From the Department of Orthopedic Surgery, Section of
Biochemistry and Molecular Biology, Rush University at
Rush-Presbyterian-St. Luke's Medical Center, Chicago, Illinois 60612 and the § Molecular and Cell Biology Laboratory, The Salk
Institute, San Diego, California 92186
Received for publication, October 18, 2002, and in revised form, December 26, 2002
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ABSTRACT |
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CD44 can function as an adhesion receptor that
mediates leukocyte rolling on hyaluronan (HA). To study the
contributions of different domains of the standard isoform of CD44 to
cell rolling, a CD44-negative mouse T lymphoma AKR1 was transfected
with wild type (WT) or mutated cDNA constructs. A parallel flow
chamber was used to study the rolling behavior of CD44 transfectants on immobilized HA. For CD44WT transfectants, the fraction of cells that
rolled and the rolling velocity was inversely proportional to the
amount of cell surface CD44. When the cytoplasmic domain distal to
Gly305 or sequences that serve as binding sites for
cytoskeletal linker proteins, were deleted or replaced with foreign
sequences, no significant changes in the rolling behavior of mutant
cells, compared with the transfectant expressing CD44WT, were observed.
Transfectants lacking 64 amino acids of the cytoplasmic tail distal to
Cys295 adhered to HA but showed enhanced rolling at low
shear forces. When 83 amino acids from the "non-conserved"
membrane-proximal region of the CD44 extracellular domain were deleted,
cells adhered firmly to the HA substrate and did not roll at any fluid
shear force tested. Unlike wild type cells that exhibited a nearly
homogenous distribution of CD44 on a smooth cell surface, cells
expressing the non-conserved region deletion mutant accumulated CD44 in
membrane protrusions. Disruption of the actin cytoskeleton with
cytochalasin B precluded the formation of membrane protrusions,
however, treated cells still adhered firmly to HA and did not roll. We
conclude that interaction between the cytoplasmic domain of CD44 and
the cytoskeleton is not required for cell rolling on immobilized
ligand. The strong effect of deletion of the non-conserved region of
the extracellular domain argues for a critical role of this region in
CD44-dependent rolling and adhesion interactions with HA
under flow.
During inflammatory reactions, recruitment of leukocytes from
circulating blood to the site of inflammation takes place in postcapillary venules. This multistep process begins with establishment of relatively weak, transient adhesive interactions between the leukocyte and the vessel wall that result in cell rolling along the
endothelium under blood flow (1). The rolling interaction is a
prerequisite for the generation of high strength intermolecular bonds
and firm adhesion of the leukocyte to the vessel wall via activation-induced binding of integrins to their ligands (2). The
sequence of cell rolling, firm adhesion, and transendothelial migration
requires cooperation among a number of adhesion receptors (1, 3,
4).
CD44 is a transmembrane glycoprotein expressed in a wide variety of
cell types (reviewed in Refs. 5 and 6). The primary physiologic ligand
of CD44 is hyaluronan (hyaluronic acid,
HA)1 (7), a component of the
extracellular matrix. CD44 is believed to be one of the key adhesion
molecules that direct the traffic of activated T cells to inflamed
tissues (8-10), in part, by its ability to mediate cell rolling on HA
(9, 11). Cell surface expression of CD44 is up-regulated in activated
lymphocytes, and cell activation also facilitates HA binding (12, 13).
HA expression is also up-regulated on the endothelial cell surface upon
pro-inflammatory stimuli (14, 15), thus providing a high local ligand
density that may enhance the CD44-mediated interactions of lymphocytes with HA.
CD44 has been implicated in metastasis formation by different types of
malignant tumors that express high amounts of this receptor (16-20).
During the hematogenous spread of some types of cancer, the initial
arrest of cancer cells, and the migration of these cells on endothelial
HA or within an HA-rich stroma, could potentially be mediated via CD44
(16, 18, 21, 22). It has not been demonstrated, however, whether tumor
cell extravasation requires a rolling interaction with endothelial
HA.
The cytoplasmic tail of CD44 has sites mediating interaction with ezrin
(23, 24), and a putative ankyrin binding site has also been reported
(25). Intracellular association of CD44 with these cytoskeletal linker
proteins could function in post-ligand binding events mediated by CD44.
The cytoplasmic domain of CD44 may be phosphorylated at one or more
serine residues (26, 27), and phosphorylation has been implicated in
mediating cell migration on HA (27) and in the interaction of CD44 with
ezrin (26).
Whether specific sequence within the CD44 cytoplasmic domain or other
regions of the standard form of CD44 are required for or modulate cell
rolling on an HA substrate remains unclear. Here we have used AKR1
lymphoma cells transfected with constructs encoding either WT or
mutated mouse CD44 molecules to examine whether mutation of the CD44
cytoplasmic, transmembrane, or extracellular domains would alter the
rolling behavior of transfected cells on an HA-coated substrate.
Antibodies and Reagents--
HA, purified from either human
umbilical cord (ICN, Costa Mesa, CA) or rooster comb (Healon®,
Pharmacia-Upjohn, Kalamazoo, MI), was used in this study. Both
preparations contain high molecular-weight HA (~1 × 103 and 4 × 103 kDa for umbilical cord HA
and Healon®, respectively). Rat monoclonal antibodies (mAb) specific
for mouse CD44 IM7.8.1 (referred to as IM7) and IRAWB14 (28-30) were
purified from ascites fluid on protein G-Sepharose (Amersham
Biosciences, Piscataway, NJ) as described (31, 32). Conjugation of
antibodies with biotin (Pierce, Rockford, IL) or Alexa Fluor 594 (Molecular Probes, Eugene, OR) was performed according to the
manufacturers' instructions. Fluorescein-conjugated HA (FL-HA) was
prepared as described by de Belder and Wik (33).
Streptavidin-phycoerythrin (PE) was obtained from Molecular Probes, and
Streptomyces hyaluronidase, chondroitin sulfate A,
cytochalasin B, and TRITC-labeled phalloidin were purchased from Sigma
(St. Louis, MO).
Mouse CD44 cDNA Construction and Cell
Transfection--
cDNA constructs, encoding wild type and mutated
forms of the standard isoform (also known as hemopoietic form, CD44H)
of mouse CD44, were generated using PCR and site-directed mutagenesis
as described previously (29, 34-36). The sequences of the PCR products were verified on an automated DNA sequencer, and the constructs were
subcloned into expression vectors pRC/RSV, pH
AKR1, a CD44-negative mouse T-cell lymphoma, was transfected by
electroporation, and stable transfectants were selected in the presence
of G418 (Calbiochem, La Jolla, CA) as described (34, 36). The cells
were cultured in Dulbecco's modified Eagle's medium (Sigma),
supplemented with 10% heat-inactivated fetal calf serum (Sigma).
