receptor,
V28 (19, 20), is a receptor for fractalkine
(CX3CR1) and have demonstrated that it is expressed on most
of CD16+ NK cells and the majority of
CD14+ monocytes (21). Since it has
been reported that a myeloid cell line, THP-1, exhibits high expression
of V28 mRNA (20), we examined the surface expression of
CX3CR1 on THP-1 cells and investigated the functional
effects of fractalkine on ß1 and
ß2 integrin-mediated adhesion of THP-1 cells to
their respective ligands. We report here that THP-1 cells express
CX3CR1 and bind to immobilized fractalkine, indicating that
fractalkine can function as an adhesion molecule in THP-1 cells. We
also observed that soluble fractalkine (s-fractalkine) enhanced the
binding of THP-1 cells to immobilized fibronectin and ICAM-1. This
enhancement was efficiently inhibited by a Gi
inhibitor, pertussis toxin (PTX), suggesting that CX3CR1
transduces signals to increase integrin avidity through G protein
activation. Furthermore, THP-1 cells adhered to a
fractalkine-transfected ECV304 cells (FRK-ECV) more efficiently than to
control ECV304 cells. THP-1 cells also adhered to TNF--activated
HUVECs, and this adhesion could be partially inhibited by s-fractalkine
as well as Abs to integrins. These results suggest that fractalkine
might be a key molecule for the interaction of monocytes and ECs
through both integrin-dependent and -independent mechanisms.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Hybridomas producing mAbs against CD18 (TS1/18), CD11a (TS1/22), CD2 (TS2/18), and CD3 (OKT3) were purchased from the American Type Culture Collection (Manassas, VA), and mAbs were purified as described previously (22, 23). Anti-CD14 and anti-CD19 mAbs were purchased from Immunotech (Marseille, France). FITC- or PE-conjugated Abs against CD3, CD14, CD16, CD21, and ICAM-1 were obtained from Becton Dickinson (Mountain View, CA). Monoclonal anti-CD106 (VCAM-1) Ab and anti-CD54 (ICAM-1) Ab were purchased from Ancell (Bayport, MN) and Serotec (Kidlington, Oxford, U.K.), respectively. Monoclonal anti-CD29 (ß1 integrin) Ab was purchased from Upstate Biotechnology (Lake Placid, NY). Monoclonal anti-alkaline phosphatase Ab and recombinant human MCP-1 were purchased from Genzyme (Cambridge, MA). Affinity-purified Ab against CX3CR1 and recombinant soluble ICAM-1 were kindly provided by Dr. Yiyang Xia (Torrey Pines Biolabs, San Diego, CA) and Dr. Takashi Kei Kishimoto (Boehringer Ingelheim Pharmaceuticals, Ridgelfield, CT), respectively. Recombinant human RANTES was purchased from Intergen (Purchase, NY). Human fibronectin, laminin, and collagen types I and IV were obtained from Becton Dickinson (Bedford, MA). Acetoxymethyl ester of calcein (calcein-AM) was purchased from Wako Pure Chemical Industries (Osaka, Japan).
Production of recombinant proteins
s-Fractalkine and fractalkine-SEAP (secreted form of placental alkaline phosphatase), CX3C-SEAP, and mucin-SEAP fusion proteins were prepared by using a baculovirus expression system as described previously (21). Briefly, to express fractalkine-SEAP, the DNA fragment encoding fractalkine was amplified from fractalkine cDNA by PCR using 5' SalI-fractalkine primer (+5'-CGCGTCGACTCAGCCATGGCTCCGATATCT-3') and 3' fractalkine-XbaI primer (-5'-CGCTCTAGAGGTGGCT GCCTGGGCGTCAGG-3') and subcloned into pDREF-SEAP (His)6-Hyg vector as described previously (24). CX3CSEAP was similarly generated using 5' SalI-fractalkine primer and 3' fractalkine-XbaI-2 primer (-5'-CGCTCTAGATAGGGCAGCAGCCTGGCGGTC-3'). For generation of mucin-SEAP, the DNA fragment encoding SalI-oncostatin M signal sequence-XbaI-fractalkine lacking the chemokine domain NheI was amplified by three-step PCR using 5' SalI-OMC-fractalkine primer (+5'-CTG TTTCCATGCATGGCGAGCATGTCT-3') and 3' fractalkine-NheI primer (-5'-CGCGCTAGCGGTGGCAGCCTGGGCGTCAGG-3'). After 3–4 days, the supernatants were collected. For one-step affinity purification, supernatants were applied to 1 ml of Hisbond resin (Qiagen, Hilden, Germany). After washing, bound recombinant proteins were eluted with 100 mM imidazol. s-Fractalkine with a tag of six histidine residues, (His)6, at its C terminus was prepared by using a baculovirus expression system. The cDNA fragment encoding the extracellular domain of fractalkine was subcloned into the SalI-XbaI sites of the modified pFastBac1 baculovirus transfer vector (Life Technologies, Rockville, MD) to express fractalkine as a soluble fusion protein with Ser-Arg-Ser-Ser-Gly-(His)6. The recombinant bacmids were generated in Escherichia coli DH10Bac and transfected into Spodoptera frugiperda Sf9 cells using Lipofectin (Life Technologies) to obtain the recombinant viruses. For expression of the recombinant fractalkine-(His)6, Trichoplusia ni BTI-TN-5B1-4 cells were infected with the recombinant viruses at a multiplicity of infection of 10–20. The culture supernatants collected 2 days after infection were applied to a 1 ml Hisbond resin (Qiagen). After washing, bound fractalkine-(His)6 was eluted with 100 mM imidazol. Protein concentration was determined by the bicinchoninic acid kit (Pierce, Rockford, IL). The concentration of each recombinant protein was determined by a sandwich-type ELISA as described previously (24).
