From the
L-selectin is a leukocyte cell surface glycoprotein involved in
carbohydrate-specific ligand binding which mediates rolling of
leukocytes along endothelial surfaces. In addition to its role in
adhesion, an intracellular signaling role for L-selectin has recently
been recognized. In particular, cross-linking L-selectin leads to
increased cytosolic Ca
Neutrophils constitute the first line of defense against
invading micro-organisms. Paradoxically their uncontrolled activation
also contributes to a variety of diseases, such as ischemia-reperfusion
injury, shock, and the adult respiratory distress syndrome(1) ,
through the unregulated release of large amounts of reactive oxygen
intermediates, proteolytic enzymes, and inflammatory cytokines. An
early step in their recruitment into sites of inflammation entails the
interaction of L-selectin on the neutrophil and P-selectin on the
endothelial cell with their respective carbohydrate ligands. This
results in rolling of the neutrophil along the endothelial surface
under conditions of flow(2) . The next phase is firm adhesion,
which requires stimulation of the leukocyte, activation of its
integrins, and their association with endothelial cell adhesion
molecules including ICAM-1(3) . Transmigration follows,
involving additional adhesion molecules, such as platelet-endothelium
cell adhesion molecule(4) . Although the function of L-selectin
in ligand binding and rolling has been extensively studied, its
potential role in neutrophil activation events has only recently been
described(5, 6, 7) .
L-selectin is the
smallest member of the selectin family of carbohydrate-specific
adhesion molecules(8) . Its extracellular region consists of a
lectin domain, an epidermal growth factor-like domain, and two repeats
of a complement regulatory protein domain. Its intracellular domain is
small (17 amino acids), and the predicted primary structure reveals no
known catalytic region or binding site consensus sequences. The natural
ligands for neutrophil L-selectin have not yet been determined,
although the lymphocyte-specific ligands, GlyCAM-1 and CD34, have
recently been identified(9, 10) . The ability to bind
L-selectin depends both on the protein backbone as well as on
post-translational modifications of these ligands(11) . On both
lymphocytes and neutrophils, the surface expression of L-selectin is
down-regulated after activation by proteolytic cleavage(12) .
Recent studies by ourselves and others have revealed a signaling
role for neutrophil L-selectin in the generation of cytosolic calcium
transients, potentiation or activation of the oxidative
burst(5, 7) , and enhanced tumor necrosis factor and
interleukin-8 gene expression (6). The signaling pathways utilized by
L-selectin have not yet been identified. Phosphorylation of proteins on
tyrosine residues is an extremely important regulatory pathway in a
wide variety of cell types (13). Tyrosine phosphorylation in
neutrophils occurs rapidly following stimulation with a variety of
agonists(14) , and tyrosine kinase inhibitors can block many
neutrophil functional responses such as chemotaxis and the oxidative
burst(15) . Because of the purported importance of these
pathways and evidence that other adhesion molecules, such as integrins,
transmit intracellular signals by pathways involving tyrosine
phosphorylation(16) , we examined whether L-selectin might
transmit intracellular signals by pathways involving tyrosine
phosphorylation in neutrophils.
MAP kinase activity was determined using a gel renaturation
assay, using myelin basic protein as the substrate(19) .
Polyacrylamide (10%) gels were cast, incorporating 0.54 mg/ml myelin
basic protein within the gel. After electrophoresis of the
immunoprecipitate (the equivalent of 2
To determine whether tyrosine phosphorylation in human
neutrophils could be modulated by L-selectin, cells were incubated with
primary anti-L-selectin (DREG 55, 56, or 200) or control mAb. This
treatment resulted in an increase in tyrosine phosphorylation of
several cellular proteins, including prominent bands at 40-42,
55-60, 70-72, and 105-120 kDa ( Fig. 1and Fig. 3A, lane 1). This response was not seen when cells
were treated with a variety of mAb which bind to the neutrophil surface
in amounts approximately equal to the anti-L-selectin mAbs, as
confirmed by flow cytometry (data not shown). To ensure that the
enhanced tyrosine phosphorylation observed after ligation of L-selectin
by Ab was not an artifact of the antibodies used, we studied the
effects of sulfatides, sulfated glycolipids which are potential ligands
of L-selectin. Sulfatides clearly can function as a ligand for the
L-selectin molecule in vitro(21) and have been shown
to induce Ca
The increase in
tyrosine phosphorylation seen after incubation of the cells with whole
anti-L-selectin mAb might have been due to binding of the Fc portion of
the mAb to Fc receptors on the neutrophil(23) . This is not
likely since isotype-matched control mAb, which also bound to the
neutrophil as confirmed by flow cytometry (data not shown), did not
result in enhanced tyrosine phosphorylation (Fig. 1A).
