Department of Bioregulation, Biomedical Research Center, Osaka University Medical School, Japan
Correspondence: Dr. Ziqiang Ding, Department of Pediatrics, IWK Grace Health Center, 5850 University Avenue, Halifax, NS, B3J 3G9, Canada. E-mail: zding{at}is.dal.ca
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Key Words: adhesion molecules lymphocytes signal transduction phorbol ester
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Sulfatide has recently been used as an L-selectin ligand to study post-receptor signal transduction after L-selectin engagement. Sulfatide can activate leukocytes [16 17 18 ] and trigger cytoplasmic calcium fluxes, tyrosine kinase activation, superoxide anion release, and cytokine production [19 20 21 ]. Sulfatide has also been reported to regulate lymphocyte proliferation and immunoglobulin production [22 ]. These sulfatide-induced events in leukocytes have been thought to be mediated via L-selectin [9 , 23 , 24 ]. However, our group has previously demonstrated that a 65-kDa sulfatide receptor (SulfR), distinguishable from L-selectin by its resistance to PMA-induced down-regulation, is also involved in lymphocyte activation [25 ].
In this study, our previous findings are extended by using various leukocyte subsets expressing different levels of L-selectin and SulfR. The signal transduction pathways activated by L-selectin and SulfR are also compared. The results demonstrate that SulfR is ubiquitously expressed by these leukocytes. From an L-selectin-negative mouse T lymphoma cell line, EL-4, a SulfR of 65 kDa has been identified. L-selectin engagement induces LFA-1-independent lymphocyte aggregation via protein tyrosine kinase (PTK) and protein kinase C (PKC). In contrast, SulfR triggers a PTK and PKC-independent signal transduction pathway that can promote LFA-1 binding to ICAM-1 and induce aggregation of EL-4 cells.
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Cells
Rat mesenteric lymph node (MLN) lymphocytes were obtained by
dicing lymph nodes with a scalpel and then gently grinding between
glass slides. The cell preparation was passed through a nylon mesh to
obtain single cell suspension. Peritoneal neutrophils were induced by
intraperitoneal injection of 10 mL of 1% casein and recovered by
peritoneal lavage 1416 h later. The purity of neutrophils obtained
was greater than 90% by crystal violet staining. Peripheral blood
leukocytes were isolated by dextran sedimentation. Briefly, rat
peripheral blood was withdrawn by cardiac puncture and was centrifuged
at 200 g for 10 min to remove platelet-rich plasma. The
pellet was mixed with two volumes of phosphate-buffered saline (PBS)
and one-third final volume of 3% Dextran T-500. The mixture was
settled for sedimentation at room temperature. The supernatant was
retrieved as leukocyte-rich plasma and centrifuged at 300 g
for 5 min. The pellet was washed and the contaminating red blood cells
were lysed with 3 mL of 0.2% NaCl at room temperature for 20 s.
The osmolarity was recovered immediately by adding 3 mL of 1.6% NaCl.
The cells were centrifuged and washed with PBS. Granulocytes and
lymphocytes were discriminated on the basis of their characteristic
forward and side light-scattering patterns in the flow cytometer. EL-4
cells (mouse T lymphoma) were cultured in RPMI 1640 medium supplemented
with 10% fetal calf serum (FCS), HEPES (10 mM), penicillin (100 U/mL),
streptomycin (100 µg/mL), L-glutamine (2 mM), sodium
pyruvate (1 mM), nonessential amino acid (0.1 mM), and
2-mercaptoethanol (2-ME; 50 µM).
Antibodies and reagents
GS5 [26
], a mouse anti-sulfatide mAb (IgM), was
used as culture supernatant. HRL2 and HRL3 are nonblocking and blocking
hamster anti-rat L-selectin mAbs (both IgG), respectively, prepared in
our laboratory [27
]. WT.1 [27
], OX50
[28
], 1A29 [29
], and TA-2
[30
] are mouse IgG mAbs to rat CD11a (LFA-1), CD44,
ICAM-1, and
4 integrin, respectively. MEL-14
[31
], KBA [32
], KAT-1
[33
], KM201 [34
], and M301
[14
] are rat IgG mAbs to mouse L-selectin, LFA-1,
ICAM-1, CD44, and ß7 integrin, respectively. HMß1
[35
] is a hamster IgG mAb against mouse ß1
integrin (PharMingen). Fluorescein isothiocyanate (FITC)-conjugated
goat anti-hamster IgG was purchased from Organon Teknika (Durham, NC).