Cells and cell culture
Hut78, Hut102, Raji, Daudi, KU812, MEG1 cells, and ECV304
cells4 were obtained
from the American Type Culture Collection. THP-1, Molt4, Jur-kat,
HL60, U937, and K562 cells were kindly provided by Dr. M. Maeda (Kyoto
University, Kyoto, Japan). PBMC were isolated from samples of venous
blood from consenting healthy volunteers by Ficoll-Paque (Pharmacia
Biotech, Uppsala, Sweden) density-gradient centrifugation. Monocytes
were isolated by negative selection using a mixture of anti-CD3,
anti-CD19, and anti-CD16 mAbs and immunomagnetic beads
(PerSeptive Diagnostics, Cambridge, MA) to deplete T cells, B cells,
and NK cells, respectively, as described previously (25, 26). For stable expression of membrane-bound fractalkine in
ECV304 cells, the expression plasmid pCAGG-Neo-fractalkine-1 was
transfected into ECV304 by LipofectAMINE (Life Technologies). After
selection with 800 µg/ml of G418 for 1–2 wk, drug-resistant cells
were pooled as described previously (21). HUVECs were
obtained from Iwaki (Chiba, Japan) and maintained in endothelial cell
growth medium (10 ng/ml human epidermal growth factor, 1.0 µg/ml
hydrocortisone, 50 µg/ml gentamicin, 50 µg/ml amphotericin B, 12
µg/ml bovine brain extract, and 2% FBS), as recommended by the
manufacturer. HUVECs were stimulated with TNF- (100 nM; Genzyme) for
16 h and used in the THP-1 adhesion assays.
Adhesion assay
The assay to measure adhesion to immobilized fractalkine was performed as described previously (21). Briefly, each well of 48-well plates was coated with 50 µl of anti-SEAP Ab (10 µg/ml) in 50 mM Tris-HCl (pH 9.5) at 4°C overnight. After washing with PBS, nonspecific binding sites were blocked with adhesion buffer (RPMI 1640, 1% BSA, and 20 mM HEPES (pH 7.4)). SEAP fusion proteins (10 nM) were added to wells, and the plates were incubated for 2 h at room temperature and washed extensively. Alternatively, to assess cell adhesion to immobilized fibronectin and ICAM-1, each well was coated with 50 µg/ml of fibronectin or 100 ng/ml of soluble ICAM-1 at 4°C overnight. THP-1 cells were fluorescently labeled by incubation with calcein-AM and added to each well (1–2 x 104 cells/well) in a final volume of 100 µl and incubated for 30 min at 37°C. After removal of nonadherent cells, fluorescence was measured using a Wallac 1420 ARVO fluoroscan (Pharmacia Biotech) using excitation and emission wavelengths of 496 and 520 nm, respectively. To assess cell adhesion to endothelial cells, FRK-ECV and control ECV were seeded at 2 x 104 cells/well in 48-well culture plates and cultured overnight to form a confluent monolayer. In blocking experiments using Abs and s-frac-talkine, THP-1 cells were pretreated with saturating amounts of mAbs or 10 nM of s-fractalkine for 30 min at 4°C before the assay.
Northern blot
Northern blots were prepared as described previously (24). Briefly, total RNAs were prepared from various cell lines using Trizol (Life Technologies). RNA samples (5 µg) were fractionated by electrophoresis on a 1% agarose gel containing 0.66 M formaldehyde. Gels were blotted onto a filter membrane (Hybond N+; Amersham Japan, Tokyo, Japan). The probe was the Sma-PstI fragment of clone D3A of CX3CR1 (27). Hybridization was conducted at 65°C in QuickHyb solution (Stratagene, La Jolla, CA) with probes labeled with 32P using Prime it II (Stratagene). After washing at 55°C with 0.2x SSC and 0.1% SDS, filters were exposed to x-ray films at -80°C with an intensifying screen.