Flow Cytometry and Confocal Microscopy--
AKR1 transfectants
expressing WT and mutated CD44 molecules were assayed for cell surface
CD44 density and for binding of FL-HA from solution. CD44 was detected
using biotinylated IRAWB14 mAb (shown in Fig. 2A) or IM7
(not shown), both mAb giving essentially identical results for each
transfectant. Following incubation with streptavidin-PE and washing,
fluorescence was analyzed using a FACScan flow cytometer and CellQuest
software (BD Biosciences, San Jose, CA). For CD44 immunostaining, cells
(with or without pretreatment with 40 µM cytochalasin B
at 37 °C for 3 h) were incubated on HA-coated culture dishes
(see below) for 10 min. Following washing and fixation with 2%
formaldehyde, cells that adhered to immobilized HA were incubated with
Alexa Fluor 594-conjugated IM7 mAb. Because the epitope recognized by
IM7 is outside of the HA binding domain of CD44 (30), binding of this
antibody to the epitope is not expected to be affected by ligand
occupancy at the membrane-substrate interface. For actin staining,
HA-adherent, formaldehyde-fixed cells were permeabilized with 0.2%
Triton X-100 and incubated with TRITC-phalloidin (32). The distribution
of CD44 or filamentous actin was examined using an Eclipse TE200 confocal microscope (Nikon, Garden City, NJ) and Metavue imaging software (Universal Imaging, West Chester, PA). For three-dimensional reconstruction of images, Z-series of 300-nm-thick slices were used.
Cell Rolling on HA under Fluid Flow in a Parallel Flow
Chamber--
The experimental conditions for assessment of cell
rolling on immobilized HA have been described in detail before (37). In
brief, HA (1 mg/ml) was adsorbed onto the bottom of 60-mm plastic Petri
dishes that constituted the bottom of a parallel plate flow chamber
(GlycoTech, Rockville, MD). The chamber was perfused with the cell
suspension using a programmable syringe pump (Harvard Apparatus,
Holliston, MA) in withdrawal mode. Cells were accumulated at a shear
force of 0.25 dyn/cm2, followed by exposure to increasing
levels of fluid shear (0.25, 0.5, 1, 2, 4, and 8 dyn/cm2,
each for 2 min). Cell movement was recorded by streamline acquisition of images using a digital camera (RS Photometrics, Trenton, NJ) attached to a Nikon Diaphot inverted phase-contrast microscope. To
assess the strength of cell adhesion, the number of adherent cells was
determined at the end of each 2-min flow period and expressed as a
percentage of the number of adherent cells at the starting shear force
(0.25 dyn/cm2). The ratio of rolling cells was expressed as
the percentage of adherent cells that moved parallel with the fluid
flow on the HA substrate (37). The rolling velocity (microns/s) was
also determined at each level of shear force. Images were collected and
analyzed using a Metaview image analysis system (Universal Imaging). At
least three fields of microscopic view and, for velocity measurements,
at least 100 cells were analyzed at each shear force.
In initial experiments, both mammalian (umbilical cord) and avian
(Healon®, prepared from rooster comb) HA was used to coat culture
dishes that constituted the bottom of the flow chamber. Although both
HA preparations supported rolling, the CD44 transfectants attached more
evenly to, and rolled more smoothly on, the surfaces coated with
umbilical cord HA than on those coated with Healon®. For this study,
therefore, HA of mammalian origin was chosen as a substrate for the
cell rolling experiments. To rule out the possibility that the rolling
interactions were mediated by contaminating proteins or chondroitin
sulfate, which could be present in this preparation, specificity
experiments were conducted. Attachment and subsequent rolling of CD44
transfectants on immobilized mammalian HA were completely abolished by
digestion of the substrate with Streptomyces hyaluronidase
(10 units/ml at 37 °C for 30 min) and were inhibited in a
dose-dependent manner by a soluble HA competitor (either
mammalian HA or Healon®), but not by chondroitin sulfate. For
example, at a concentration of 200 µg/ml, Healon® reduced the
fraction of cells that bound to immobilized mammalian HA to 15%,
whereas 99% of the cells attached to, and rolled on, the same
substrate when chondroitin sulfate was present at 200 µg/ml. These
results indicate that the rolling interactions are dependent on HA and
not on contaminants in the immobilized substrate.
Normalization of CD44 Expression--
The capacity of cells to
bind HA is dependent on the amount of CD44 on the cell surface (34).
Because a change in the rolling properties of the cells transfected
with mutant CD44 constructs could be caused by either the specific
mutation or a change in cell surface CD44 density, the amount of CD44
expressed by each mutant cell line had to be "normalized" to CD44
expression in a wild type transfectant. This normalization was done for
each set of experiments by pairing each mutant with a wild type
counterpart that expressed a similar amount of CD44. Two strategies
were employed to achieve this: 1) AKR1 cells transfected with mutant
CD44 constructs were cultured on immobilized HA for several days prior
to the rolling assays. Regular removal of non-adherent cells from the HA-coated dishes resulted in enrichment for a homogenous population of
HA-binding cells with relatively high densities of CD44, comparable with CD44 expression in CD44WT transfectants. 2) Using the limiting dilution method, several subclones of AKR1/CD44WT cells were generated, each with a different amount of CD44 on the cell surface (see Fig. 2).
Subclones with lower amounts of CD44WT served as "controls" for
mutant lines in which high CD44 expression could not be achieved.
For flow chamber experiments, transfectants were characterized for CD44
expression, and the results of rolling assays for each mutant line were
compared with those of an AKR1/CD44WT clone with similar CD44
expression. All experiments were repeated at least three times under
similar conditions. Although differences between mutant and wild type
lines were consistently observed, the measured values showed a
relatively large variation from experiment to experiment that could be
ascribed to fluctuations in CD44 expression of wild type and mutant
transfectants. Some measurements also had a statistically non-normal
(left-skewed) distribution. Therefore, several statistical tests,
including the paired Student's t test, repeated measures
analysis of variance and the non-parametric Friedman test (SPSS,
Chicago, IL), were employed to determine significant differences
between mutant and wild type cells in their rolling parameters.
Structure and HA Binding Properties of WT and Mutant CD44 Expressed
in AKR1 Lymphoma Cells--
Fig. 1 shows
the schematic structure of WT CD44 (standard isoform) and seven CD44
mutants. "ERM" below the first boxed
area in the cytoplasmic (CY) domain of the WT schematic in Fig. 1
indicates the location of sequences
(Arg292-Lys300) required for binding ERM
(ezrin, radixin, moesin) proteins (23, 24), and "Ank"
below the second box indicates the location of sequences (Asn304-Leu318) reported to mediate
ankyrin binding (25). P after Ank indicates the
location of Ser325, which is constitutively phosphorylated
and whose phosphorylation is required for CD44-mediated cell migration
on HA (27). The numbering of amino acids is according to that of a
previous study (38).