FITC-conjugated donkey anti-mouse IgM was obtained from Jackson
ImmunoResearch Laboratories (West Grove, PA). FITC-conjugated goat
anti-mouse Ig (IgG + IgM) and FITC-conjugated goat anti-rat Ig
were from Southern Biotechnology Associates (Birmingham, AL). Phorbol
myristate acetate (PMA), herbimycin A, genistein, staurosporine,
cytochalasin B, EGTA, sulfatide, fucoidan, and bovine serum albumin
(BSA; fraction V) were all obtained from Sigma (St. Louis, MO).
Galactosylceramide was from Funakoshi (Tokyo, Japan). 2,
7-Bis-(2-carboxyethyl)-5-carboxyfluorescein acetoxymethyl
ester (BCECF-AM) was from Dojindo (Osaka, Japan). Dextran T-500 (mol wt
523,000) was from Pharmacia (Uppsala, Sweden).
Flow cytometry
Sulfatide binding to leukocytes was detected by
immunofluorescence flow cytometry with anti-sulfatide mAb GS5.
Sulfatide was first dissolved in dimethyl sulfoxide (DMSO) at 50 mg/mL
and diluted with PBS to appropriate concentrations just before use.
Leukocytes were either left untreated or stimulated with 50 ng/mL PMA
at 37°C for 1030 min, unless otherwise indicated. Cells were washed
and incubated with 50300 µg/mL sulfatide in Ca2+-,
Mg2+-free PBS on ice for 30 min. Then cells were washed
twice with PBS and incubated with anti-sulfatide mAb GS5, using a 1:50
dilution of culture supernatant, on ice for 30 min. Cells were then
stained with FITC-conjugated donkey anti-mouse IgM at 5 µg/mL on ice
for 30 min. Finally, the cells were washed twice again, and resuspended
in PBS for flow cytometric analysis in an Epics XL flow cytometer
(Coulter Electronics, Hialeah, FL).
Adhesion assay
Adhesion of EL-4 cells to immobilized sulfatide or
galactosylceramide (GalCer) was determined on 96-well enzyme-linked
immunosorbent assay (ELISA) plates (SUMILON, H type, Japan). Sulfatide
or GalCer dissolved in methanol was immobilized onto plates by drying
at 37°C for 1.5 h. The nonspecific binding sites were then
blocked with 3% BSA in PBS overnight at 4°C. The wells were washed
three times with 0.05% Tween 20 in PBS. Cultured EL-4 cells were
washed twice with RPMI 1640 medium, labeled with 5 µM BCECF-AM at
37°C for 30 min. Then the cells were washed three times and
resuspended in RPMI medium containing 10% FCS. Cells were left
untreated or preincubated with 1001000 µg/mL sulfatide on ice for
20 min, washed, and resuspended in RPMI + 10% FCS at 2 x
106 cells/mL. Next, 50 µL of the cell suspension was
added in quadruplicate to wells coated with sulfatide or
galactosylceramide. After 40 min incubation at 7°C, the wells were
filled with RPMI medium and sealed with parafilm. The plates were
inverted at 7°C for 40 min to allow the nonadherent cells to detach
from the bottom surface of the wells. The medium containing unbound
cells was aspirated. Bound cells were lysed with 50 µL of 1%
NP-40/PBS, and fluorescence intensity was determined in a Fluoroskan II
microplate fluorometer (Labsystem, Tokyo, Japan) at an excitation
wavelength of 485 nm and an emission wavelength of 538 nm. Percent
bound cells represented the percentage of fluorescence intensity of
bound cells over the fluorescence intensity of total added cells. The
results were expressed as means ± standard deviation of
quadruplicate determinations.
Affinity purification and sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) analysis of SulfR
Sulfatide was hydrophobically conjugated to octyl-Sepharose
CL-4B (Pharmacia) as reported previously [25
].
