| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J Biol Chem, Vol. 274, Issue 15, 10053-10058, April 9, 1999
,, and§¶
From the Gladstone Institute of Cardiovascular
Disease, San Francisco, CA 94141-9100, the § Cardiovascular
Research Institute, and the ¶ Department of Medicine, University
of California, San Francisco, California 94143
 |
ABSTRACT |
|---|
 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
Fractalkine is a novel multidomain protein
expressed on the surface of activated endothelial cells. Cells
expressing the chemokine receptor CX3CR1 adhere to
fractalkine with high affinity, but it is not known if adherence
requires G-protein activation and signal transduction. To investigate
the cell adhesion properties of fractalkine, we created mutated forms
of CX3CR1 that have little or no ability to transduce
intracellular signals. Cells expressing signaling-incompetent forms of
CX3CR1 bound rapidly and with high affinity to immobilized
fractalkine in both static and flow assays. Video microscopy revealed
that CX3CR1-expressing cells bound more rapidly to
fractalkine than to VCAM-1 (60 versus 190 ms). Unlike VCAM-1, fractalkine did not mediate cell rolling, and after capture on
fractalkine, cells did not dislodge. Finally, soluble fractalkine induced intracellular calcium fluxes and chemotaxis, but it did not
activate integrins. Taken together these data provide strong evidence
that CX3CR1, a seven-transmembrane domain receptor,
mediates robust cell adhesion to fractalkine in the absence of
G-protein activation and suggest a novel role for this receptor as an
adhesion molecule.
The interaction of leukocytes with the vascular wall and their
subsequent migration into surrounding tissues are central components of
the inflammatory response. This leukocyte trafficking is directed by
chemokines (chemotactic cytokines), a rapidly
growing family of low molecular weight, soluble proteins. There are two
major subfamilies of chemokines, classified by the sequence of a
conserved di-cysteine motif near the amino-terminal end of the protein. The CC or Fractalkine (CX3C) is a recently discovered chemokine that
contains three amino acids between the first two cysteines and may thus
be the first member of a new family of chemokines. Fractalkine possesses a number of structural characteristics and biological activities not previously found within the chemokine family (4, 5). In
contrast to soluble chemokines, fractalkine is anchored to vascular
wall cells by an extended mucin stalk linked to a transmembrane domain
(5). This domain may serve to position fractalkine for efficient
interactions with leukocytes expressing CX3CR1, the
receptor for fractalkine (6, 7). Unlike most chemokines, fractalkine is
not synthesized by leukocytes. Instead, it is expressed on activated
endothelial cells in the vasculature and on neurons in the central
nervous system, where it appears to be involved in regulating the
activity of microglia (8, 9). Soluble forms of fractalkine have been
detected and may be the result of proteolytic cleavage at a di-basic
consensus sequence located next to the transmembrane domain (5).
The location of fractalkine at the luminal surface of endothelial cells
suggested a potential role in the arrest of cells from flowing blood.
Consistent with this idea was the observation that
CX3CR1-expressing cells bound to fractalkine-coated glass and to cells expressing fractalkine on their surface (6, 10). A unique
feature of this interaction was the failure of pertussis toxin
(PTX)1 to block cell adhesion
to fractalkine (6, 10). Other chemokines induce cell adhesion but do so
indirectly by up-regulating integrins, a process that is dependent upon
G-protein activation (3, 11, 12). The finding that fractalkine mediated
cell adhesion in the presence of PTX raised the intriguing possibility
that CX3CR1 acts primarily as an adhesion molecule, rather
than as a signaling molecule. To test this hypothesis, we used an
in vitro system to study the binding of flowing cells to
fractalkine. Here, we report that fractalkine promotes the rapid arrest
of CX3CR1-expressing cells under conditions of
physiological shear stress. Unlike adhesion mediated by integrins,
fractalkine-induced adhesion was independent of G-protein activation.
Adhesion of CX3CR1-expressing cells to fractalkine did
not enhance integrin-dependent cell adhesion, suggesting
that the presence of fractalkine may provide an alternative to
integrin-mediated cell adhesion.