Several deletion and substitution mutants (34, 36) were used to examine
the importance of specific regions of the CD44 cytoplasmic domain in
mediating cell rolling on HA. CD44G305 contains a cytoplasmic domain
truncated at Gly305 and lacks the putative ankyrin binding
motif (25). The CD44
Two mutants were used to examine the importance of features of the CD44
extracellular domain. CD44mArg41 has the Arg41 residue,
which is located in the ligand binding domain and is critical for HA
binding (40, 41), mutated to Ala. In CD44
As described under "Experimental Procedures," for each series of
rolling assays, AKR1/CD44WT cells were subcloned, and a WT clone was
chosen as a control for each mutant line on the basis of similar
amounts of CD44 expression. Binding of FL-HA was determined by flow
cytometry, and the transfectants expressing mutant CD44 were compared
with WT controls matched for CD44 expression (data not shown). With the
exception of AKR1/CD44 Relationship between Cell Surface CD44 Density and Rolling Behavior
of AKR1/CD44WT Cells--
The cellular avidity of
AKR1/CD44WT transfectants for soluble HA increases with increasing
expression of the receptor on the cell surface (34). To determine
whether CD44 density also affects rolling behavior, we examined the
rolling of CD44 WT transfectants expressing different amounts of CD44.
Fig. 2A shows CD44 expression and FL-HA binding (mean fluorescence intensity at saturating
concentrations of mAb and FL-HA, respectively) for five AKR1/CD44WT
subclones with different levels of CD44 expression. As shown previously (34), the greater the amount of CD44 on the surface, the greater is the
capacity of the cells to bind FL-HA. When these clones were exposed to
increasing fluid shear forces, the fraction of AKR1/CD44WT cells that
remained adherent to HA (Fig. 2B) was roughly proportional
to the amount of CD44 on the cell surface. At any given shear force,
fewer cells with high than low CD44 density detached from the
substrate. The percentage of adherent cells that rolled on HA (Fig.
2C) and the rolling velocity (Fig. 2D) were
inversely related to the level of receptor expression. Cells expressing
the greatest amounts of CD44 only began to roll on HA at higher shear
forces (at or above 1 dyn/cm2) and exhibited lower rolling
velocities.
Rolling of AKR1 CD44 Cytoplasmic/Transmembrane Domain
Deletion/Replacement Mutants on HA--
Cells expressing
the CD44G305 truncation mutant rolled in a manner similar to CD44WT
transfectants at all shear forces tested (0.25-8 dyn/cm2)
(Fig. 3A). Two transfectants
expressing CD44 with a cytoplasmic tail truncated more distally
(Thr345 and Ser323 (34)) also displayed rolling
kinetics similar to cells expressing WT CD44 (results not shown).
Although AKR1/CD44
For both the AKR1/CD44 Rolling of AKR1 CD44 Extracellular Domain
Replacement/Deletion Mutants on HA--
AKR1/CD44mArg41
cells expressing CD44 with the Arg41/Ala mutation did not
bind FL-HA and were also unable to attach to, and roll on, immobilized
HA (results not shown). The membrane-proximal region of the CD44
extracellular domain, which is poorly conserved across species, has no
established function in ligand binding (35). AKR1/CD44
To determine whether AKR1/CD44 Differential Distribution and Presentation of CD44 on the Surface
of AKR1/CD44WT, AKR1CD44/ The roles of the cytoplasmic, transmembrane, and extracellular
domains of CD44 in HA binding and membrane localization have been
studied by several laboratories using site-directed mutagenesis and
domain swapping techniques (24, 25, 29, 34-36, 40, 41, 43, 45, 50). In
this study, we utilized a number of well characterized mutants of the
standard form of mouse CD44, expressed in AKR1 lymphoma cells (which,
when transfected with CD44WT, bind HA constitutively), to ask how
specific mutations within these functional domains influence the
ability of CD44 to mediate cell rolling on HA under flow.
The amount of CD44 expressed on the cell surface is one of the most
important factors regulating ligand binding function (34, 36, 37). Here
we have demonstrated that cell surface receptor density is also a
critical factor for CD44-dependent cell rolling on HA. As
shown in Fig. 2, each of the rolling parameters (adhesion strength,
rolling fraction, and rolling velocity) are affected by the net amount
of CD44 expressed. At low levels of receptor expression (where the
cells are virtually unable to bind HA from solution), the capture of
AKR1/CD44WT cells on immobilized HA is poor. Almost all cells that
remain attached, however, engage in relatively high speed rolling
interactions with the substrate. AKR1 cells with high CD44 densities
adhere strongly to HA under flow, and they begin to roll with low
velocity only at or above 1 dyn/cm2 (Fig. 2). Thus,
depending on the density on the cell surface, standard CD44 (the most
abundant isoform) is capable of mediating low, moderate, and high
strength adhesion to HA in a cellular environment that is permissive
for constitutive ligand binding.
The versatility of standard CD44 in its ability to support either
rolling or strong cell adhesion has not been fully appreciated, although a large body of literature suggests that this molecule plays
important roles in a variety of pathologic processes (12, 18, 20-22,
31, 51-54). As shown in Fig. 2, the density of cell surface CD44 is an
important factor regulating HA-dependent cell adhesion and
rolling in vitro. It seems likely that CD44 density is also
important in regulating these processes in vivo. Activated leukocytes may represent the cells, which, at moderate levels of CD44
expression, can participate in rolling interactions with endothelial HA
prior to extravasation into injured tissues in acute inflammation and
inflammatory disease (8, 12, 14, 20). Metastasis formation by cancer
cells, which often express CD44 at high densities, represents a
pathologic process in which CD44 is thought to play a detrimental role.
By mediating attachment (not necessarily rolling) of tumor cells to HA
on endothelium, CD44 can facilitate tumor cell extravasation, thus
contributing to the hematogenous dissemination of several types of
cancer (18, 20-22, 53).
The experiments summarized in Figs. 3 and 4 demonstrate that mutations
in the cytoplasmic domain have little or no effect on CD44-mediated
adhesion and rolling interactions with immobilized HA. AKR1 cells
expressing the CD44 truncation mutant Gly305, the internal
deletion mutant In this study, we found only three mutations within the CD44 molecule
that resulted in significant deviations from the normal (WT-like)
rolling behavior of AKR1 transfectants on immobilized ligand: 1)
truncation of the cytoplasmic tail to 6 amino acids ( For the CD44 The CD44 has been shown to localize to the planar cell membrane (as seen in
AKR1/CD44WT) and, unlike L-selectin or The extracellular domain of CD44 is substituted with carbohydrate side
chains, often including sulfated glycosaminoglycans (GAGs) (20), that
may confer negative electrostatic charge and rigidity to the receptor.