Receptor-binding assay
Cells were incubated for 1 h at 16°C with increasing concentrations of SEAP fusion proteins in the presence or absence of 200 nM s-fractalkine in 200 µl of RPMI 1640 containing 20 mM HEPES (pH 7.4), 1% BSA, and 0.02% sodium azide. After incubation, cells were washed, lysed in 50 µl of 10 mM Tris-HCl (pH 8.0), 1% Triton X-100, and heated at 65°C for 10 min to inactivate cellular phosphatases. Lysates were collected by centrifugation, and AP activity in 10 µl of lysate was determined by the chemiluminescent assay (21). All assays were done in duplicate. Binding data were analyzed using the Ligand program (GraphPad Software, San Diego, CA).
FACS analysis
For assessment of membrane markers by immunofluorescence, control-ECV or FRK-ECV cells were stained directly with FITC-labeled reagents or with an unlabeled Ab and FITC-goat [F(ab)2'] anti-mouse IgG (Becton Dickinson, Mountain View, CA). Cells were then analyzed on a FACSCaliber (Becton Dickinson).
Statistical analysis
All data were expressed as means ± SEM. Differences between groups were examined for statistical significance using the Student’s t test for unpaired data and paired t test for paired data. A p value <0.05 denoted the presence of a statistically significant difference.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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First, we examined the expression of CX3CR1 mRNA (V28)
in various types of human hematopoietic cell lines. As shown in Fig. 1
, CX3CR1 mRNA was highly
expressed by THP-1 cells and at lower levels by U937 and HL60 cells,
all of which are believed to represent cells of the monocyte lineage.
In contrast, expression was not detectable in any of the seven lymphoid
cell lines examined nor in a basophilic line or in K562, an
undifferentiated myelomonocytic cell line. Since it has been reported
that chemokines fused with SEAP retain their ability to bind
specifically to their cell surface receptors, whereas assay of the
phosphatase activity allows quantitative determination of specific
binding (24, 28), we conducted fractalkine-SEAP-binding
experiments using increasing concentrations of fractalkine-SEAP to
characterize CX3CR1 on THP-1 cells. As shown in Fig. 2, a single class of receptors was
revealed on THP-1 cells. It was expressed at about 400 sites per cell
and exhibited a Kd of 30 pM.
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Next, we examined the adhesion of THP-1 cells to immobilized
fractalkine using plates on which fractalkine-SEAP, mucin-SEAP lacking
the CX3C chemokine domain and CX3C-SEAP lacking
the mucin domain, or SEAP alone were immobilized using wells pretreated
with anti-SEAP Ab. Calcein-AM-labeled THP-1 cells (1–2 x
104 cells/well) were applied to each well for 30
min at room temperature. After removal of nonadherent cells, the
fluorescence was measured. As shown in Fig. 3A, THP-1 cells efficiently
adhered to immobilized fractalkine-SEAP, but not to mucin-SEAP,
CX3C-SEAP, or SEAP alone. Since specific
anti-fractalkine-neutralizing mAbs are not available at present, we
exploited inhibitory actions of s-fractalkine to dissect the
interaction of fractalkine and CX3CR1 in inducing cell
adhesion (21). We found that s-fractalkine as well as
CX3C-SEAP and fractalkine-SEAP, but not MCP-1 and RANTES
nor Abs against integrins (CD18 and CD29), efficiently inhibited
adhesion of THP-1 cells to immobilized fractalkine (Fig. 3, B and C), supporting the conclusion that adhesion
of THP-1 cells to fractalkine is specific to the interaction between
CX3CR1 and fractalkine. These results indicate that
CX3CR1 on THP-1 cells recognize the intact fractalkine and
that fractalkine can function as an adhesion molecule on THP-1 cells,
as previously reported in other cell systems (21, 29, 30).
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The regulation of integrin-dependent adhesion and de-adhesion is
important in leukocyte cell to cell and cell to matrix interactions
in immunity and inflammation. For firm cell to cell adhesion
through ß1 or ß2
integrins and their ligands, triggering of integrin activation by
selectins and/or chemokines is required (31, 32). Since it
has been reported that CC chemokines, including MCP-1, RANTES,
and MIP-1, modulate the avidity of
ß1 and ß2 integrins on
T cells, monocytes, and eosinophils (33, 34, 35, 36), we examined
whether fractalkine also modulates adhesion of THP-1 cells to integrin
ligands. THP-1 cells bind to fibronectin and ICAM-1 in a dose- and
time-dependent manner up to 60 min, but not to laminin and collagen
type I and IV (data not shown). As shown in Fig. 4, s-fractalkine as well as MCP-1 and
RANTES markedly enhanced adhesion of THP-1 cells to fibronectin and
ICAM-1 in a dose-dependent manner, and the maximum response was
observed at 10 nM of fractalkine. These data indicate that fractalkine
also functions as a regulator of integrin avidity, as reported in other
chemokines (33, 34, 36, 37).