Materials--
The recombinant human chemokines secondary
lymphoid tissue chemokine (SLC) and fractalkine were obtained from
R & D Systems (Minneapolis, MN). Two forms of fractalkine were used.
The full-length extracellular domain, including a carboxyl-terminal
polyhistidine epitope tag, was used for cell adhesion studies. A
second form consisting of the chemokine domain portion only (designated
soluble fractalkine) was used in signaling assays and in competitive
cell binding studies. The antihistidine monoclonal antibody was from R & D Systems. Soluble VCAM-1 (13) was kindly provided by Dr. Ted A. Yednock (Athena Neurosciences, South San Francisco, CA). Minimal
essential medium, Optim-MEM, and RPMI were from Life Technologies, Inc.
Fetal calf serum was from Hyclone Laboratories (Logan, UT). myo-[2-3H]Inositol was obtained from NEN Life
Science Products. PTX was from List Biological Laboratories (Eugene,
OR) or Sigma. All other chemicals were from Sigma.
DNA Constructs--
A genomic clone of murine CX3CR1
was isolated by a PCR-based technique, in which genomic DNA from
embryonic stem cells was used as a template. The following
primers were used: 5'-GCGTCGACTCCACCTCCTTCCCTGAACTGG and
5'-ATGCGGCCGCTCAGAGCAGGAGAGACCCATCTC (receptor-coding
regions are underlined) and added 5' SalI and 3'
NotI sites to the amplified cDNA. The receptor-coding
region was sequenced, and the nucleotide sequence matched that recently
deposited in GenBankTM (accession number AF102269). The
cDNA was ligated into an expression vector that added the prolactin
signal sequence followed by a FLAG epitope to the amino terminus (14).
Murine and human CX3CR1 showed equivalent binding affinity
and signaling in response to human fractalkine (data not shown), and
the murine form of the receptor was used in all experiments reported.
Mutations were generated with the QuikChange site-directed mutagenesis
kit (Stratagene, La Jolla, CA) and overlapping primers coding for the
desired mutated sequences. Human CCR7 was isolated from HUT-78 mRNA
with a reverse transcription PCR-based technique and subcloned into the
expression vector described above. All constructs were confirmed by
sequencing of both strands. The G-protein construct
(G Cell Culture and Transfection--
Human embryonic kidney cells
(HEK-293) were purchased from the American Type Culture Collection
(Manassas, VA), and the murine pre-B cell line (300-19) (16, 17) was a
generous gift of Dr. G. La Rosa (LeukoSite, Cambridge, MA). Cells were
grown and transfected as described (17). To further select for cells
expressing high numbers of receptors, cells were labeled with the
anti-FLAG epitope antibody (M1), as described below, and sorted on a
fluorescence-activated cell sorter (Vantage, Becton Dickinson, Franklin
Lakes, NJ). In some of the assays described below, cells were
pretreated with PTX (100 ng/ml) to block signaling through
G Assay of Inositol Phosphate Formation--
The formation of
inositol phosphate was assayed as described (18).
Assessment of Surface Expression of the Receptor--
The
surface expression of the wild-type and mutated forms of
CX3CR1 was assessed either by enzyme-linked immunosorbent
assay or by flow cytometry as described previously (14, 17, 19).
Static Adhesion Assay--
Static adhesion assays were performed
as described previously (6).
Slide Preparation--
Sixteen-well glass chamber slides (Nunc,
Naperville, IL) were coated overnight with a monoclonal antihistidine
antibody (final concentration, 10 µg/ml) in coating buffer (50 mM Tris, pH 9.5) at 4 °C. The wells were washed twice
with phosphate-buffered saline and incubated with 200 µl of static
adhesion buffer (RPMI, 1.0 mg/ml bovine serum albumin, 10 mM HEPES, pH 7.4) for 1 h at room temperature. The
adhesion buffer was removed, and human fractalkine, including the
mucin stalk and a carboxyl-tail polyhistidine epitope tag, was added at
a final concentration of 10 nM in adhesion buffer (50 µl/well) and incubated for 1 h at room temperature. The final step in slide preparation was three washes with 200 µl of adhesion buffer.