However, to exclude this possibility, cells were treated with
F(ab`)
The recruitment of neutrophils to inflammatory sites is
important physiologically in host defense and pathophysiologically in
disease states involving inflammatory injury. Recruitment proceeds via
several distinct steps, which involve separate adhesion receptor-ligand
interactions between the neutrophil and the endothelial cell. In
certain circumstances, engagement of L-selectin may be the first signal
encountered by the migrating neutrophil. In this study, additional
evidence for such a signaling role for L-selectin is provided. This may
be in addition to the signaling or co-signaling role for tethered
platelet activating factor which, when present, may lead to priming or
activation of the neutrophil(29) .
Using both monoclonal
anti-L-selectin antibodies and a possible ligand for L-selectin
(sulfatides), we have shown that ligation of L-selectin on the surface
of the neutrophil results in tyrosine phosphorylation of a variety of
cellular proteins. Prominent tyrosine-phosphorylated bands were found
at molecular masses 40-42, 55-60, 70-72, and
105-120 kDa. The binding of antibodies against other surface
antigens did not enhance tyrosine phosphorylation, indicating the
specificity of this response. The magnitude of enhanced tyrosine
phosphorylation after antibody-mediated ligation of L-selectin was less
than that observed in cells treated with the chemoattractant, fMLP. One
possible explanation for this observation is that fMLP may be a more
potent stimulus than L-selectin ligation. Alternatively, ligation of
L-selectin by mAb may only partially mimic the interaction of
L-selectin with its natural ligand. Sulfatides induced larger increases
in tyrosine phosphorylation and MAP kinase activity than did ligation
of L-selectin by mAb. For P-selectin the binding sites for natural
ligand and sulfatides are overlapping(30) . This mimicry of the
natural ligand by sulfatides might explain the larger response to
sulfatides.
In the current study, we have carefully tried to exclude
the potential confounding effects of Fc receptor activation, since
tyrosine phosphorylation is a known consequence of Fc receptor
engagement in neutrophils(31) . Indeed, preliminary experiments
using whole IgG rabbit anti-mouse secondary Ab to cross-link
anti-L-selectin resulted in large increases in tyrosine phosphorylation
(data not shown). However, this response was not specific for
L-selectin as it was also seen after cross-linking a variety of other
cell surface antigens and was dependent upon the presence of the Fc
portion of the secondary antibody. Therefore, to rule out Fc-mediated
signaling, F(ab`)
In previous studies, binding of
anti-L-selectin antibody alone was insufficient to induce
Ca
In the current
study, treatment of cells with cytochalasin did not prevent
L-selectin-induced tyrosine phosphorylation. These results, on first
consideration, appear to be contrary to other reports in the
literature. However, using the hypothesis advanced by Fuortes et
al.(33) , it may be possible to reconcile our data with
reports that integrin cross-linking in platelets results in
cytochalasin-sensitive tyrosine
phosphorylation(24, 34) . In the study by Fuortes et
al.(33) , neutrophils adherent to physiologic substrates
and stimulated with tumor necrosis factor underwent rapid and prolonged
increases in tyrosine phosphorylation. This effect required
integrin-dependent adherence to physiologic substrates. Most of the
tyrosine-phosphorylated proteins were concentrated at points of contact
with the substratum as determined by immunofluorescence microscopy. In
their study, tyrosine phosphorylation was not inhibited by cytochalasin
but rather tyrosine dephosphorylation was enhanced. They speculated
that this was because an intact cytoskeleton served to sequester
phosphoproteins and protect them from phosphatases. Returning to the
studies with platelets(24, 34) , ligation of integrins
may have activated tyrosine kinases and subsequent aggregation and
cytoskeletal reorganization may have protected tyrosine-phosphorylated
proteins from phosphatases. Treatment with cytochalasin, by preventing
cytoskeletal reorganization and protection from phosphatases, would be
predicted to result in diminished tyrosine phosphorylation. In our
studies with suspended neutrophils, any effect of cytochalasin on
dephosphorylation was not apparent (perhaps because the cells were in
suspension at low density which did not allow aggregation or adherence
to a substrate). This might explain our findings that tyrosine
phosphorylation was not affected by cytochalasin. Alternatively, the
signaling pathways after L-selectin ligation and integrin cross-linking
may be distinct and differ in their sensitivity to cytochalasins.