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It has been reported that chemokine receptors identified to date,
including CX3CR1, all manifest a seven-transmembrane G
protein-linked architecture and transduce signals that lead to
cytoskeletal reorganization, integrin activation, and other functions
leading to increased adhesion and migration of the cells
(12, 13, 14, 15). To examine the involvement of G
protein-dependent signaling in fractalkine-mediated enhancement of
THP-1 cell adhesion to fibronectin and ICAM-1, cells were pretreated
with a Gi inhibitor, PTX, for 30 min at room
temperature, and then allowed to interact with immobilized fibronectin
and ICAM-1 in the presence of either MCP-1, RANTES, or s-fractalkine.
As shown in Fig. 5, enhancement of THP-1
cell adhesion induced by s-fractalkine as well as RANTES and MCP-1 was
completely prevented by PTX.
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-stimulated HUVECs
The membrane-bound fractalkine can be markedly induced on primary
endothelial cells by proinflammatory cytokines such as TNF- and
IL-1 (17); this form supports the robust adhesion of
monocytes and T cells (21). However, cytokine-activated
HUVECs express high levels of ICAM-1 and VCAM-1, as well as
exhibiting de novo expression of fractalkine. To assess the role of
membrane-bound fractalkine in the adhesion of THP-1 cells to
endothelial cells in a more direct manner, we used the ECV304 cell line
(control-ECV) to establish a fractalkine-transfected subline (FRK-ECV),
which constitutively expressed the membrane-bound fractalkine without
significant changes in the expression of other adhesion molecules (Fig. 6). We then used this subline to examine
adhesion of THP-1 cells to FRK-ECV and control-ECV. As shown in Fig. 7A, THP-1 cells adhered to
FRK-ECV more efficiently than to control-ECV. Moreover, s-fractalkine,
used as a competitor, markedly reduced the adhesion between THP-1 cells
and FRK-ECV cells to the basal levels of adhesion between THP-1 cells
and control-ECV, whereas mAbs against CD18 and CD29 only partially
inhibited adhesion of THP-1 cells to FRK-ECV. Similarly, MCP-1 and
RANTES, but not s-fractalkine, have no inhibitory effects on the
interaction between THP-1 cells and FRK-ECV (Fig. 7B). These
findings support the conclusion that physical interaction of the
membrane-bound fractalkine on ECs and CX3CR1 on THP-1 cells
can directly mediate adhesion between ECs and THP-1 cells independent
of integrins, as previously reported (21, 29, 30).
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-stimulated HUVECs. As shown in Fig. 8, THP-1 cells markedly adhered to
TNF--stimulated HUVECs and s-fractalkine significantly inhibited the
adhesion. All combinations of anti-CD18, anti-CD29 mAbs, and
s-fractalkine dramatically decreased THP-1 adhesion to near the levels
of resting HUVECs (Fig. 8), suggesting involvement of fractalkine as
well as integrins in the adhesion.
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Finally, we examined whether the interaction of fractalkine and
CX3CR1 supported cell adhesion and whether s-fractalkine
enhanced integrin-mediated cell adhesion in more natural setting using
freshly isolated monocytes. As shown in Fig. 9A, fresh monocytes
specifically adhered to the immobilized fractalkine, but not to
the truncated forms of fractalkine and control-SEAP.
s-fractalkine also enhanced monocyte adhesion to fibronectin and ICAM-1
(Fig. 9B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and 2), and
that THP-1 cells adhere to immobilized intact fract-alkine with no
requirement for the participation of other adhesion molecules, such as
integrins, as previously reported in fresh monocytes, NK cells, and
CD8+ T cells (Fig. 3).
Since CC chemokines, including MCP-1, MIP-1, and RANTES,
have been reported to increase the avidity of
ß1 and ß2 integrins in
T cells and monocytes (33, 34, 35, 36), we next investigated
whether s-fractalkine also modulates the avidity of integrins on THP-1
cells. s-Fractalkine as well as MCP-1 and RANTES enhanced adhesion of
THP-1 cells to fibronectin and ICAM-1 in a dose-dependent manner (Fig. 4) without altering surface expression of other adhesion molecules
(data not shown), suggesting that s-fractalkine stimulates the
development of a high-affinity state in the integrin molecules. It has
been reported that chemokine receptors identified to date, including
CX3CR1, all manifest a seven-transmembrane G protein-linked
architecture and transduce signals that lead to cytoskeletal
reorganization, integrin activation, and other functions leading to
increased adhesion and migration of the cells (12, 13, 14, 15).