Because the shortened core protein of CD44
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
APr-1, or p304SR
(34-37). Schematic structures of CD44 proteins, encoded by these constructs, are shown in Fig. 1.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Schematic structure of WT and mutant CD44
proteins expressed in AKR1 cells. EC, TM,
and CY refer to the extracellular (amino acids (aa) 1-269),
transmembrane (aa 270-289), and cytoplasmic (aa 290-361) domains,
respectively, of the standard isoform of mouse CD44, encoded by exons
1-5, 16-18, and 20 (amino acid nomenclature is as for human CD44
(38); exon nomenclature is as in Ref. 39). ERM below the WT
cytoplasmic domain indicates the location of clusters of basic aa
residues (Arg292-Lys300) required for ezrin
binding (24), and Ank indicates the location of the putative
ankyrin binding region (Asn304-Leu318) (25).
P denotes the position of the constitutively phosphorylated
Ser325 residue (27). Compared with CD44WT, cytoplasmic
domain truncation mutants CY and G305 lack all but 6 and 16 aa
residues, respectively, of the 72-aa-long cytoplasmic tail.
292-301
lacks the binding site for the cytoskeletal linker protein ezrin. SG5N
has a tandem insertion of 5 Ser-Gly dimers following the transmembrane
domain, followed by the 6 membrane-proximal aa of the cytoplasmic tail.
In the
5TM+CY construct both the transmembrane and cytoplasmic
domains of mouse CD44 are replaced by the homologous domains of the
human
5 integrin. The mArg41 mutant has a single aa
Arg41 within the HA binding domain mutated to Ala
("X"). The region of the CD44 extracellular domain
(between aa 161 and 244) that is relatively non-conserved among species
has been removed in
161-244 by a loop-out deletion between
nucleotides 540 and 790 of the CD44 cDNA (35).
CY ("tailless") mutant lacks all amino acid
residues distal to Cys295, resulting in loss of the ankyrin
binding sequence together with one of the two clusters of positively
charged amino acids reported to be necessary for ezrin (ERM) binding
(24). In CD44
292-301, the entire ezrin binding site (24) has been
deleted. CD44SG5N has 5 Ser-Gly repeats inserted between the
transmembrane and cytoplasmic domains, followed by the 6 membrane-proximal residues of the cytoplasmic domain (amino acids
290-295). In CD44
5TM+CY the entire transmembrane and cytoplasmic
regions of CD44 have been replaced by homologous domains of the human
5 integrin. In summary, the ERM binding region is
absent or disrupted in all of the cytoplasmic domain mutants except
CD44G305, the putative ankyrin binding region is absent in all of these
transfectants except CD44
292-301, and all cytoplasmic tail mutants,
except CD44
292-301, lack Ser325.
161-244, 83 amino acids of
the non-conserved membrane-proximal region of the CD44
ectodomain have been deleted (35).
CY, all of the cell lines expressing CD44 with
mutations in the cytoplasmic domain bound FL-HA similarly to WT
controls, consistent with previous reports (34, 36). As reported
previously (29), the line expressing
CY (tailless) CD44, despite
fairly high CD44 density on the cell surface, bound FL-HA very poorly
(data not shown). Cells expressing CD44
161-244 with a deletion in
the ectodomain bound FL-HA similarly to CD44WT controls, as reported
before (35). The mArg41 mutant did not bind HA at all, a finding that
is also consistent with previous reports (40, 41). Although binding of
FL-HA to the tailless transfectant increased dramatically in the
presence of "inducing" mAb IRAWB14 (29, 42), no ligand binding to
the mArg41 mutant could be induced by IRAWB14 treatment (results not shown).
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Fig. 2.
Rolling of AKR1 transfectants expressing
different amounts of WT CD44 on immobilized HA under fluid flow.
A, relationship between cell surface CD44 density and FL-HA
binding by AKR1/CD44WT subclones (denoted WT1-WT5),
selected for different levels of receptor expression. The mean
fluorescence intensity values of cell surface-bound CD44-specific mAb
or FL-HA were expressed relative to the fluorescence of unstained cells
(-fold background). B-D, rolling properties of AKR1
transfectants, expressing different amounts of CD44WT, on immobilized
HA under increasing shear forces of fluid flow. B,
percentage of adherent cells represents the fraction of cells that
remained adherent to the HA-coated surface at different levels of shear
stress, following their initial accumulation under low fluid shear
(0.25 dyn/cm2). The net numbers of cells per microscopic
field that remained adherent to HA after the accumulation period
(representing 100% adherence) were very similar and were as follows:
WT1, 224; WT2, 204; WT3, 201; WT4, 209; and WT5, 202 cells/field.
C, percentage of rolling cells (rolling fraction) is shown
as the fraction of adherent cells that display rolling movement on the
HA-coated substrate under flow. D, rolling velocity is the
speed of rolling cells (mean ± S.E.), expressed in microns/s. The
data shown are the results of one of three experiments, done under
similar conditions.
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Fig. 3.
HA-mediated rolling of AKR1 CD44 cytoplasmic
domain truncation mutants G305 and CY.
A, HA-mediated rolling of AKR1/CD44G305 mutants
versus AKR1/CD44WT cells. B, rolling of
AKR1/CD44
CY cells in comparison with the control CD44WT
transfectant. The parameters measured are the same as described in Fig.
2. Starting net cell numbers in the experiment shown here were:
254/field for G305, 248/field for the expression-matched WT control;
281/field for
CY, 269/field for the paired WT control. Statistical
analysis of data from three experiments for each pair did not reveal
significant differences between the mutant and WT lines in rolling
behavior, except for AKR1/CD44
CY, where the difference from wild
type in the percentage of cells rolling at or below 1 dyn/cm2 shear force approached significance
(p = 0.083; Friedman test).
CY cells bound soluble FL-HA very poorly (Refs.
29, 34, and 36 and data not shown), cells with WT and tailless CD44
rolled with similar kinetics on immobilized HA (Fig. 3B),
except for one consistent difference. At low shear forces, the fraction
of AKR1/CD44
CY cells that rolled was greater than the fraction of
AKR1/CD44WT cells that rolled (Fig. 3B, middle panel), however, the rolling velocity of the mutant at any given shear force was not appreciably greater (Fig. 3B,
bottom panel).
292-301 mutant with an internal
("loop-out") deletion that encompasses the entire sequence of the
ezrin binding motif, and the AKR1/CD44SG5N mutant, the interaction
between CD44 and ezrin should be impaired (24). CD44SG5N lacks, in
addition, the putative ankyrin-binding motif (25). The CD44
5TM+CY
chimera, with all of the CD44 transmembrane and cytoplasmic domain
sequences replaced by the corresponding sequences of human
5 integrin, should not be able to interact with
intracellular linker, adaptor, or signaling molecules in a
CD44-specific manner. This chimera lacks all CD44-specific
phosphorylation sites (26) as well as Cys286, which has
been reported to have the potential to "bypass" a requirement for
the CD44 cytoplasmic domain in binding HA by mediating CD44
dimerization via the transmembrane region (43, 44). When matched with
AKR1/CD44WT cells showing the same level of receptor expression,
however, transfectants for all three of these mutants showed rolling
behavior that was indistinguishable from that of the CD44WT
transfectants (Fig. 4).