An
intact cytoskeleton may be required for some L-selectin functions, such
as those occurring later or distally in the signaling pathways but may
not required for others, such as very early or proximal events. In
previous studies, cytochalasin did not abrogate the potentiation of the
oxidative burst that occurred after L-selectin
cross-linking(5) . However, cytochalasin was able to inhibit
leukocyte rolling and binding to HEV cells(25) . In the latter
study, mutated L-selectin with a deletion of the cytoplasmic tail was
similarly unable to mediate rolling or cell adhesion to HEV
endothelium. At least two possible explanations for these data exist:
that there is an interaction of components of the cytoskeleton with the
cytoplasmic tail of L-selectin (or associated molecule) or that the
cytoplasmic domain of L-selectin has a role in signaling and there is a
later requirement for an intact cytoskeleton during rolling and
adhesion. In this regard, there is data to support association between
selectins and signaling molecules: the CD3
Ligation of L-selectin by monoclonal antibodies or by sulfatides
resulted in similar patterns of enhanced tyrosine phosphorylation.
Sulfatides were used in these experiments largely to show that the
observed effects were not an artifact of the mAb used. However, the
potential significance of sulfatide-induced responses warrants further
consideration. Sulfatides are a heterogeneous group of sulfated
galactosyl-ceramides, with fatty acyl groups of varying lengths. They
are found in a variety of tissues, including brain, tumors, and on
leukocytes themselves. They are related to CD57, which is found on
endothelial cells. Although both CD57 and sulfatides are ligands for
L-selectin in vitro(21, 37) , their function in vivo is not yet known. They are almost certainly not the
inducible ligand on non-lymphoid endothelium. There is another
incompletely characterized ligand for L-selectin in the central nervous
system which has been localized to myelin(38) . It is noteworthy
that the central nervous system ligand is chemically quite distinct
from the endothelial ligand. The L-selectin ligand in the central
nervous system is resistant to sialidase, but sensitive to organic
solvents, suggesting that it has a lipid rather than a protein
backbone(38) . Sulfatide or a closely related sulfated
glycolipid might fulfil these criteria. Sulfatides on or released by
the neutrophil are also ligands for P-selectin. Sulfatides will inhibit
rolling of neutrophils on purified P-selectin, suggesting that
sulfatides bind to P-selectin on or close to the functional
site(39) . Indeed, the sulfatide binding site on P-selectin has
recently been mapped to a separate but overlapping site from the
carbohydrate binding site(30) . It has even been suggested that
binding of sulfatides may cause a conformational change in P-selectin
leading to ligand release. Thus, the physiological role for sulfatides
may be to regulate selectin disengagement that would allow cellular
transmigration. Whether sulfatides are indeed important ligands for
L-selectin in vivo and whether they function in an adhesion
promoting or inhibiting fashion is yet to be determined. It is
plausible that changes in tyrosine phosphorylation lead to decreased
rather than increased selectin-mediated adhesion.
As seen in Fig. 3B, the responses to fMLP and ligation of
L-selectin differ. Following fMLP treatment there is more prominent
phosphorylation of protein substrates at approximately 30, 49, and 55
kDa, when compared with L-selectin ligation. The identity of the
specific protein substrates, other than MAP kinase, and the
significance of these different patterns of phosphorylation remains
unknown. The magnitude of increased tyrosine phosphorylation was
generally smaller following L-selectin ligation when compared with
chemoattractant stimulation. However, it is possible that the amount of
tyrosine phosphorylation induced by interaction of L-selectin with its
natural ligand under physiologic conditions is larger. A more complete
understanding of this will depend upon the isolation and
characterization of the inducible endothelial ligand for neutrophil
L-selectin.
The significance of the activation of MAP kinase by
L-selectin ligation is as yet unknown. MAP kinase has been directly
linked to long term responses, such as proliferation(40) , but
its role in acute events in neutrophils is unclear. Recent evidence
suggests that MAP kinase activation does not occur early enough to
account for rapid responses, such as triggering the oxidative burst
(41). Activation of MAP kinase leads to phosphorylation, activation,
and translocation of cPLA
It is not possible to demonstrate the physiologic relevance
of MAP kinase activation in neutrophils, which would require specific
inhibition of the enzyme such as by overexpression of dominant negative
MAP kinase mutants. However, it does appear that tyrosine
phosphorylation is required for downstream signaling events leading to
functional neutrophil responses. The tyrosine kinase inhibitors,
genistein and herbimycin, significantly inhibited the cytosolic calcium
transient induced by sulfatides. We speculate that L-selectin is linked
to tyrosine kinases which activate phospholipase C
In summary,
ligation of L-selectin by either monoclonal antibodies or the natural
ligand sulfatides led to increased tyrosine phosphorylation of several
cellular proteins, including MAP kinase. These data confirm the
important role of L-selectin as a signaling molecule. Signaling
pathways, initiated by the engagement of L-selectin, may lead to
enhancement of a variety of neutrophil functions, such as
integrin-mediated adhesion, cytokine production, and the respiratory
burst.