Although the interaction between fractalkine and CX3CR1 is
G protein independent, one possible explanation for
fractalkine-mediated enhancement of THP-1 cell adhesion to integrin
ligands is that fractalkine might activate G proteins to enhance
integrin avidity. Our data demonstrating that PTX, an inhibitor of
Gi, completely inhibits the fractalkine-mediated
enhancement of THP-1 cell adhesion to fibronectin and ICAM-1 (Fig. 5)
support this possibility. Recently, Campbell et al. (30)
and Haskell et al. (38) reported that fractalkine failed
to increase the attachment of CX3CR1-expressing cells to
ICAM-1 and VCAM-1. The discrepancy with the current findings may be
attributable to the differences in the cells and ligands used in the
assays. Campbell et al. (30) used peripheral lymphocytes
after depletion of monocytes and Haskell et al. (38) has
examined the adhesion of CX3CR1-transfected embryonic
kidney cells to VCAM-1.
Endothelial cells lining venules at sites of inflammation respond to
inflammatory mediators, which leads to expression of adhesion molecules
on their cell surface (4, 5, 6, 7, 39). Since the membrane-bound
fractalkine is expressed on EC membranes after stimulation by TNF-
or IL-1, we hypothesized that fractalkine may be involved in adhesion
between monocytes and ECs. However, the cytokine-activated HUVECs
express high levels of ICAM-1 and VCAM-1 as well as exhibiting de novo
expression of fractalkine, producing a highly complex pattern of
adhesion pathways between monocytes and activated HUVECs. To assess the
role of membrane-bound fractalkine in the adhesion of THP-1 cells to
endothelial cells in a nonconfounded system, we have established a
fractalkine-transfected subline (FRK-ECV), which constitutively
expressed membrane-bound fractalkine without significant changes in
the expression of other adhesion molecules (Fig. 6). We observed that
THP-1 cells adhered to FRK-ECV more efficiently than to control-ECV.
Moreover, s-fractalkine, used as competitor, but not MCP-1 and RANTES,
markedly inhibited adhesion of THP-1 cells to FRK-ECV (Fig. 7B), whereas mAbs against ICAM-1 and VCAM-1 only partially
inhibited THP-1 cell adhesion to FRK-ECV (Fig. 7A).
Furthermore, we examined adhesion of THP-1 cells to resting or
TNF--stimulated HUVECs to evaluate the relative role of fractalkine
under more physiological conditions and found results consistent with
those we observed in FRK-ECV. Interestingly, all combinations of
anti-CD18, anti-CD29 mAbs, and s-fractalkine dramatically
decreased THP-1 adhesion to TNF--stimulated HUVECs to near the
levels of resting HUVECs, supporting the use of multiple adhesion
pathways between activated HUVECs and THP-1 cells (Fig. 8).
The recruitment and activation of leukocytes at sites of various kinds of pathologic processes or injury is a hallmark of inflammation, in which the endothelium plays a prominent role in the recruitment and emigration of circulating effector cells into inflammatory sites. For this reason, fractalkine was assayed for chemotactic activity on THP-1 cells. Although MCP-1 was found to be chemotactic for THP-1 cells, s-fractalkine has no significant effect on THP-1 cell chemotaxis (data not shown). Bazan et al. (17) have reported that s-fractalkine can mediate both chemotaxis and adhesion of monocytes, whereas Pan et al. (18) have reported that neither the chemokine-like domain nor the entire extracellular domain of fractalkine have chemotactic effects on human monocytes and THP-1 cells. We have previously reported that s-fractalkine induces transendothelial migration of monocytes with a smaller maximal effect than that seen with MCP-1 (1.5% vs 30.7%) (21). Since multiple classes of G proteins exist and each chemokine receptor couples to different types of G proteins, it is possible that activation of G proteins differ between cell types and classes of chemokines. In this regard, Al-Aoukaty et al. (40) have reported that fractalkine receptors are coupled to Gi and Gz in human NK cells. Overall, fractalkine seems to act to modulate adhesion of monocytes both through its own adhesion function and through the activation of integrins rather than as a haptotactic factor. The same effects of chemokines have been reported by Gerszten et al. (41), who reported that IL-8 triggers the firm adhesion of monocytes to vascular endothelium through integrin activation but has only minor chemotactic effects.
Endothelial cell damage has been identified in a variety of pathologic
states, including infectious diseases, atherosclerosis, transplantation
rejection, and autoimmunity, coinciding with a chronic macrophage
accumulation in the inflamed tissues (5, 39). Our data
suggest that fractalkine may be involved in vascular injury by
strengthening the adhesion between monocytes and ECs through at least
two pathways: an intrinsic adhesion function of fractalkine itself and
activation of integrin avidity for their respective ligands. Finally,
we demonstrated that s-fractalkine enhanced adhesion of freshly
separated monocytes to fibronectin and ICAM-1 (Fig. 9). Recently, Chen
et al. (42) have reported that a viral protein, vMIP-II,
encoded by human herpesvirus 8, has antagonistic activity for CC, CXC,
and CX3C chemokine receptors and anti-inflammatory
activity in experimental glomerulonephritis in a rat model system.