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Fig. 4.
Rolling properties of AKR1 CD44 cytoplasmic
domain deletion mutants 292-301 and SG5N and
the transmembrane/cytoplasmic domain replacement mutant
5TM+CY. HA-mediated rolling of
AKR1/CD44
292-301 (A), AKR1/CD44SG5N (B), and
AKR1/CD44
5TM+CY (C) mutants in comparison with
AKR1/CD44WT controls matched for CD44 expression. The results of one
experiment are shown in which the net starting numbers of adherent
cells varied between 290 and 301 per field for the mutants and between
302 and 318 per field for the WT controls. Statistical analysis of data
from three experiments for each pair did not reveal significant
differences between mutant and WT lines in any of the rolling
parameters.
161-244
cells, expressing CD44 in which 83 amino acids of this non-conserved
region were deleted, did not roll on immobilized HA like the control
CD44WT transfectant. Instead, AKR1 cells with the CD44
161-244
mutation remained firmly adherent to the substrate under fluid flow.
These mutant cells failed to detach from or roll on the HA-coated
surface under a wide range of shear forces (Fig.
5A).
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Fig. 5.
HA-mediated rolling of AKR1 cells expressing
the CD44 extracellular domain (non-conserved region) deletion
mutant 161-244. A, comparison
of the rolling properties of AKR1/CD44
161-244 mutant and
corresponding WT control. The results of one typical experiment are
shown. The net starting cell numbers were 310/field for the
161-244
mutant and 292/field for the WT control. Analysis of data from three
experiments, in which CD44 expression was relatively stable on the
mutant cells, revealed statistically significant differences between
the mutant and WT control at or above 1 dyn/cm2 shear
(p < 0.05 for velocity and rolling cells; repeated
measures analysis of variance, and p = 0.083 "borderline" difference in the percentage of adherent cells;
Friedman test). B, relationship between CD44 density and
adhesion/rolling behavior in AKR1/CD44
161-244 and CD44WT
transfectants. The ratios of adherent cells (upper panel)
and rolling cells (lower panel) at 2 dyn/cm2
shear are shown as a function of CD44 expression. C, the
ratios of AKR1/CD44WT and AKR1/CD44
161-244 cells that adhered to
and rolled on HA at 2 dyn/cm2 fluid shear without
(black bars) or with (gray bars) treatment with
cytochalasin B (Cyto B). Data shown are the mean ± S.E. (three experiments).
161-244 cells lose firm adhesion to
HA at lower densities of the receptor on the cell surface, we generated
subclones of the mutant line that expressed varying amounts of CD44 and
compared their adhesion/rolling behavior on HA. When exposed to a shear
force of 2 dyn/cm2, the fraction of firmly adherent cells
increased and the fraction of rolling cells decreased roughly in
proportion to receptor density for the AKR1/CD44WT clones, whereas the
majority of AKR1/CD44
161-244 cells remained firmly adherent and
failed to roll on HA at all levels of CD44 expression (Fig.
5B). We then asked whether the extreme "stickiness" of
AKR1/CD44
161-244 cells was a consequence of secondary mechanisms
that enhanced the interaction of CD44 with the actin cytoskeleton.
However, similarly to AKR1/CD44WT cells, treatment of the
AKR1/CD44
161-244 transfectant with the cytoskeleton-disrupting drug
cytochalasin B for 3 h before the rolling assay did not result in
any change in the adhesion or rolling behavior of the cells (Fig.
5C), although, as discussed below, this treatment resulted
in the collapse of the actin cytoskeleton.
CY, and
AKR1/CD44
161--
244 Cells
The avidity of
leukocyte adhesion receptors (including CD44) for ligand depends to a
considerable extent on their distribution and presentation on the cell
surface (45-47). Both the cytoplasmic and extracellular domains of
adhesion molecules have been found to play a role in determining
membrane localization (45, 48, 49). Drastic truncation of the CD44
cytoplasmic tail in AKR1/CD44
CY and deletion of a large region of
the extracellular domain in AKR1/CD44
161-244 could affect the
membrane topology of the receptor, thus contributing to the HA binding
and rolling properties of these mutants. We used confocal microscopy to
examine and compare the cell surface localization of CD44 (visualized by immunofluorescence) in the WT control with that in the tailless and
161-244 mutants. In AKR1/CD44WT cells that attached to HA, CD44 was
present in a small circular area in the focal plane of membrane contact
with immobilized ligand (Fig.
6A), was evenly distributed
along the membrane as seen from the middle section of the cell (Fig.
6B), and was nearly homogenously dispersed on the entire
cell surface (Fig. 6, C and D). In AKR1/CD44
CY
cells (Fig. 6, E-H), the membrane attachment site was
larger than in the CD44WT control (Fig. 6, compare A with
E, and D with H) and contained CD44
dispersed in small clusters (Fig. 6E). Also, CD44 showed a
patchy distribution over the entire cell surface (Fig. 6,
F-H). HA-adherent AKR1/CD44
161-244 cells demonstrated a
fundamentally different distribution of CD44 (Fig. 6, I-L).
The area of membrane attachment in this mutant was much larger than the
contact area in the CD44WT (Fig. 6, compare I with
A) and markedly larger than that in the tailless
transfectant (Fig. 6, compare I with E). This
difference was also obvious from the side view of the cells (Fig. 6,
compare L with D and H). Furthermore,
unlike in the other two transfectants where CD44 showed a homogenous or
patchy distribution within the planar cell membrane, in the
AKR1/CD44
161-244 mutant CD44 was organized into thin filamentous
structures reminiscent of microvilli, protruding from virtually the
entire cell surface (Fig. 6, I-L). Staining of the actin
cytoskeleton with TRITC-phalloidin revealed the presence of actin
filaments in the membrane projections of AKR1/CD44
161-244 (Fig.
7, C and D),
confirming that they were, indeed, microvilli. These microvilli were
not present in the wild type transfectant (Fig. 7, A and
B). Following incubation with cytochalasin B, the cortical
cytoskeleton collapsed (Fig. 7, F and H), and the
microvilli disappeared from the membrane contact site (Fig.
7G) and from the cell surface (Fig. 7H) in the
AKR1/CD44
161-244 mutant. CD44 showed an even distribution in the
planar plasma membrane of both the wild type transfectant (Fig. 7,
I and J) and the CD44
161-244 mutant (Fig. 7,
K and L) following treatment with cytochalasin
B.
View larger version (68K):
[in a new window]
Fig. 6.