Cell Preparation and Adhesion Assay--
HEK-293 cells were
removed from the tissue culture dish by trypsinization and filtered
through a 35-µm cell strainer cap tube (Becton Dickinson, Franklin
Lakes, NJ) to eliminate cell aggregates. Cells were washed with
adhesion buffer and resuspended to a density of 3.2 × 106 cells/ml. Fifty microliters of the cell suspension was
added to each well, and adhesion was allowed to progress for 30 min at
room temperature. Nonadherent cells were washed from the slide by
dipping the entire chamber in phosphate-buffered saline, removing the
snap-off wells, and dipping the slide twice more. The cells were fixed
in 1% glutaraldehyde for 10 min and rinsed in phosphate-buffered saline. Adherent cells were counted with an inverted microscope and a
×40 objective.
Flow Chamber Adhesion Assay--
Cells were perfused over a
20 × 3-mm area of a glass slide (Corning Glass Works, Corning,
New York) in a laminar flow chamber (Glycotech, Rockville, MD) as
described previously (12).
Slide Preparation--
A template and a solvent-resistant pen
were used to mark a 20 × 3-mm region, which was subdivided into
two sections. The slide was then inverted and the coating reagents were
"drawn" onto the slide with an Eppendorf pipettor, with the
markings on the opposite side serving as a guide. One of the two
regions was coated with the "test" ligand, and the other was left
uncoated as a control. The initial coating (9 µl) was either
anti-polyhistidine antibody (100 µg/ml) or VCAM-1 (2 µg/ml) in flow
adhesion buffer (150 mM NaCl, 10 mM HEPES, 1 mM MgCl2, 1 mM CaCl2,
pH 7.4). The slides were incubated in a humidified box at 4 °C
overnight. Before the next reagent was added, the slides were washed by
applying 150 µl of adhesion buffer with a pipettor and then
aspirating the buffer until the slide was almost dry. The second
reagent (fractalkine-stalk-polyhistidine (100 nM) or SLC (2 µM)) was added in the same manner. The slides were
incubated for 1 h at 37 °C and washed. The entire marked area
was then coated with fetal bovine serum for 15 min at 37 °C. Before
use in the assay, the entire slide was washed by dipping into adhesion
buffer, and the flow chamber was aligned with the coated area.
Cell Preparation and Adhesion Assay--
Cells were washed once,
resuspended in flow adhesion buffer (2 × 106
cells/ml), and perfused through the chamber. The calculated wall shear
stress was 1.5 dynes/cm2 (assuming viscosity equaled 0.01 poise), unless otherwise noted. Images were captured with a TMS
microscope (Nikon, Garden City, NJ), a charged-coupling device video
camera (Sony SSC-S20, Park Ridge, NJ), and a videotape recorder
(Panasonic, Secaucus, NJ).
Analysis of Number of Cells Adhering--
After a 3-min
perfusion, the entire substrate-coated region, equaling 10 fields (0.5 mm2/field), was captured on video. The number of adherent
cells was determined by counting the cells in these fields during video playback.
Analysis of Cell Behavior and Time to Capture--
Time to
capture was determined by replaying the video frame by frame (17 ms/frame = 60 frames/s). Cells that interacted with the substrate
and subsequently stopped were counted. The "zero" time was defined
as the time at which the cell first entered the focal plane of the
substrate-coated glass, indicating an interaction with the substrate.
Cells transiently stopping were not included in this analysis. These
transient interactions, which we observed commonly in
integrin-dependent adhesion to VCAM-1, were never seen in
fractalkine-mediated adhesion. Therefore, to compare stable adhesion,
we included in the data set only cells that remained attached for more
than 30 s. Cell position as a function of time was determined by
analysis of videotape with NIH Image 1.61 (National Institutes of
Health). These data were analyzed at 20 frames/s.