Although further studies are needed, our data suggest agents with
antagonistic activity for fractalkine may provide therapeutic benefit
for treating endothelial injury in pathologic states.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Hisanori Umehara, Department of Internal Medicine, Osaka Dental University, 8-1 Kuzuha Hanazono-cho, Hirakata-shi, Osaka 573-1121, Japan.
3 Abbreviations used in this paper: EC, endothelial cell; PTX, pertussis toxin; s, soluble, FRK-ECV; fractalkine-transfected ECV304 cell; MCP, monocyte chemoattractant protein; MIP, macrophage-inflammatory protein; SEAP, secreted form of placental alkaline phosphatase; calcein-AM, acetoxymethylester of calcein.
4 After completing the experiments using ECV304 cell lines and FRK-ECV as the endothelial cell line, American Type Culture Collection announced that ECV304 should be considered a variant of human bladder cancer line T-24 derived by cross-contamination.
Received for publication September 20, 1999. Accepted for publication February 8, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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4ß1 and 5ß1 integrin activity by CC chemokines in monocytes: implications for transendothelial chemotaxis. J. Cell Biol. 134:1063., MIP-3ß and fractalkine induce the locomotion and the mobilization of intracellular calcium, and activate the heterotrimeric G proteins in human natural killer cells. Immunology 95:618.[Medline]
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M. Popovic, Y. Laumonnier, L. Burysek, T. Syrovets, and T. Simmet Thrombin-induced expression of endothelial CX3CL1 potentiates monocyte CCL2 production and transendothelial migration J. Leukoc. Biol., July 1, 2008; 84(1): 215 - 223. [Abstract] [Full Text] [PDF]
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 C. Combadiere, S. Potteaux, M. Rodero, T. Simon, A. Pezard, B. Esposito, R. Merval, A. Proudfoot, A. Tedgui, and Z. Mallat Combined Inhibition of CCL2, CX3CR1, and CCR5 Abrogates Ly6Chi and Ly6Clo Monocytosis and Almost Abolishes Atherosclerosis in Hypercholesterolemic Mice Circulation, April 1, 2008; 117(13): 1649 - 1657. [Abstract] [Full Text] [PDF]
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W. L. Jamieson, S. Shimizu, J. A. D'Ambrosio, O. Meucci, and A. Fatatis CX3CR1 Is Expressed by Prostate Epithelial Cells and Androgens Regulate the Levels of CX3CL1/Fractalkine in the Bone Marrow: Potential Role in Prostate Cancer Bone Tropism Cancer Res., March 15, 2008; 68(6): 1715 - 1722. [Abstract] [Full Text] [PDF]
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C. Schulz, A. Schafer, M. Stolla, S. Kerstan, M. Lorenz, M.-L. von Bruhl, M. Schiemann, J. Bauersachs, T. Gloe, D. H. Busch, et al. Chemokine Fractalkine Mediates Leukocyte Recruitment to Inflammatory Endothelial Cells in Flowing Whole Blood: A Critical Role for P-Selectin Expressed on Activated Platelets Circulation, August 14, 2007; 116(7): 764 - 773. [Abstract] [Full Text] [PDF]
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 C. Auffray, D. Fogg, M. Garfa, G. Elain, O. Join-Lambert, S. Kayal, S. Sarnacki, A. Cumano, G. Lauvau, and F. Geissmann Monitoring of Blood Vessels and Tissues by a Population of Monocytes with Patrolling Behavior Science, August 3, 2007; 317(5838): 666 - 670. [Abstract] [Full Text] [PDF]
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 M. V. Ramos, G. C. Fernandez, N. Patey, P. Schierloh, R. Exeni, I. Grimoldi, G. Vallejo, C. Elias-Costa, M. del Carmen Sasiain, H. Trachtman, et al. Involvement of the fractalkine pathway in the pathogenesis of childhood hemolytic uremic syndrome Blood, March 15, 2007; 109(6): 2438 - 2445. [Abstract] [Full Text] [PDF]
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 A. M. Durkan, R. T. Alexander, G.-Y. Liu, M. Rui, G. Femia, and L. A. Robinson Expression and Targeting of CX3CL1 (Fractalkine) in Renal Tubular Epithelial Cells J. Am. Soc. Nephrol., January 1, 2007; 18(1): 74 - 83. [Abstract] [Full Text] [PDF]