Nonspecific binding sites on Sepharose beads were blocked by incubation
with 1% BSA for 1 h at room temperature. EL-4 cells were
surface-biotinylated with 50 µg/mL NHS-LC-biotin (Pierce, IL) and
lysed with 0.5% NP-40 in Ca2+-, Mg2+-free PBS
supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF). The cell
lysate was applied first to a nonconjugated octyl-Sepharose column for
preclearance. The pass-through fraction was subsequently applied to a
sulfatide-conjugated octyl-Sepharose column. The column was washed
consecutively with PBS, 1 M NaCl in PBS, and 3 M NaSCN in PBS as
described previously [25
]. The NaSCN eluate was dialyzed
against PBS overnight and precipitated first with
galactosylceramide-conjugated octyl-Sepharose and then with
sulfatide-conjugated octyl-Sepharose. Precipitated proteins were eluted
by boiling the Sepharose beads for 3 min in SDS sample buffer
supplemented with 2-ME. The eluted proteins were then resolved by
SDS-PAGE in 420% gradient gels (Daiichi Pure Chemicals, Tokyo,
Japan), transferred to IPVH membrane (Millipore, Japan), and detected
by a horseradish peroxidase-conjugated avidin-biotin complex kit (ABC
kit, Funakoshi, Tokyo, Japan) and enhanced chemiluminescence (ECL,
Amersham), according to the manufacturers instructions.
Aggregation assay
MLN lymphocytes and EL-4 cells were suspended at 2 x
106 cells/mL in RPMI medium containing 10% FCS. Next, 100
µL of cells were added in duplicate wells to 96-well culture plates
(IWAKI, Japan). Reagents for cell pretreatment were added at the
indicated concentrations 30 min before cell stimulation with
anti-L-selectin mAbs (1 µg/mL) or sulfatide (100 µg/mL). Ten
microliters of MEL-14 (anti-mouse L-selectin mAb) culture supernatant
was used for EL-4 cell stimulation. Cell aggregation was allowed to
proceed at 37°C for 3 h and was scored under a phase-contrast
microscope (Olympus, Japan) on a scale of 05, according to Rothlein
and Springer [36
], as modified by Ruegg et al.
[37
]. Zero indicates no cells were aggregated; 1
indicates up to 20% of the cells were in aggregates; 2, 2040%; 3,
4060%; 4, 6080%; and 5, 80100%.
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![]() View larger version (44K): [in a new window] |
Figure 1. Sulfatide binds to leukocytes through L-selectin-dependent and
-independent pathways. Peripheral blood leukocytes, peritoneal
neutrophils, and MLN lymphocytes were left untreated or stimulated with
50 ng/mL PMA at 37°C for 1030 min. Cells were washed and incubated
with 50300 µg/mL sulfatide on ice for 30 min. Sulfatide binding was
detected by anti-sulfatide mAb GS5 and FITC-conjugated anti-mouse IgM
with a flow cytometer. Expression of L-selectin was also assessed at
the same time. The ordinates represent relative mean channel
fluorescence intensity in log values. A representative result of three
similar experiments is presented. The mean fluorescence intensity was
lower than 0.5 when an irrelevant antibody was used.
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Figure 2. L-selectin expression and sulfatide binding on peritoneal neutrophils.
Peritoneal infiltrating neutrophils were induced by casein injection.
Neutrophils were unstimulated or stimulated with 50 ng/mL PMA at 37°C
for 1030 min. Sulfatide binding at 100 µg/mL and L-selectin
expression were detected as described in Figure 1
. Solid lines
represent the flow cytometric profiles obtained by GS5 (anti-sulfatide
mAb) and by HRL2 (anti-rat L-selectin mAb), respectively. Dotted lines
represent the flow cytometric profiles obtained by control irrelevant
Ab. A representative result from three similar experiments is
presented.
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![]() View larger version (27K): [in a new window] |
Figure 3. Sulfatide binds to L-selectin-negative EL-4 cells. (A) Representative
histograms of L-selectin expression on EL-4 cells (a) and mouse lymph
node lymphocytes (b). Solid and dotted lines represent the flow
cytometric profiles obtained by MEL-14 (anti-L-selectin) and control
irrelevant Ab, respectively. (B) Effect of PMA on sulfatide binding to
EL-4 cells. EL-4 cells were incubated with or without 50 ng/mL PMA at
37°C for 1060 min. Cells were washed and incubated with 50300
µg/mL sulfatide on ice for 30 min. Sulfatide binding was detected by
monoclonal anti-sulfatide mAb GS5 and FITC-conjugated anti-mouse IgM
with a flow cytometer. The ordinate represents relative mean channel
fluorescence intensity in log values. A representative result of three
similar experiments is presented.