Statistical Analysis--
Statistics for all assays were done
with Instat software (GraphPad Software, San Diego, CA) for the
Macintosh. The Mann-Whitney test or Welch's t test was
used, where appropriate.
To determine if fractalkine-mediated adhesion is dependent upon
signal transduction, we changed critical intracellular domains of
CX3CR1 to uncouple it from G proteins. CX3CR1,
like many seven-transmembrane domain receptors, has in its second
intracellular loop a highly conserved aspartate-arginine-tyrosine (DRY)
sequence that is required for G-protein activation (20-23). Receptors
with both single (R128N) and double (D127N plus R128N, denoted DR/NN)
point mutations were generated because the double mutant tended to be
expressed at lower levels than wild-type CX3CR1. All
receptors were constructed with the FLAG epitope at the amino terminus
to facilitate quantitation of surface expression.
The three receptor isoforms (wild-type, R128N, and DR/NN) were
transiently transfected into HEK-293 cells, and the transfected cells
were assayed for their ability to signal in response to saturating
levels of fractalkine. HEK-293 cells expressing wild-type CX3CR1 gave a robust signal, as measured by an increase in
inositol phosphate release (Fig. 1). In
contrast, cells expressing the R128N or DR/NN form of
CX3CR1 failed to signal. Similar results were obtained when
signaling was assayed by measuring agonist-dependent intracellular calcium fluxes or chemotaxis (data not shown).
Quantitative enzyme-linked immunosorbent assays confirmed that each
form of the receptor was expressed at the cell surface (Fig. 1). These findings show that mutation of the "DRY" region of
CX3CR1 prevents G-protein-dependent
signaling.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
chemokines contain a sequential cysteine-cysteine motif
and are predominately mononuclear cell agonists. The CXC or 
chemokines contain a single amino acid between these two cysteines and
are agonists for polymorphonuclear leukocytes. The mechanism of
leukocyte migration to sites of inflammation involves the sequential
actions of selectins, which mediate rolling along the vessel wall, and
integrins, which mediate firm adhesion and diapedesis (1, 2).
Chemokines appear to play a key role in this process by providing a
chemotactic gradient to direct cell migration and by activating
integrins (3).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
qi5) (15) was kindly provided by Dr. Bruce R. Conklin
(Gladstone Institute of Cardiovascular Disease).
i. This incubation was at 37 °C in growth medium for
16 h before the assay.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (27K):
[in a new window]
Fig. 1.
Signaling and expression of
CX3CR1-transfected HEK-293 cells. HEK-293 cells were
transiently transfected with wild-type (0.25 µg/ml) or mutant
CX3CR1 (2 µg/ml) plus the chimeric G protein
G qi5 (0.5 µg/ml). Error bars represent S.D.
values, and each data point was determined in triplicate. A,
[3H]inositol phosphate (IP) release was
measured in the presence or absence of soluble fractalkine
(FK) (100 nM). The asterisk indicates
p < 0.05 versus unstimulated. B,
cell surface expression of the receptor was measured by enzyme-linked
immunosorbent assay. Representative results from one of three assays
are shown.
We next examined the ability of the wild-type and mutated forms of
CX3CR1 to mediate binding to fractalkine under static
conditions. Addition of a polyhistidine tail to the carboxyl terminus
allowed us to "tether" fractalkine to an antipolyhistidine antibody
previously coated onto glass slides. HEK-293 cells expressing wild-type
CX3CR1 bound well to fractalkine; this binding was
completely blocked by the addition of soluble fractalkine (chemokine
domain only) (Fig. 2). Preincubating the
cells with PTX (10 ng/ml, 16 h) did not block adhesion, although
this concentration of PTX completely eliminated the
fractalkine-induced increase in intercellular calcium (data not
shown). Although unable to signal in response to soluble fractalkine,
the DR/NN form of CX3CR1 bound as well as the wild-type receptor to tethered fractalkine (Fig. 2). Similar results were obtained when the wild-type or mutated forms of CX3CR1 were
expressed in a 300-19 pre-B cells (Fig. 2). These data indicate that
receptor-mediated signaling is not required for cells expressing
CX3CR1 to bind to fractalkine.
|
We next asked whether the expression of CX3CR1 would cause
rapidly flowing cells to adhere to a surface coated with fractalkine. Under flow conditions that produced a wall shear stress of 1.5 dyn/cm2, cells expressing wild-type CX3CR1
adhered well to immobilized fractalkine; this adherence was completely
blocked by soluble fractalkine (Fig. 3).