levels and potentiation of the
oxidative burst. As several cell surface glycoproteins have been shown
to be linked to tyrosine kinases, we examined the hypothesis that
L-selectin may be linked to pathways involving tyrosine phosphorylation
in human neutrophils. Ligation of L-selectin by three different
antibodies recognizing separate epitopes led to increased tyrosine
phosphorylation of several cellular proteins as judged by
anti-phosphotyrosine immunoblots of whole cell lysates with prominent
bands at 40-42, 55-60, 70-72, and 105-120 kDa.
The 42-kDa band co-migrated with mitogen- activated protein (MAP)
kinase as determined by immunoblotting with anti-MAP kinase antibody.
This effect was specific for L-selectin, because antibodies against
CD18, CD45, and CD10 did not increase tyrosine phosphorylation.
Phosphorylation was not due to Fc binding, since F(ab`)
fragments of the anti-L-selectin antibodies were similarly
effective, and the response was unaffected by Fc receptor blockade.
Cross-linking of L-selectin was not required for enhanced tyrosine
phosphorylation, because monovalent Fab fragments also increased
tyrosine phosphorylation. The response to L-selectin antibodies was not
inhibited by cytochalasin, suggesting that reorganization of the actin
cytoskeleton was not required for this response. Sulfatides, sulfated
glycolipids which may be natural ligands for L-selectin, also induced a
rapid, dose-dependent increase in tyrosine phosphorylation. In
addition, sulfatides, but not control glycolipids, resulted in enhanced
tyrosine phosphorylation of MAP kinase. Both sulfatides and
anti-L-selectin antibodies increased kinase activity of MAP kinase as
determined by gel renaturation assay. The tyrosine kinase inhibitor,
genistein, blocked the transient increase in intracellular
Ca
and the oxidative burst induced by sulfatides,
suggesting that this tyrosine phosphorylation is functionally
important. We conclude that L-selectin is able to transmit
intracellular signals, including increased tyrosine phosphorylation and
activation of MAP kinase in neutrophils. We speculate that these events
may contribute to the activation of neutrophils during adhesion.
Reagents
Percoll and dextran T500 were obtained
from Pharmacia Biotech Inc. (Baie D'Urfe, Quebec, Canada).
KRPD(
)buffer was prepared using reagents from
Mallinckrodt (Paris, KY). Gelatin was from Bio-Rad (Hercules, CA).
Nonidet P-40 and phenylmethylsulfonyl fluoride were obtained from
Boehringer Mannheim (Laval, Quebec, Canada). Leupeptin, pepstatin A,
and aprotinin were obtained from ICN Biochemicals (Mississauga,
Ontario, Canada). Ethanolamine, 2-mercaptoethanol, Trizma (Tris base),
HEPES-buffered RPMI 1640, cytochalasin B and D, fMLP, herbimycin A,
bovine serum albumin, superoxide dismutase, cytochrome c, PMA,
sulfatides, and Type I and Type II cerebrosides were purchased from
Sigma. EDTA was obtained from BDH (Toronto, Ontario, Canada). DFP was
from Calbiochem (La Jolla, CA). Genistein was from Upstate
Biotechnology, Inc. (Lake Placid, NY). Paraformaldehyde was from J. B.
E. M. Services (Pointe Claire-Dorval, Quebec, Canada).
Antibodies
The murine anti-human L-selectin
antibodies, DREG 200, DREG 56 (whole molecule and F(ab`) fragment), and DREG 55 (all IgG
) were prepared as
described previously(17) . DREG 200 was proteolytically cleaved
using immobilized papain (Calbiochem) to generate monovalent Fab
fragments, which were purified by protein A affinity chromatography
(Bio-Rad Affi-Gel MAPS II System). Control mAb included: IB4, an
IgG
mouse anti-human CD18 provided by Dr. D. Chambers (San
Diego Regional Cancer Center); 4B2, an IgG
anti-CD45
provided by R. Sutherland (The Toronto Hospital); B-E3, an IgG
anti-CD10 (Serotec Canada, Toronto, Ontario, Canada); and an
IgG
anti-human PKC (Upstate Biotechnology, Inc.).