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![]() View larger version (22K): [in a new window] |
Figure 4. Adhesion of EL-4 cells to immobilized sulfatide is specific and
saturable. (A) Adhesion of EL-4 cells to immobilized sulfatide (Sulf)
and galactosylceramide (GalCer). Various concentrations of sulfatide
and GalCer were adsorbed to 96-well plates in quadruplicate.
BCECF-labeled EL-4 cells were added to the wells and incubated at 7°C
for 40 min. The fluorescence intensity of the bound cells was
determined in a microplate fluorometer. (B) Inhibition of EL-4 cell
adhesion to immobilized sulfatide by preincubating cells with sulfatide
in a concentration-dependent fashion. Sulfatide was immobilized in
96-well plates at 300 ng/well. Fluorescence-labeled EL-4 cells were
preincubated with 0.11.0 mg/mL sulfatide in an ice bath for 20 min,
washed, and resuspended in RPMI medium containing 10% FCS. The
subsequent procedures for adhesion assay were the same as in panel A.
Each column is expressed as mean ± SE of
quadruplicate experiments. Results from one of three experiments are
presented.
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Figure 5. Sulfatide binding to L-selectin-negative EL-4 cells is rapid,
dose-dependent, and saturable. (A) Sulfatide binding to EL-4 cells as a
function of time. EL-4 cells were incubated with 300 µg/mL of
sulfatide on ice for 560 min. Cells were washed and sulfatide binding
was detected by mAb GS5 and FITC-conjugated anti-mouse IgM with a flow
cytometer. (B) Concentration-dependent binding of sulfatide by EL-4
cells. EL-4 cells were incubated with 0.052 mg/mL sulfatide on ice
for 30 min, washed, and stained by GS5 and FITC-conjugated anti-mouse
IgM. The ordinate represents relative mean channel fluorescence
intensity in log values. A representative result from three experiments
is presented.
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![]() View larger version (52K): [in a new window] |
Figure 6. A 65-kDa molecule is detected as a sulfatide binding protein from EL-4
cells. EL-4 cells were surface biotinylated and lysed with NP-40 in PBS
with PMSF. The cell lysate was first precleared by passing through an
octyl-Sepharose column, then applied to an affinity column with
sulfatide-conjugated octyl-Sepharose CL-4B. The column was washed
consecutively with PBS, 1 M NaCl, and 3 M NaSCN. The NaSCN eluate was
dialyzed, and precipitated with galactosylceramide (GalCer)-conjugated
octyl-Sepharose beads (lane 1). The supernatant after GalCer-beads
precipitation was separated into three aliquots. One aliquot was
directly precipitated with sulfatide-conjugated Sepharose beads (lane
2). The other two were first preincubated with either 0.5 mg/mL
sulfatide (lane 3) or 0.5 mg/mL fucoidan (lane 4) on ice for 30 min,
then precipitated with sulfatide-conjugated beads. The precipitates
were subjected to SDS-PAGE under reducing conditions, transferred to
IPVH membranes, and detected by ABC kit and ECL as described in
Materials and Methods. The molecular mass markers (kDa) are indicated
on left.
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4 integrin did not appear to be required for the
aggregation of lymphocytes, because none of the blocking mAbs to these
adhesion molecules was inhibitory for either anti-L-selectin mAb or
sulfatide-induced lymphocyte aggregation. Combined blockade of LFA-1
and ICAM-1 or LFA-1 and
4 integrin also did not inhibit
the aggregation of MLN lymphocytes. Compared to lymphocyte aggregation,
EL-4 cell aggregation was not inhibited by herbimycin A and
staurosporine, but the size of individual aggregation was substantially
inhibited by EDTA and EGTA, although the percentage of cells in
aggregates was not substantially altered (Fig. 7)
. EL-4 cell
aggregation was also strongly inhibited by anti-LFA-1 and partially by
anti-ICAM-1 (Table 1
and Fig. 8
), suggesting that LFA-1/ICAM-1 interaction is activated by
sulfatide stimulation and that this activation is essential in
sulfatide-induced EL-4 cell aggregation. LFA-1 activation on EL-4 cells
was also suggested by the finding that sulfatide-treated EL-4 cells
adhered more efficiently than untreated cells to immobilized ICAM-1
(data not shown). |
View this table: [in a new window] |
Table 1. Mechanisms Involved in Sulfatide-Induced Lymphocyte Aggregation
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![]() View larger version (83K): [in a new window] |
Figure 7. Effect of various inhibitors on sulfatide-induced EL-4 cell
aggregation. EL-4 cells resuspended in RPMI medium containing 10% FCS
were added in duplicate to 96-well culture plates. Reagents for cell
pretreatment at concentrations described in Table 1
were added 30 min
before sulfatide (100 µg/mL) stimulation. Photographs were taken
under a phase-contrast microscope after 3 h of incubation.