Membrane distribution of CD44 on AKR1 cells
expressing CD44WT, CD44 CY, and
CD44
161-244 during adhesion to HA. To
visualize surface distribution of CD44 in cells that attached to
immobilized HA, the substrate-bound cells were fixed with formaldehyde
and then stained with Alexa Fluor 594-conjugated IM7 mAb. Using
confocal microscopy, the cells were optically sectioned A,
E, and I, at the plane of membrane attachment,
and B, F, and J, in a position midway
between the bottom and the top (middle
sections). Three-dimensional view of the cells in C,
G, and K, from the bottom, and in
D, H, and L, from the side,
was reconstructed from Z-series of optical sections. One typical cell
of each CD44 transfectant is shown.
View larger version (38K):
[in a new window]
Fig. 7.
Actin cytoskeleton structure and CD44
distribution in AKR1/CD44WT and
AKR1/CD44 161-244 cells with and without
cytochalasin B treatment. Actin filaments were visualized by
TRITC-phalloidin staining of cells that attached to HA.
A-D, actin structure in untreated cells; E-H,
actin structure; and I-L, CD44 distribution following
treatment with cytochalasin B (Cyto B). Top
panels, AKR1/CD44WT; bottom panels,
AKR1/CD44
161-244 transfectants. As described in Fig. 6, the cells
were sectioned optically A, C, E,
G, I, and K, at the membrane
attachment site, and B, D, F,
H, J, and L, in the middle of the cell
body.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
292-301, the CD44SG5N chimera, and the
CD44
5TM+CY chimera all showed rolling interactions with immobilized
ligand that were similar to those of AKR1 cells transfected with wild
type CD44. These results indicate that CD44-mediated rolling of AKR1
cells on HA is not regulated via associations between the cytoplasmic
tail and the cytoskeletal linker proteins ezrin or ankyrin and that
specific phosphorylated serine residues (26, 27) are unlikely to be
involved in mediating rolling. It is important to note that, in
contrast to cell attachment or rolling under flow, there is evidence
that cytoskeletal signaling and coordination are required for
HA-dependent cell migration under static conditions (16,
26, 27, 55, 56).
CY), 2)
substitution of Arg41 within the HA binding domain with Ala
(mArg41), and 3) deletion of 83 amino acids from the non-conserved
membrane-proximal region of the extracellular domain (
161-244).
Because the mArg41 mutation affects a critical amino acid in the Link
module (40), the failure of AKR1/CD44mArg41 cells to bind to
immobilized HA (and thus to roll) was expected.
CY mutant, a greater fraction of AKR1 cells transfected
with this construct rolled on HA compared with transfectants expressing
similar amounts of CD44WT. In this respect, the tailless mutant behaved
like a WT transfectant with a lower level of receptor expression,
consistent with the known properties of this mutant, extremely
inefficient binding of soluble HA and a reduced, but still reasonably
efficient, binding to immobilized HA (16, 29, 34, 36). An altered
distribution of tailless, relative to wild type, CD44 on the cell
surface might change the rolling behavior on HA. In this connection,
confocal microscopy of HA-adherent cells revealed that the distribution
of CD44 in the
CY mutant was, indeed, different from that in cells
expressing CD44WT (Fig. 6). AKR1/CD44WT cells showed a nearly
homogenous distribution of CD44 on the cell membrane, including the
contact site with the immobilized substrate. In contrast,
AKR1/CD44
CY cells demonstrated a patchy distribution of the receptor
over the cell body as well as in the substrate contact site. It is
possible that cell contact with immobilized HA, but not soluble HA,
stabilizes weak receptor-ligand interactions by promoting clustering of
tailless CD44. This receptor clustering could increase cellular avidity
for ligand sufficiently to enable adhesion to and rolling on the
immobilized substrate.
161-244 deletion (35) does not involve the Link module, which
contains residues responsible for HA recognition (6, 40, 41). Instead,
this mutation removes a portion of the extracellular domain that is
relatively non-conserved among species, including the site where the
products of alternatively spliced exons can be inserted, giving rise to
variant isoforms of CD44 in certain types of cells (6). Deletion of
this region results in no significant change in soluble HA binding
(Ref. 35 and data not shown). Unexpectedly, AKR1/CD44
161-244 cells
adhered to HA much more strongly than the corresponding AKR1/CD44WT
transfectant, because the mutant cells did not detach from or roll on
the HA substrate at any level of shear force tested (Fig.
5A).
4
integrin, is excluded from the tips of the microvilli in leukocytes
that possess such protrusions (45, 49). Adhesion receptors such as
L-selectin and
4 integrins that localize to
the tips of microvilli can initiate attachment to and rolling on
immobilized ligands under flow, whereas several other adhesion
receptors, localized to the planar cell body (with the notable
exception of CD44), are unable to do so (48, 49). Also, concentration
of L-selectin on the tips of microvilli has been shown to
enhance the resistance of leukocytes to shear stress upon initial
attachment to native ligand under flow (45, 48). In
AKR1/CD44
161-244 cells, whereas CD44 was also present on the cell
body, a large proportion of receptors was localized to microvillus-like
membrane protrusions (Fig. 6). The questions arise as to what mechanism
is responsible for directing CD44 (with a shortened ectodomain) to
microvilli, and whether this particular distribution of CD44 molecules
contributes to the increased strength of cell adhesion to HA,
characteristic of this mutant. Treatment with cytochalasin B resulted
in a WT-like homogenous distribution of CD44
161-244 on the cell
surface (Fig. 7), yet, this treatment did not result in weakening of
the adhesive interactions of mutant cells with HA (Fig. 5). These
observations suggest that, although cytoskeletal integrity is required
for the formation of microvilli in this particular transfectant (Fig. 7), neither the microvilli nor the actin cytoskeleton appear to contribute to the stability of the bond between CD44
161-244 and immobilized HA under shear stress.
161-244 lacks a number of
these carbohydrate attachment sites, the intermolecular repulsion
afforded by the GAG chains (57) might be reduced in the mutant. It is
tempting to speculate that poorly glycosylated CD44
161-244
molecules would readily undergo clustering with each other or with
other adhesion receptors at the ligand contact site, thus increasing
the strength of adhesion. However, treatment of AKR1/CD44WT cells with
sodium chlorate (an inhibitor of sulfation (58)), chondroitinase ABC
(which removes the GAG chondroitin sulfate), or
p-nitrophenyl-
-D-xylopyranoside (which
prevents addition of GAG side chains to the CD44 core protein (59))
under conditions that have been reported to affect soluble HA binding
to CD44 (59), failed to increase adhesion of wild type cells to
immobilized HA.2 In addition,
although AKR1 cells express significant amounts of the leukocyte
integrin
4
1 (VLA-4), which could
potentially amplify the strength of CD44-initiated cell adhesion
(60-62), we found no evidence of co-clustering or other type of direct
interaction between CD44
161-244 (or CD44WT) and the VLA-4 adhesion
molecule in the transfected cells upon exposure to immobilized
HA.3 Although enhanced
receptor clustering is not definitely ruled out by these experiments,
the results suggest that the increased stability of binding between the
mutant receptor and immobilized ligand could reflect a conformational
change in the ectodomain of CD44, caused by deletion of 83 amino acids.