Secondary, cross-linking goat anti-mouse IgG antibodies were
affinity-purified cross-adsorbed F(ab`)
fragments from
Cappel Research Products (Organon Teknika, Durham, NC).
Anti-phosphotyrosine antibodies were from two sources: (i)
affinity-purified polyclonal anti-phosphotyrosine from Transduction
Laboratories (Lexington, KY) and (ii) affinity-purified monoclonal
anti-phosphotyrosine (4G10 hybridoma) from Upstate Biotechnology, Inc.
A monoclonal anti-MAP kinase antibody (raised against a peptide
corresponding to amino acids 325-345 of
p44
) from Zymed (San Francisco, CA) was used
for immunoblotting. Polyclonal anti-MAP kinase antibody, anti-ERK-2
(SC-154), obtained from Santa Cruz Biotechnology (Santa Cruz, CA) was
used for immunoprecipitation. Blocking antibodies against the
neutrophil Fc receptor were obtained from Medarex (Annandale, NJ). Fab
fragments of the monoclonal IV.3, a mouse IgG2b against human FcRII,
and F(ab`)
fragments of 3G8, a mouse IgG1 against FcRIII,
were used. ST-AR 9, which is a F(ab`)
fragment of rabbit
anti-mouse labeled with FITC, was used to stain unconjugated primary
antibodies (Serotec Canada).
Cell Isolation
Human neutrophils (>98% pure)
were isolated from citrated whole blood obtained by venipuncture, using
dextran sedimentation and discontinuous plasma-Percoll gradient
techniques as described previously(18) . The separation
procedure required 2 h, and the cells were used immediately after
isolation at a cell density of 8 10
/ml. The
functional integrity and nonactivated state of neutrophils isolated in
this manner have been extensively validated in previous
publications(18) .
Flow Cytometry
Surface expression of L-selectin,
CD18, FcRII, and HLA class I was measured using indirect
immunofluorescence and flow cytometry. Unless otherwise specified,
cells were labeled with primary mAb for 30 min on ice, washed, and
resuspended in PBS containing 0.5% bovine serum albumin and 20
mM glucose with FITC-labeled secondary antibody (1:500). After
a 30-min incubation on ice, cells were washed and resuspended in PBS
with 1.5% paraformaldehyde. Stained cells were analyzed on a FACScan
(Becton-Dickinson, Palo Alto, CA) using FL1 detector (488 nm excitation
and 530 nm emission wavelengths). Cells were gated on the forward and
right angle light scatter to exclude debris and cell clumps. Typically,
10,000 cells were analyzed per condition.
SDS-PAGE and Immunoblotting
Aliquots of cells were
either stimulated with sulfatides or control lipids in KRPD at 37
°C or resuspended in HEPES-buffered RPMI 1640 and incubated with
anti-L-selectin or control mAb for 20 min at 37 °C. The latter
cells were washed and resuspended in KRPD (which contained, in
mM, NaCl 122, NaHPO
3.1,
Na
HPO
12.5, KCl 4.8, MgSO
1.2,
CaCl
1, glucose 11). After stimulation, the cells were
rapidly sedimented, resuspended in boiling 2% SDS sample buffer, and
boiled for 15 min. Thirty µl of cell lysate (the equivalent of 8
10
cells) of each sample and molecular weight
standards were then subjected to SDS-polyacrylamide gel electrophoresis
through a 4-20% gradient polyacrylamide gel (Novex Experimental
Technology, San Diego, CA). Following electrophoresis, the proteins
were transferred to a nitrocellulose membrane (Schleicher and Schuell,
Keene, NH) and blocked overnight at 4 °C in blocking buffer (0.25%
gelatin, 10% ethanolamine, in 0.1 M Tris, pH 9.0). The
membrane was then incubated in antibody buffer (0.25% gelatin and 0.05%
Nonidet P-40 in 0.15 M NaCl, 5 mM EDTA, and 0.05 M Tris, pH 7.5) containing 0.2 µg/ml polyclonal
anti-phosphotyrosine Ab (Transduction Laboratories) for 2 h at room
temperature. Next, the membrane was washed three times in antibody
buffer, incubated with a 1:5000 dilution of peroxidase-conjugated
polyclonal anti-rabbit secondary Ab (Amersham, Oakville, Ontario,
Canada) for 1 h at room temperature, and washed three additional times
with antibody buffer alone. The membrane was then visualized using ECL
(Amersham Corp.) according to the manufacturer's instructions.