Original magnification x100.
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![]() View larger version (116K): [in a new window] |
Figure 8. Adhesion molecules involved in sulfatide-induced EL-4 cell aggregation.
EL-4 cells resuspended in RPMI medium containing 10% FCS were added in
duplicate to 96-well culture plates. Blocking mAbs to LFA-1 (KBA),
ICAM-1 (KAT-1), ß1 integrin (HMß1), ß7
integrin (M301), and CD44 (KM201) at 30 µg/mL were added to the cells
30 min before sulfatide (100 µg/mL) stimulation. Photographs were
taken under a phase-contrast microscope after 3 h of incubation.
Original magnification x100.
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To more clearly distinguish SulfR from L-selectin, leukocytes of various sources were treated with PMA for an extended time course. Peripheral blood lymphocytes and granulocytes expressed comparable levels of L-selectin, which was almost completely lost after 10 min treatment with PMA and was not reexpressed within 30 min. In the context of a dramatic decline in L-selectin expression, sulfatide binding detected by GS5 was only moderately decreased after 10 min treatment with PMA. It is of note that sulfatide bindings recovered to levels as high as the baselines after 30-min stimulation with PMA when L-selectin expression was still undetectable. These findings suggest that L-selectin can mediate sulfatide binding to peripheral blood leukocytes but L-selectin appears to be only one of the binding sites for sulfatide. The dissociation between the levels of L-selectin expression and sulfatide binding was further demonstrated in peritoneal infiltrating neutrophils, which expressed much less surface L-selectin compared with blood neutrophils. Sulfatide binding to peritoneal neutrophils was accordingly less decreased after 10 min treatment with PMA. This decrease in sulfatide binding corresponded to the loss of L-selectin expression. However, sulfatide binding to peritoneal neutrophils that had been treated with PMA for 30 min was increased to approximately twofold the initial level. These findings indicate that an L-selectin-independent molecule was also involved in sulfatide binding to leukocytes. This novel sulfatide binding receptor (SulfR) can be up-regulated by PMA in expression or affinity.
L-selectin-independent sulfatide binding was also demonstrated in L-selectin-negative EL-4 cells. In addition, sulfatide binding to PMA-treated EL-4 cells did not show the initial decrease as seen in the other leukocytes, but rather was gradually increased as the time of PMA stimulation was extended. This result further supports our conclusion that the newly identified SulfR is distinct from L-selectin.
The protein structure of SulfR and its relationship with other cell adhesion molecules remains to be determined. Several proteins have been reported to bind sulfatide in vitro. The adhesive proteins in extracellular matrix, laminin, thrombospondin, and von Willebrand factor (vWF) have been shown to bind sulfatide [2 ]. In addition, antistasin [39 ] and properdin [40 ] also bind sulfatide. Antistasin is a leech salivary protein and an inhibitor of coagulation and metastasis. Properdin is a complement alternative pathway protein. Based on the structure of these proteins, a consensus sequence (Cys-Ser-Val-Thr-Cys-Gly-X-Gly-X-X-X-Arg-X-Arg) has been proposed to represent a sulfatide binding domain [39 ]. However, there are no reports indicating that proteins bearing this consensus sequence are also expressed on the leukocyte surface and are up-regulated by PMA. Therefore, it is unlikely that these proteins are candidates for our proposed SulfR. Several other proteins lacking the above consensus sequence have also been reported to bind to sulfatide. For instance, P-selectin can bind to sulfatide in vitro [41 ]. Sulfatide can block P-selectin-mediated leukocyte adhesion and migration in vivo [11 , 12 ]. However, P-selectin is only expressed on activated endothelial cells and platelets but not on neutrophils and lymphocytes. Amphoterin [42 ] and cytotactin [43 ] have also been demonstrated to be sulfatide binding proteins. These two proteins are present in the neural tissues and seem to be important in neural cell adhesion, differentiation, and myelin formation. Although it is not known whether these proteins are also expressed on the leukocyte surface, their reported molecular weights are different from that of SulfR we have demonstrated in this study.