Changes in ectodomain length, frequently seen under pathologic
conditions, can occur by insertion of variant sequences (by inclusion
of one or more of ten alternatively spliced exons between exons 5 and
16) in the region deleted in CD44
161-244 (35, 63). We did not
investigate whether insertion of variant sequences in the non-conserved
region of the extracellular domain would have the same or opposite
effect on cell rolling as the deletion of this region. It is known that modification in CD44 structure by insertion of variant exon products within the non-conserved region can have either negative or positive influence on receptor-ligand interactions (18, 35, 63-65). Our results
extend these findings by demonstrating that the non-conserved region of
CD44 ectodomain is critically involved in the regulation of
CD44-mediated cell rolling under flow.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Susan Shott, Nicole M. English, and Sonja Velins for expert assistance.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Grants AI-31613 (to R. H.) and AR-45652 (to K. M.) from the National Institutes of Health.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: Dept. of Orthopedic Surgery, Rush University at Rush-Presbyterian-St. Lukes Medical Center, 1735 W. Harrison St., Cohn Bldg., Chicago, IL 60612. Tel.: 312-942-5767; Fax: 312-942-8828; E-mail: Katalin_Mikecz@rsh.net.
Published, JBC Papers in Press, January 22, 2003, DOI 10.1074/jbc.M210661200
2 I. Gál, T. T. Glant, and K. Mikecz, unpublished data.
3 I. Gál and K. Mikecz, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: HA, hyaluronan; FL-HA, fluorescein-conjugated HA; aa, amino acid(s); CY, cytoplasmic; Cyto B, cytochalasin B; EC, extracellular; ERM, ezrin, radixin, and moesin; GAG, glycosaminoglycan; mAb, monoclonal antibody; PE, phycoerythrin; TM, transmembrane; TRITC, tetramethylrhodamine isothiocyanate; VLA-4, very late activation antigen-4; WT, wild type.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Springer, T. A. (1994) Cell 76, 301-314[Medline] [Order article via Infotrieve] |
2. | Lawrence, M. B., and Springer, T. A. (1991) Cell 65, 859-873[Medline] [Order article via Infotrieve] |
3. | von Andrian, U. H., Chambers, J. D., McEvoy, L. M., Bargatze, R. F., Arfors, K. E., and Butcher, E. C. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 7538-7542[Abstract] |
4. |
Siegelman, M.
(2001)
J. Clin. Invest.
107,
159-160 |
5. | Lesley, J., Hyman, R., and Kincade, P. W. (1993) Adv. Immunol. 54, 271-335[Medline] [Order article via Infotrieve] |
6. | Lesley, J., and Hyman, R. (1998) Frontiers Biosci. 3, 616-630 |
7. | Aruffo, A., Stamenkovic, I., Melnick, M., Underhill, C. B., and Seed, B. (1990) Cell 61, 1303-1313[Medline] [Order article via Infotrieve] |
8. |
DeGrendele, H. C.,
Estess, P.,
and Siegelman, M. H.
(1997)
Science
278,
672-675 |
9. | DeGrendele, H. C., Estess, P., Picker, L. J., and Siegelman, M. H. (1996) J. Exp. Med. 183, 1119-1130[Abstract] |
10. | Stoop, R., Kotani, H., McNeish, J. D., Otterness, I. G., and Mikecz, K. (2001) Arthritis Rheum. 44, 2922-2931[CrossRef][Medline] [Order article via Infotrieve] |
11. | Clark, R. A., Alon, R., and Springer, T. A. (1996) J. Cell Biol. 134, 1075-1087[Abstract] |
12. | Lesley, J., Howes, N., Perschl, A., and Hyman, R. (1994) J. Exp. Med. 180, 383-387[Abstract] |
13. | DeGrendele, H. C., Kosfiszer, M., Estess, P., and Siegelman, M. H. (1997) J. Immunol. 159, 2549-2553[Abstract] |
14. |
Mohamadzadeh, M.,
DeGrendele, H.,
Arizpe, H.,
Estess, P.,
and Siegelman, M.
(1998)
J. Clin. Invest.
101,
97-108 |
15. |
Nandi, A.,
Estess, P.,
and Siegelman, M. H.
(2000)
J. Biol. Chem.
275,
14939-14948 |
16. | Thomas, L., Byers, H. R., Vink, J., and Stamenkovic, I. (1992) J. Cell Biol. 118, 971-977[Abstract] |
17. | Bartolazzi, A., Peach, R., Aruffo, A., and Stamenkovic, I. (1994) J. Exp. Med. 180, 53-66[Abstract] |
18. | Naor, D., Sionov, R. V., and Ish-Shalom, D. (1997) Adv. Cancer Res. 71, 241-319[Medline] [Order article via Infotrieve] |
19. | Sy, M. S., Liu, D., Schiavone, R., Ma, J., Mori, H., and Guo, Y. (1996) Curr. Top. Microbiol. Immunol. 213, 129-153[Medline] [Order article via Infotrieve] |
20. | Lesley, J., Hyman, R., English, N., Catterall, J. B., and Turner, G. A. (1997) Glycoconj. J. 14, 611-622[CrossRef][Medline] [Order article via Infotrieve] |
21. |
Kogerman, P.,
Sy, M.-S.,
and Culp, L. A.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
13233-13238 |
22. |
Drillenburg, P.,
and Pals, S. T.
(2000)
Blood
95,
1900-1910 |
23. |
Yonemura, S.,
Hirao, M.,
Doi, Y.,
Takahashi, N.,
Kondo, T.,
and Tsukita, S.
(1998)
J. Cell Biol.
140,
885-895 |
24. | Legg, J. W., and Isacke, C. M. (1998) Curr. Biol. 8, 705-708[Medline] [Order article via Infotrieve] |
25. | Lokeshwar, V. B., Fregien, N., and Bourguignon, L. Y. (1994) J. Cell Biol. 126, 1099-1109[Abstract] |
26. | Legg, J. W., Lewis, C. A., Parsons, M., Ng, T., and Isacke, C. M. (2002) Nat. Cell Biol. 4, 399-407[CrossRef][Medline] [Order article via Infotrieve] |
27. |
Peck, D.,
and Isacke, C. M.