Similar results were obtained using the 4G10 monoclonal
anti-phosphotyrosine Ab (Upstate Biotechnology, Inc.) and
peroxidase-conjugated polyclonal anti-mouse Ab (Amersham) (data not
shown). To strip the membrane for reprobing with anti-MAP kinase, the
membrane was washed in water and then incubated in stripping buffer
(62.5 mM Tris, pH 6.8, 2 mM EDTA, and 100 mM
2-mercaptoethanol) for 30 min at 50 °C. The membrane was again
blocked overnight and blotted as described previously, except that the
primary antibody used was a 1:5000 dilution of anti-MAP kinase (Zymed,
San Francisco, CA) which is reactive against both the p42 and p44
isoforms of MAP kinase and the secondary Ab was peroxidase-conjugated
anti-mouse (Amersham).
MAP Kinase Immunoprecipitation and Gel Renaturation
Kinase Assay
Cells were pretreated with 2.5 mM DFP for
30 min at room temperature, washed, and resuspended at a concentration
of 2 10
cells/ml in HEPES-buffered RPMI. After
stimulation, the cells were sedimented and resuspended in boiling lysis
buffer (0.15 M NaCl, 5 mM EGTA, 10 mM sodium
pyrophosphate, 10 mM NaF, 1 mM sodium vanadate, 1
mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10
µg/ml leupeptin, 1 µM pepstatin A, 10% glycerol, 25
mM Tris, pH 7.5) containing 1% SDS and boiled for 15 min. The
lysate was diluted with 9 parts lysis buffer without SDS but containing
1% Triton X-100, 0.5% Nonidet P-40, and 1% sodium deoxycholate. The
lysate was sonicated for 15 min and then centrifuged in a
microcentrifuge for 15 min at 4 °C. The lysate was precleared by
incubation with 50 µl prewashed protein A/G-agarose beads (Santa
Cruz catalog number SC-2003) for 1 h at 4 °C. The supernatant was
transferred to a fresh tube, and 50 µl of polyclonal anti-MAP
kinase Ab (Santa Cruz) was added for 2 h at 4 °C. This mixture was
then added to 50 µl of prewashed protein A/G beads and incubated
overnight at 4 °C. The immune complexes were washed twice with
washing buffer (25 mM Tris, pH 7.4, 2 mM EDTA, 150
mM NaCl) and then boiled for 5 min in Laemmli sample buffer.
The beads were sedimented and the supernatants analyzed by SDS-PAGE as
described previously except that the equivalent of 2
10
cells were loaded per lane. For immunoblotting, the monoclonal
anti-phosphotyrosine (4G10) from Upstate Biotechnology, Inc. was used
because of diminished nonspecific binding compared with the polyclonal
Ab.
10
cells/lane) or whole cell lysate (the equivalent of 5
10
cells/lane), the SDS was removed by washing the gel
twice in 50 mM Tris, pH 8.0, 20% isopropyl alcohol for 30 min
each time. This was followed by a 1-h incubation in 50 mM Tris, pH 8.0, 5 mM 2-mercaptoethanol and then two 30-min
incubations in 6 M guanidine HCl, 50 mM Tris, pH 8.0,
5 mM 2-mercaptoethanol. The gel was renatured by incubation in
five changes of buffer (50 mM Tris, pH 8.0, 5 mM 2-mercaptoethanol, and 0.04% Tween 40) over 12-18 h at 4
°C. The kinase reaction was carried out by incubation of the gel in
kinase buffer (25 mM HEPES, pH 8.0, 2 mM dithiothreitol, 0.1 mM EGTA, 5 mM MgCl
, 25 µM ATP, and 25 µCi
[
-
P] ATP) for 1 h at 21 °C. The gel was
then washed repeatedly with 5% trichloroacetic acid containing 1%
sodium pyrophosphate until the washes contained minimal radioactivity.
The gel was then dried and exposed using a PhosphorImager (Molecular
Dynamics, Sunnyvale, CA) followed by conventional autoradiography using
KODAK X-Omat film.