Cells expressing the DR/NN mutation of CX3CR1 adhered as
well as those expressing the wild-type receptor; this adherence was not
reduced by pretreatment with PTX. These data suggest that fractalkine
induces the capture of cells flowing at physiologically relevant shear
rates and that cell capture is not dependent upon receptor
activation.
|
Next, we used the flow assay to compare cell adhesion to fractalkine
with adhesion to VCAM-1. Cell adhesion to VCAM-1 is mediated by the
integrin 41. To induce adhesion to
VCAM-1, we used the chemokine SLC to activate
41 on the cell surface. Upon coming into
contact with VCAM-1, the cells slowed and rolled for varying periods of
time before coming to a full stop (Fig.
4). These cells often adhered transiently
and reentered the flow before stopping. In contrast, cells expressing
CX3CR1 came to a rapid and complete stop virtually
coincident with encountering the fractalkine-coated surface. Rolling
was rarely, if ever, observed on the fractalkine-coated slides (Fig.
4). Next, we quantitated the time necessary for cells to stop after
their initial contact with fractalkine or VCAM-1. Cells were captured
after a mean of 60 ms (±42 ms) when the slide was coated with
fractalkine; in contrast, cells were captured after a mean of 190 ms
(±192 ms) when the slide was coated with VCAM-1 (Fig.
5). These data indicate that the cell
adhesion to fractalkine is qualitatively and quantitatively different
from adhesion mediated by integrins.
|
|
To determine whether adhesion to fractalkine up-regulated the cell's
integrins, we examined the adherence of CX3CR1-expressing cells to slides coated with VCAM-1, fractalkine, or VCAM-1 and fractalkine in the flow assay. The results are shown in Fig.
6. On VCAM-1-coated slides, there was a
low level of binding. On fractalkine coated-slides, increased cell
binding was observed. The cell binding to fractalkine was specific
binding because it was virtually eliminated by the addition of soluble
fractalkine (data not shown; see Fig. 2). On slides coated with VCAM-1
and fractalkine, the number of cells bound was equal to the sum of the
cells binding to each substrate individually. Thus, adhesion to
fractalkine did not induce integrin-dependent adhesion. Rather, cells appeared to bind either to fractalkine or to VCAM-1. We next
asked whether the soluble form of fractalkine, which induces robust
CX3CR1-dependent signaling (Fig. 1), also
induces integrin-dependent adhesion. Soluble fractalkine
did not increase integrin-dependent adhesion to VCAM-1. In
control studies, we showed that integrin activation by the chemokine
SLC did induce binding of these cells to VCAM-1; as expected, this
binding was blocked by EDTA.
|
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
This study demonstrates that immobilized fractalkine mediates the rapid arrest of cells flowing at physiological shear rates. In mediating cell adhesion, the fractalkine receptor, CX3CR1, acts as a primary adhesion molecule rather than as a signaling molecule. Mutation of intracellular residues critically required for G-protein coupling demonstrated that fractalkine-dependent signaling by CX3CR1 was not required for cell arrest. Furthermore, activation of CX3CR1 by soluble fractalkine did not up-regulate integrin-dependent binding to VCAM-1. The dissociation of cell adhesion from signal transduction suggests a novel role for CX3CR1, a G-protein coupled receptor, as an adhesion molecule.