It is of interest to assume that sulfatide, which is widely expressed in the extravascular tissue, can promote leukocyte locomotion, on the basis of haptotaxis, in the extravascular space. Leukocytes that migrate through the vascular wall have undergone activation and shed surface L-selectin. Therefore, SulfR may become more important in leukocyte interactions with sulfatide in the tissue. More studies are required to address the importance of tissue-associated sulfatide in leukocyte activation and interstitial movement.
Sulfatide can activate MLN lymphocytes and induce intercellular aggregation. This process was dependent on temperature, energy, and an intact cytoskeleton. At 4°C, sulfatide did not induce cellular proximity and aggregation, suggesting that sulfatide-induced cell aggregation is an active process requiring signal transduction and active metabolism. Mechanisms for the aggregation of MLN lymphocytes and EL-4 cells were apparently different. Sulfatide-induced aggregation of lymphocytes was substantially but not completely inhibited after PMA treatment, which removed cell-surface L-selectin [25 ]. Therefore, both L-selectin and SulfR appeared to be involved in lymphocyte aggregation. PTK and PKC were also involved in this aggregation. In contrast, sulfatide-induced aggregation of EL-4 cells was mediated solely through the stimulation of SulfR because L-selectin was not expressed by EL-4 cells. PTK and PKC did not seem to play a role. These results together suggest that the intracellular signals induced by L-selectin and SulfR are different.
Adhesion molecules involved in the aggregation of MLN lymphocytes and EL-4 cells also appeared to be different. Blocking one of the adhesion molecules LFA-1, ICAM-1, and VLA-4 was not inhibitory to sulfatide-induced aggregation of MLN lymphocytes. Blocking both LFA-1 and ICAM-1, or both LFA-1 and VLA-4, also did not inhibit this aggregation. However, EDTA almost completely blocked lymphocyte aggregation, suggesting that divalent cation-dependent molecular interactions or Ca2+ influx are essential. In contrast to the aggregation of MLN lymphocytes, EL-4 cell aggregation was substantially inhibited by blocking LFA-1 and ICAM-1 adhesion pathway. It is interesting that EDTA decreased the size of EL-4 cell aggregates but did not alter the percentage of cells in aggregates, suggesting that cation-independent molecules were also involved in the initiation of EL-4 cell aggregation. These results together suggest that lymphocytes and EL-4 cells may have different mechanisms for sulfatide-induced signal transduction and cellular interaction. However, it cannot be excluded that the signal transduction pathways in normal MLN lymphocytes can be different from those in EL-4 cells.
An unexpected finding in the present study was the prerequisite of serum for intercellular aggregation. For both MLN lymphocytes and EL-4 cells, sulfatide-induced intercellular aggregation failed to occur without fetal calf serum in the medium. The molecular mechanism involved in this phenomenon is currently not clear. It has been reported that fibrinogen can promote leukocyte adhesion to endothelium by bridging CD11b/CD18 and ICAM-1 in vitro and also bridging two ICAM-1 molecules on the opposing lymphocytes [44 ]. However, in our experiment, addition of albumin, fibrinogen, fibronectin, or hyaluronic acid to the serum-free culture medium did not increase sulfatide-induced cell aggregation (data not shown), suggesting that some other serum components may be important in the intercellular aggregation.
In conclusion, we have demonstrated a novel sulfatide receptor of 65 kDa on leukocytes, which is distinct from the sulfatide binding proteins identified to date. Signals from SulfR engagement can activate LFA-1, promoting LFA-1-dependent aggregation of EL-4 cells. The molecular structure and function of the SulfR are currently under investigation.
Received September 18, 1999; revised February 16, 2000; accepted February 17, 2000.
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