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C. Lauro, M. Catalano, F. Trettel, F. Mainiero, M. T. Ciotti, F. Eusebi, and C. Limatola The Chemokine CX3CL1 Reduces Migration and Increases Adhesion of Neurons with Mechanisms Dependent on the beta1 Integrin Subunit J. Immunol., December 1, 2006; 177(11): 7599 - 7606. [Abstract] [Full Text] [PDF]
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V. Faure, C. Cerini, P. Paul, Y. Berland, F. Dignat-George, and P. Brunet The uremic solute p-cresol decreases leukocyte transendothelial migration in vitro Int. Immunol., October 1, 2006; 18(10): 1453 - 1459. [Abstract] [Full Text] [PDF]
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J. Barlic, Y. Zhang, J. F. Foley, and P. M. Murphy Oxidized Lipid-Driven Chemokine Receptor Switch, CCR2 to CX3CR1, Mediates Adhesion of Human Macrophages to Coronary Artery Smooth Muscle Cells Through a Peroxisome Proliferator-Activated Receptor {gamma}-Dependent Pathway Circulation, August 22, 2006; 114(8): 807 - 819. [Abstract] [Full Text] [PDF]
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S. R. Green, K. H. Han, Y. Chen, F. Almazan, I. F. Charo, Y. I. Miller, and O. Quehenberger The CC Chemokine MCP-1 Stimulates Surface Expression of CX3CR1 and Enhances the Adhesion of Monocytes to Fractalkine/CX3CL1 via p38 MAPK. J. Immunol., June 15, 2006; 176(12): 7412 - 7420. [Abstract] [Full Text] [PDF]
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 C. Sonnet, P. Lafuste, L. Arnold, M. Brigitte, F. Poron, F. Authier, F. Chretien, R. K. Gherardi, and B. Chazaud Human macrophages rescue myoblasts and myotubes from apoptosis through a set of adhesion molecular systems J. Cell Sci., June 15, 2006; 119(12): 2497 - 2507. [Abstract] [Full Text] [PDF]
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J.-C. Gevrey, B. M. Isaac, and D. Cox Syk Is Required for Monocyte/Macrophage Chemotaxis to CX3CL1 (Fractalkine) J. Immunol., September 15, 2005; 175(6): 3737 - 3745. [Abstract] [Full Text] [PDF]
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 O. Quehenberger Thematic Review Series: The Immune System and Atherogenesis. Molecular mechanisms regulating monocyte recruitment in atherosclerosis J. Lipid Res., August 1, 2005; 46(8): 1582 - 1590. [Abstract] [Full Text] [PDF]
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T. Shimaoka, T. Nakayama, N. Fukumoto, N. Kume, S. Takahashi, J. Yamaguchi, M. Minami, K. Hayashida, T. Kita, J. Ohsumi, et al. Cell surface-anchored SR-PSOX/CXC chemokine ligand 16 mediates firm adhesion of CXC chemokine receptor 6-expressing cells J. Leukoc. Biol., February 1, 2004; 75(2): 267 - 274. [Abstract] [Full Text] [PDF]
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A. Schafer, C. Schulz, M. Eigenthaler, D. Fraccarollo, A. Kobsar, M. Gawaz, G. Ertl, U. Walter, and J. Bauersachs Novel role of the membrane-bound chemokine fractalkine in platelet activation and adhesion Blood, January 15, 2004; 103(2): 407 - 412. [Abstract] [Full Text] [PDF]
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S. Vitale, A. Schmid-Alliana, V. Breuil, M. Pomeranz, M.-A. Millet, B. Rossi, and H. Schmid-Antomarchi Soluble Fractalkine Prevents Monocyte Chemoattractant Protein-1-Induced Monocyte Migration via Inhibition of Stress-Activated Protein Kinase 2/p38 and Matrix Metalloproteinase Activities J. Immunol., January 1, 2004; 172(1): 585 - 592. [Abstract] [Full Text] [PDF]
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 H. Umehara, E. T. Bloom, T. Okazaki, Y. Nagano, O. Yoshie, and T. Imai Fractalkine in Vascular Biology: From Basic Research to Clinical Disease Arterioscler Thromb Vasc Biol, January 1, 2004; 24(1): 34 - 40. [Abstract] [Full Text] [PDF]
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T. Minami, A. Sugiyama, S.-Q. Wu, R. Abid, T. Kodama, and W. C. Aird Thrombin and Phenotypic Modulation of the Endothelium Arterioscler Thromb Vasc Biol, January 1, 2004; 24(1): 41 - 53. [Abstract] [Full Text] [PDF]
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 M. E. T. Penfold, T. L. Schmidt, D. J. Dairaghi, P. A. Barry, and T. J. Schall Characterization of the Rhesus Cytomegalovirus US28 Locus J. Virol., October 1, 2003; 77(19): 10404 - 10413. [Abstract] [Full Text] [PDF]