Evidence from many groups supports a multistep model for leukocyte emigration from the bloodstream (1, 2). The initial step in this model is the rolling of unactivated cells along the vessel wall, which is thought to be mediated by the interaction of leukocyte selectins with glycosylated proteins or lipids on the surface of endothelial cells. In the next steps, firm adhesion to the endothelium and migration into the tissues are dependent upon activation of leukocyte integrins. Recent data suggest that integrin activation is an important physiological end point of chemokine receptor activation and that specific chemokines play an important role in recruiting lymphocytes to secondary lymphoid organs (3, 11, 12). Because PTX blocks chemokine-induced chemotaxis (24) and cell adherence (3), the observation that it failed to block fractalkine-dependent cell adhesion (6) was surprising and suggested a novel role for fractalkine as an adhesion molecule.
PTX does not uncouple chemokine receptors from all classes of G
proteins, however, and so a role for signaling in mediating adhesion
was not ruled out. We (18) and others (25) have shown that CCR2 and
CXCR1 couple to multiple G proteins, including Gq and
G16, which are not sensitive to PTX. Evidence that intracellular signal transduction might be important in fractalkine-mediated adhesion came from the observation that cells did not attach to fractalkine-coated surfaces at 4 °C (6). Since it is not known which
G proteins bind to CX3CR1, we chose to uncouple it from second messengers by mutating highly conserved intracellular amino acids known to be critically involved in G-protein interactions (20-23).
Several lines of evidence indicated that the mutated CX3CR1 failed to activate signaling pathways. First, we were unable to detect an increase in fractalkine-dependent hydrolysis of phosphoinositol. Second, fractalkine did not mobilize intracellular calcium in these cells. Third, cells transfected with the mutated receptor failed to undergo chemotaxis in response to fractalkine. The failure to support chemotaxis was especially significant because it provided a functional end point, independent of specific second messengers. In both static and flow assays, the signaling-incompetent form of CX3CR1 mediated cell adhesion as well as wild-type fractalkine. These results indicate that the interaction between full-length fractalkine and CX3CR1 leads to rapid cell arrest independent of receptor activation and G-protein coupling. These properties make fractalkine unique among the chemokines.
Fractalkine captured flowing cells extremely rapidly. In the parallel-plate assay, cells expressing CX3CR1 adhered to fractalkine in less than 60 ms. In contrast, cells required approximately 190 ms to adhere to VCAM-1. Videomicroscopy clearly showed that cells exhibited rolling and transient binding to VCAM-1, whereas they stopped almost instantaneously after encountering a fractalkine-coated surface. This rapid and firm adhesion of cells is consistent with the hypothesis that fractalkine, which is expressed on activated endothelial cells (5), may serve to capture cells from flowing blood.
Although signaling by CX3CR1 was not required for cell
adhesion to fractalkine, it was unclear whether activation of this receptor would up-regulate integrins, as is the case for the chemokines SLC and SDF-1 (11), and thus further increase cell attachment to
VCAM-1. Cells expressing CX3CR1 exhibited robust,
fractalkine-dependent signaling as measured by
phosphoinositol turnover, intracellular calcium mobilization, and
chemotaxis, indicating that the wild-type receptor coupled well to G
proteins. Up-regulation of integrins did not, however, appear to be an
end point of fractalkine-mediated signal transduction, since soluble
fractalkine did not increase the binding of cells to VCAM-1. Thus,
unlike SLC, fractalkine does not activate the integrins. This result is
consistent with the data of Campbell et al. (11), who
reported that soluble fractalkine failed to increase the attachment of
lymphocytes to ICAM-1, an adhesive interaction that is dependent upon
the integrin M2. It was still possible, however, that
when presented on the end of the mucin stalk, fractalkine could
activate integrins. Our data suggest that this is not the case,
however, because full-length fractalkine also failed to induce any
additional binding of 41-expressing cells
to VCAM-1. Taken together, these data suggest a model in which
high-affinity binding of cells to fractalkine obviates the need for
these cells to adhere through integrin-dependent mechanisms.