(1998)
J. Cell Sci.
111,
1595-1601 |
28. | Trowbridge, I. S., Lesley, J., Schulte, R., Hyman, R., and Trotter, J. (1982) Immunogenetics 15, 299-312[Medline] [Order article via Infotrieve] |
29. | Lesley, J., He, Q., Miyake, K., Hamann, A., Hyman, R., and Kincade, P. W. (1992) J. Exp. Med. 175, 257-266[Abstract] |
30. | Zheng, Z., Katoh, S., He, Q., Oritani, K., Miyake, K., Lesley, J., Hyman, R., Hamik, A., Parkhouse, R. M. E., Farr, A. G., and Kincade, P. W. (1995) J. Cell Biol. 130, 485-495[Abstract] |
31. | Mikecz, K., Brennan, F. R., Kim, J. H., and Glant, T. T. (1995) Nat. Med. 1, 558-563[Medline] [Order article via Infotrieve] |
32. | Kim, J. H., Glant, T. T., Lesley, J., Hyman, R., and Mikecz, K. (2000) Exp. Cell Res. 256, 445-453[CrossRef][Medline] [Order article via Infotrieve] |
33. | de Belder, A. N., and Wik, K. O. (1975) Carbohydr. Res. 44, 251-257[CrossRef][Medline] [Order article via Infotrieve] |
34. | Perschl, A., Lesley, J., English, N., Trowbridge, I., and Hyman, R. (1995) Eur. J. Immunol. 25, 495-501[Medline] [Order article via Infotrieve] |
35. | He, Q., Lesley, J., Hyman, R., Ishihara, K., and Kincade, P. W. (1992) J. Cell Biol. 119, 1711-1719[Abstract] |
36. | Lesley, J., English, N., Charles, C., and Hyman, R. (2000) Eur. J. Immunol. 30, 245-253[CrossRef][Medline] [Order article via Infotrieve] |
37. |
Lesley, J.,
English, N. M.,
Gal, I.,
Mikecz, K.,
Day, A. J.,
and Hyman, R.
(2002)
J. Biol. Chem.
277,
26600-26608 |
38. | Stamenkovic, I., Amiot, M., Pesando, J. M., and Seed, B. (1989) Cell 56, 1057-1062[Medline] [Order article via Infotrieve] |
39. |
Bell, M. V.,
Cowper, A. E.,
Lefranc, M.,
Bell, J. I.,
and Screaton, G. R.
(1998)
Mol. Cell. Biol.
18,
5930-5941 |
40. | Peach, R. J., Hollenbaugh, D., Stamenkovic, I., and Aruffo, A. (1993) J. Cell Biol. 122, 257-264[Abstract] |
41. |
Bajorath, J.,
Greenfield, B.,
Munro, S. B.,
Day, A. J.,
and Aruffo, A.
(1998)
J. Biol. Chem.
273,
338-343 |
42. | Lesley, J., Kincade, P. W., and Hyman, R. (1993) Eur. J. Immunol. 23, 1902-1909[Medline] [Order article via Infotrieve] |
43. | Liu, D., and Sy, M. S. (1996) J. Exp. Med. 183, 1987-1994[Abstract] |
44. | Liu, D. C., and Sy, M. S. (1997) J. Immunol. 159, 2702-2711[Abstract] |
45. | von Andrian, U. H., Hasslen, S. R., Nelson, R. D., Erlandsen, S. L., and Butcher, E. C. (1995) Cell 82, 989-999[Medline] [Order article via Infotrieve] |
46. |
Stein, J. V.,
Cheng, G.,
Stockton, B. M.,
Fors, B. P.,
Butcher, E. C.,
and von Andrian, U. H.
(1999)
J. Exp. Med.
189,
37-50 |
47. | Stewart, M., and Hogg, N. (1996) J. Cell. Biochem. 61, 554-561[CrossRef][Medline] [Order article via Infotrieve] |
48. |
Li, X.,
Steeber, D. A.,
Tang, M. L.,
Farrar, M. A.,
Perlmutter, R. M.,
and Tedder, T. F.
(1998)
J. Exp. Med.
188,
1385-1390 |
49. |
Abitorabi, M. A.,
Pachynski, R. K.,
Ferrando, R. E.,
Tidswell, M.,
and Erle, D. J.
(1997)
J. Cell Biol.
139,
563-571 |
50. | Liu, D. C., Liu, T., and Sy, M. S. (1998) Cell. Immunol. 190, 132-140[CrossRef][Medline] [Order article via Infotrieve] |
51. | Camp, R. L., Scheynius, A., Johansson, C., and Puré, E. (1993) J. Exp. Med. 178, 497-507[Abstract] |
52. |
Weiss, L.,
Slavin, S.,
Reich, S.,
Cohen, P.,
Shuster, S.,
Stern, R.,
Kaganovsky, E.,
Okon, E.,
Rubinstein, A. M.,
and Naor, D.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
285-290 |
53. |
Weber, G. F.,
Bronson, R. T.,
Ilagan, J.,
Cantor, H.,
Schmits, R.,
and Mak, T. W.
(2002)
Cancer Res.
62,
2281-2286 |
54. |
Estess, P.,
DeGrendele, H. C.,
Pascual, V.,
and Siegelman, M. H.
(1998)
J. Clin. Invest.
102,
1173-1182 |
55. |
Goebeler, M.,
Kaufmann, D.,
Bröcker, E. B.,
and Klein, C. E.
(1996)
J. Cell Sci.
109,
1957-1964 |
56. |
Oliferenko, S.,
Kaverina, I.,
Small, J. V.,
and Huber, L. A.
(2000)
J. Cell Biol.
148,
1159-1164 |
57. |
Esford, L. E.,
Maiti, A.,
Badar, S. A.,
Tufaro, F.,
and Johnson, P.
(1998)
J. Cell Sci.
111,
1021-1029 |
58. |
Maiti, A.,
Maki, G.,
and Johnson, P.
(1998)
Science
282,
941-943 |
59. | Lesley, J., English, N., Perschl, A., Gregoroff, J., and Hyman, R. (1995) J. Exp. Med. 182, 431-437[Abstract] |
60. | Alon, R., Kassner, P. D., Carr, M. W., Finger, E. B., Hemler, M. E., and Springer, T. A. (1995) J. Cell Biol. 128, 1243-1253[Abstract] |
61. | Berlin, C., Bargatze, R. F., Campbell, J. J., van Andrian, U. H., Czabo, M. C., Hasslen, S. R., Nelson, R. D., Berg, E. L., Erlandsen, S. L., and Butcher, E. C. (1995) Cell 80, 413-422[Medline] [Order article via Infotrieve] |
62. |
Siegelman, M. H.,
Stanescu, D.,
and Estess, P.
(2000)
J. Clin. Invest.
105,
683-691 |
63. | Gunthert, U. (1993) Curr. Top. Microbiol. Immunol. 184, 47-63[Medline] [Order article via Infotrieve] |
64. | Stamenkovic, I., Aruffo, A., Amiot, M., and Seed, B. (1991) EMBO J. 10, 343-348[Abstract] |
65. | Ponta, H., Wainwright, D., and Herrlich, P. (1998) Int. J. Biochem. Cell Biol. 30, 299-305[CrossRef][Medline] [Order article via Infotrieve] |