Measurement of Cytosolic Calcium
Concentration
Neutrophils were loaded by incubation for 20 min
at 37 °C in HEPES-buffered RPMI containing 2 µM Fura-2
AM. Cells were washed and resuspended in Ca measuring
buffer (in mM, NaCl 140, KCl 5, HEPES 10, glucose 10,
MgCl
1, pH 7.4, and CaCl
1). Fura-2
fluorescence was measured in a Hitachi F2000 fluorometer using an
excitation wavelength of 335 nm and an emission wavelength of 495 nm.
The fluorescence was calibrated using the ionomycin/Mn
technique(20) .
Measurement of Superoxide Production
Superoxide
production by neutrophils was determined as described(5) .
Neutrophils were treated with the indicated stimuli at 37 °C in
KRPD containing cytochrome c (75 µM) for 10 min.
Reference tubes were treated identically except superoxide dismutase
(30 units/ml) was added before stimulation. The absorbance at 550 nm
was used to calculate the superoxide dismutase-inhibitable reduction of
cytochrome c.
transients and increased tumor necrosis
factor and interleukin-8 mRNA in intact human neutrophils(6) . Fig. 1B illustrates that exposure of neutrophils to
sulfatides resulted in a dose-dependent increase in total cellular
tyrosine phosphorylation. This effect was not seen after treatment with
Type I or Type II cerebrosides which have the same glycolipid structure
but lack the sulfate group and therefore do not bind
L-selectin(21) . Tyrosine phosphorylation was markedly
diminished (Fig. 2A) if neutrophils were pretreated with
chymotrypsin, which cleaves L-selectin (6, 22) but not
other neutrophil surface antigens from the cell surface (Fig. 2B), prior to sulfatides treatment. Although the
magnitude of the response to sulfatides was greater than that due to
mAb-mediated L-selectin ligation, the phosphorylation patterns and the
kinetics of the response after mAb-mediated ligation of L-selectin or
stimulation with sulfatides were indistinguishable. In both cases the
response was rapid, occurring within 30 s, and persisted for at least
20 min (data not shown).
Figure 1:
Ligation of L-selectin increases
tyrosine phosphorylation of several cellular proteins. A,
human neutrophils (8 10
/ml) were incubated with or
without mAb at 30 µg/ml for 20 min at 37 °C, washed, and
resuspended. After 2 min the cells were sedimented and resuspended in
boiling sample buffer and analyzed by SDS-PAGE and anti-phosphotyrosine
immunoblotting as described under ``Experimental
Procedures.'' Increased tyrosine phosphorylation is seen after
treatment with the anti-L-selectin mAbs, DREG 56 and DREG 200, but not
after treatment with IB4 (anti-CD18), 4B2 (anti-CD45), or B-E3
(anti-CD10). B, sulfatides increase tyrosine phosphorylation
in human neutrophils. Neutrophils were incubated alone or with 400
µg/ml of either Type I or Type II cerebrosides (nonsulfated
glycolipids) or with increasing amounts of sulfatides (sulfated
glycolipids). Sulfatides caused a dose-dependent increase in tyrosine
phosphorylation, not seen even after treatment with 400 µg/ml of
the control lipids.
Figure 3:
The increased response after L-selectin
ligation is specific for phosphotyrosine and is inhibited by the
tyrosine kinase inhibitor, genistein. A, neutrophils were
incubated with either the anti-L-selectin Ab, DREG 56, or the
L-selectin ligand, sulfatides, before analysis by SDS-PAGE and
phosphotyrosine immunoblotting as described under ``Experimental
Procedures.'' After immunoblotting of parallel samples, the
nitrocellulose membrane was divided and incubated in the primary
anti-phosphotyrosine Ab in the presence or absence of 1 mMO-phosphotyrosine. While some nonspecific bands are
visible at 15, 20, 29, and 50 kDa in all lanes, the majority of the
higher molecular mass bands observed above 32.5 kDa were specific and
were not seen in the presence of excess phosphotyrosine. B,
neutrophils were incubated with genistein at the indicated dose for 30
min at 37 °C, washed, and treated with 400 µg/ml sulfatides or
cytochalasin B and fMLP for 2 min. Cells were sedimented, boiled in
sample buffer, and analyzed by SDS-PAGE and immunoblotting. The
response to sulfatides is inhibited in a dose-dependent fashion by
genistein.