The molecular basis for the unique cell-adhesion properties of
fractalkine is not yet clear. Other chemokines, such as MCP-1 and
MIP-1 (11) and TARC and ELC (10), do not support the rapid capture
of cells expressing the appropriate cognate receptors. However, it is
possible that the these chemokines would have to be expressed at the
end of a stalk, in a manner similar to the chemokine domain of
fractalkine, in order for this to be a fair comparison. While no other
known chemokines contain a mucin stalk or similar structure, the
binding of chemokines to proteoglycans could be functionally equivalent
to the presentation of fractalkine at the end of a stalk. Whether the
remarkable ability of fractalkine to arrest cells flowing at high-shear
rates is due to a unique property of the chemokine itself or to the
manner in which it is presented is currently under investigation.
While this work was in progress, Fong et al. (10) showed that fractalkine mediated the capture of leukocytes under physiologic flow. Our results are consistent with those of Fong et al. (10) and further show that after mutation to remove all G-protein coupling, CX3CR1 functioned as well as the wild-type receptor in mediating cell adhesion. In addition, we show that activation of the wild-type receptor by fractalkine does not lead to up-regulation of integrins. Finally, we show that the adhesion of cells expressing CX3CR1 to fractalkine is much more rapid, and qualitatively different, from that mediated by integrins.
In summary, we have shown that fractalkine, a structurally unique
chemokine, mediates the rapid capture of cells under physiological flow. The adhesion is not dependent upon cellular activation or signal
transduction. Fractalkine is up-regulated on activated endothelial
cells and is ideally placed to capture cells from rapidly flowing
blood, such as is found in the coronary arteries or the renal
glomerulus. Whether fractalkine plays a role in the pathophysiology of
diseases affecting these organs remains to be determined.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Dr. David Erle for assistance with the flow assays and Dr. T. Schall and Dr. Daniel Dairaghi for helpful discussions. We also thank Stephen Ordway and Gary Howard for editorial assistance, John Carroll and Neile Shea for preparation of the figures, and Kerry Humphrey for manuscript preparation.
| |
FOOTNOTES |
|---|
* This work was supported in part by National Institutes of Health Grant HL52773 (to I. F. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Gladstone
Institute of Cardiovascular Disease, P. O. Box 419100, San Francisco, CA 94141-9100. Tel.: 415-826-7500; Fax: 415-285-5632; E-mail: icharo{at}gladstone.ucsf.edu.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: PTX, pertussis toxin; SLC, secondary lymphoid tissue chemokine; VCAM-1, vascular cell adhesion molecule 1; DR/NN, D127N/R128N double mutant.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
This article has been cited by other articles:
|
|
 |
|
  C. A. Bolovan-Fritts and S. A. Spector Endothelial damage from cytomegalovirus-specific host immune response can be prevented by targeted disruption of fractalkine-CX3CR1 interaction Blood, January 1, 2008; 111(1): 175 - 182. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A. Bolovan-Fritts, R. N. Trout, and S. A. Spector High T-cell response to human cytomegalovirus induces chemokine-mediated endothelial cell damage Blood, September 15, 2007; 110(6): 1857 - 1863. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
 |
|
 P. Liu, S. Patil, M. Rojas, A. M. Fong, S. S. Smyth, and D. D. Patel CX3CR1 Deficiency Confers Protection From Intimal Hyperplasia After Arterial Injury Arterioscler. Thromb. Vasc. Biol., September 1, 2006; 26(9): 2056 - 2062. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Furuichi, J.-L. Gao, and P. M. Murphy Chemokine Receptor CX3CR1 Regulates Renal Interstitial Fibrosis after Ischemia-Reperfusion Injury Am. J. Pathol., August 1, 2006; 169(2): 372 - 387. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S.-O. Moon, W. Kim, M. J. Sung, S. Lee, K. P. Kang, D. H. Kim, S. Y. Lee, J.-N. So, and S. K. Park Resveratrol Suppresses Tumor Necrosis Factor-{alpha}-Induced Fractalkine Expression in Endothelial Cells Mol. Pharmacol., July 1, 2006; 70(1): 112 - 119. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 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]