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C. Hundhausen, D. Misztela, T. A. Berkhout, N. Broadway, P. Saftig, K. Reiss, D. Hartmann, F. Fahrenholz, R. Postina, V. Matthews, et al. The disintegrin-like metalloproteinase ADAM10 is involved in constitutive cleavage of CX3CL1 (fractalkine) and regulates CX3CL1-mediated cell-cell adhesion Blood, August 15, 2003; 102(4): 1186 - 1195. [Abstract] [Full Text] [PDF]
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M. Nishimura, H. Umehara, T. Nakayama, O. Yoneda, K. Hieshima, M. Kakizaki, N. Dohmae, O. Yoshie, and T. Imai Dual Functions of Fractalkine/CX3C Ligand 1 in Trafficking of Perforin+/Granzyme B+ Cytotoxic Effector Lymphocytes That Are Defined by CX3CR1 Expression J. Immunol., June 15, 2002; 168(12): 6173 - 6180. [Abstract] [Full Text] [PDF]
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 A. M. Fong, S. M. Alam, T. Imai, B. Haribabu, and D. D. Patel CX3CR1 Tyrosine Sulfation Enhances Fractalkine-induced Cell Adhesion J. Biol. Chem., May 24, 2002; 277(22): 19418 - 19423. [Abstract] [Full Text] [PDF]
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 K. Balabanian, A. Foussat, P. Dorfmuller, I. Durand-Gasselin, F. Capel, L. Bouchet-Delbos, A. Portier, A. Marfaing-Koka, R. Krzysiek, A.-C. Rimaniol, et al. CX3C Chemokine Fractalkine in Pulmonary Arterial Hypertension Am. J. Respir. Crit. Care Med., May 15, 2002; 165(10): 1419 - 1425. [Abstract] [Full Text] [PDF]
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A. Ludwig, T. Berkhout, K. Moores, P. Groot, and G. Chapman Fractalkine Is Expressed by Smooth Muscle Cells in Response to IFN-{gamma} and TNF-{alpha} and Is Modulated by Metalloproteinase Activity J. Immunol., January 15, 2002; 168(2): 604 - 612. [Abstract] [Full Text] [PDF]
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 M. V. Volin, J. M. Woods, M. A. Amin, M. A. Connors, L. A. Harlow, and A. E. Koch Fractalkine: A Novel Angiogenic Chemokine in Rheumatoid Arthritis Am. J. Pathol., October 1, 2001; 159(4): 1521 - 1530. [Abstract] [Full Text]
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H. Nomiyama, K. Hieshima, T. Nakayama, T. Sakaguchi, R. Fujisawa, S. Tanase, H. Nishiura, K. Matsuno, H. Takamori, Y. Tabira, et al. Human CC chemokine liver-expressed chemokine/CCL16 is a functional ligand for CCR1, CCR2 and CCR5, and constitutively expressed by hepatocytes Int. Immunol., August 1, 2001; 13(8): 1021 - 1029. [Abstract] [Full Text] [PDF]
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 D. N. Cook, S.-C. Chen, L. M. Sullivan, D. J. Manfra, M. T. Wiekowski, D. M. Prosser, G. Vassileva, and S. A. Lira Generation and Analysis of Mice Lacking the Chemokine Fractalkine Mol. Cell. Biol., May 1, 2001; 21(9): 3159 - 3165. [Abstract] [Full Text]
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B. Cambien, M. Pomeranz, H. Schmid-Antomarchi, M.-A. Millet, V. Breittmayer, B. Rossi, and A. Schmid-Alliana Signal transduction pathways involved in soluble fractalkine-induced monocytic cell adhesion Blood, April 1, 2001; 97(7): 2031 - 2037. [Abstract] [Full Text] [PDF]
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A. D. Lucas, N. Chadwick, B. F. Warren, D. P. Jewell, S. Gordon, F. Powrie, and D. R. Greaves The Transmembrane Form of the CX3CL1 Chemokine Fractalkine Is Expressed Predominantly by Epithelial Cells in Vivo Am. J. Pathol., March 1, 2001; 158(3): 855 - 866. [Abstract] [Full Text] [PDF]
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C. A. Haskell, M. D. Cleary, and I. F. Charo Unique Role of the Chemokine Domain of Fractalkine in Cell Capture. KINETICS OF RECEPTOR DISSOCIATION CORRELATE WITH CELL ADHESION J. Biol. Chem., October 27, 2000; 275(44): 34183 - 34189. [Abstract] [Full Text] [PDF]
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 D. H. McDermott, J. P.J. Halcox, W. H. Schenke, M. A. Waclawiw, M. N. Merrell, N. Epstein, A. A. Quyyumi, and P. M. Murphy Association Between Polymorphism in the Chemokine Receptor CX3CR1 and Coronary Vascular Endothelial Dysfunction and Atherosclerosis Circ. Res., August 31, 2001; 89(5): 401 - 407. [Abstract] [Full Text] [PDF]
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