|
|
|
|
 |
|
 K. J. Garton, P. J. Gough, and E. W. Raines Emerging roles for ectodomain shedding in the regulation of inflammatory responses J. Leukoc. Biol., June 1, 2006; 79(6): 1105 - 1116. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Chen, S. R. Green, F. Almazan, and O. Quehenberger The Amino Terminus and the Third Extracellular Loop of CX3CR1 Contain Determinants Critical for Distinct Receptor Functions Mol. Pharmacol., March 1, 2006; 69(3): 857 - 865. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. F. Charo and R. M. Ransohoff The Many Roles of Chemokines and Chemokine Receptors in Inflammation N. Engl. J. Med., February 9, 2006; 354(6): 610 - 621. [Full Text] [PDF]
|
|
|
|
|
|
J. Harcourt, R. Alvarez, L. P. Jones, C. Henderson, L. J. Anderson, and R. A. Tripp Respiratory Syncytial Virus G Protein and G Protein CX3C Motif Adversely Affect CX3CR1+ T Cell Responses J. Immunol., February 1, 2006; 176(3): 1600 - 1608. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 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]
|
|
|
|
 |
|
 E. A. Heller, E. Liu, A. M. Tager, S. Sinha, J. D. Roberts, S. L. Koehn, P. Libby, E. R. Aikawa, J. Q. Chen, P. Huang, et al. Inhibition of Atherogenesis in BLT1-Deficient Mice Reveals a Role for LTB4 and BLT1 in Smooth Muscle Cell Recruitment Circulation, July 26, 2005; 112(4): 578 - 586. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Cammas, P. Reinaud, O. Dubois, N. Bordas, G. Germain, and G. Charpigny Identification of Differentially Regulated Genes During Elongation and Early Implantation in the Ovine Trophoblast Using Complementary DNA Array Screening Biol Reprod, April 1, 2005; 72(4): 960 - 967. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Puneet, S. Moochhala, and M. Bhatia Chemokines in acute respiratory distress syndrome Am J Physiol Lung Cell Mol Physiol, January 1, 2005; 288(1): L3 - L15. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Nanki, Y. Urasaki, T. Imai, M. Nishimura, K. Muramoto, T. Kubota, and N. Miyasaka Inhibition of Fractalkine Ameliorates Murine Collagen-Induced Arthritis J. Immunol., December 1, 2004; 173(11): 7010 - 7016. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Barlic, D. H. McDermott, M. N. Merrell, J. Gonzales, L. E. Via, and P. M. Murphy Interleukin (IL)-15 and IL-2 Reciprocally Regulate Expression of the Chemokine Receptor CX3CR1 through Selective NFAT1- and NFAT2-dependent Mechanisms J. Biol. Chem., November 19, 2004; 279(47): 48520 - 48534. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. F. Charo and M. B. Taubman Chemokines in the Pathogenesis of Vascular Disease Circ. Res., October 29, 2004; 95(9): 858 - 866. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Daoudi, E. Lavergne, A. Garin, N. Tarantino, P. Debre, F. Pincet, C. Combadiere, and P. Deterre Enhanced Adhesive Capacities of the Naturally Occurring Ile249-Met280 Variant of the Chemokine Receptor CX3CR1 J. Biol. Chem., May 7, 2004; 279(19): 19649 - 19657. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. R. Latchney, M. A. Fallon, D. J. Culp, H. A. Gelbard, and S. Dewhurst Immunohistochemical Assessment of Fractalkine, Inflammatory Cells, and Human Herpesvirus 7 in Human Salivary Glands J. Histochem. Cytochem., May 1, 2004; 52(5): 671 - 682. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. J. Gough, K. J. Garton, P. T. Wille, M. Rychlewski, P. J. Dempsey, and E. W. Raines A Disintegrin and Metalloproteinase 10-Mediated Cleavage and Shedding Regulates the Cell Surface Expression of CXC Chemokine Ligand 16 J. Immunol., March 15, 2004; 172(6): 3678 - 3685. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