Figure 2:
Chymotrypsin pretreatment inhibits the
response to sulfatides and selectively cleaves L-selectin. A,
neutrophils were incubated with 10 units/ml chymotrypsin at 37 °C
for 10 min. After washing, the cells were untreated or stimulated with
either sulfatides or fMLP. Pretreatment with chymotrypsin by itself has
no effect on tyrosine phosphorylation (lanes 1 and 2)
but does significantly decrease the response to sulfatides (lanes 3 and 4). The response to fMLP is unaffected (lanes 5 and 6). B, neutrophils were incubated with
chymotrypsin as in A and then incubated with saturating
amounts of the indicated primary antibody for 30 min on ice. Next the
cells were washed, stained with FITC-labeled secondary antibody, and
fixed before analysis on a FACScan. Decreased binding of
anti-L-selectin antibody is seen after chymotrypsin treatment, whereas
the binding of antibodies against CD18, FcRII, and HLA class I is
unaffected. Without chymotrypsin treatment is shown as lightly
stippled, whereas chymotrypsin-pretreated cells are shown with the dark line.
To confirm that the detection system was
specific for tyrosine phosphorylation, the nitrocellulose membrane was
incubated with primary Ab in the presence of 1 mMO-phosphotyrosine. As shown in Fig. 3A, excess
phosphotyrosine significantly reduced the intensity of the majority of
higher molecular weight bands, demonstrating that the response to both
anti-L-selectin antibodies and the L-selectin ligand, sulfatides,
indeed represents phosphorylation of tyrosine residues. The nonspecific
reaction with lower M proteins was due to
nonspecificity of the primary Ab, since no bands were observed when the
blot was developed in the absence of primary Ab (data not shown).
Similar increases in tyrosine phosphorylation, especially at
40-42 kDa, were also observed when an alternate
anti-phosphotyrosine antibody, 4G10, was used (data not shown). The
response to sulfatides was inhibited, in a dose-dependent manner, by
the tyrosine kinase inhibitor, genistein, suggesting that the tyrosine
phosphorylation response was due, at least in part, to activation of
protein tyrosine kinases (Fig. 3B).
fragment of DREG 56. Fig. 4A demonstrates that binding of F(ab`)
fragments resulted
in a clear increase in tyrosine phosphorylation with a pattern similar
to that induced by whole Ab, although the magnitude of this response
was smaller. Both whole immunoglobulin and F(ab`)
fragments
are bivalent molecules and are theoretically capable of cross-linking
two adjacent L-selectin molecules which might lead to intracellular
signals. Alternatively, univalent ligation of L-selectin might be
sufficient. To distinguish between these possibilities, DREG 200 was
subjected to proteolytic cleavage into monovalent Fab fragments and
then purified to remove contaminating Fc fragments and whole IgG. Fig. 4B illustrates that binding of these Fab fragments
was also able to induce an increase in tyrosine phosphorylation, albeit
of lesser magnitude than after L-selectin ligation by whole mAb. In
addition, pretreatment of neutrophils with saturating amounts of
blocking antibodies to both neutrophil Fc receptors, FcRII and FcRIIIB,
did not inhibit tyrosine phosphorylation induced by anti-L-selectin
mAbs (Fig. 4C). Taken together, these data suggest that
univalent ligation of L-selectin is sufficient to induce tyrosine
phosphorylation and that neither cross-linking nor Fc-mediated events
are required.
Figure 4:
Increased tyrosine phosphorylation is not
due to FcR binding. A, neutrophils were incubated without
antibody or with either whole IgG or the F(ab`) fragment of
DREG 56 (anti-L-selectin). Increased tyrosine phosphorylation was still
observed after treatment with the F(ab`)
fragment. B, Fab fragments also increase tyrosine phosphorylation. The
anti-L-selectin mAb, DREG 200, was proteolytically cleaved by papain to
yield Fab fragments. Neutrophils were incubated without mAb or with
either whole DREG 200 or the Fab fragment before analysis by SDS-PAGE
and immunoblotting. C, blockade of Fc receptors does not
prevent anti-L-selectin-induced tyrosine phosphorylation. Neutrophils
were preincubated with saturating amounts of antibodies against FcRII
(IV.3 Fab) and/or FcRIII (3G8 F(ab`)
) for 30 min on ice
before stimulation with the anti-L-selectin mAb DREG 200. Treatment
with the anti-FcR antibodies alone does not increase tyrosine
phosphorylation, and the response to anti-L-selectin mAb is
unchanged.
In previous studies(5) , we observed that
binding of primary antibody alone was insufficient to increase
intracellular calcium or enhance the oxidative burst: cross-linking
with secondary Ab was required. Accordingly, the effect of
cross-linking L-selectin on tyrosine phosphorylation was examined.
Cells were treated with primary anti-L-selectin mAb, washed, and
resuspended at 37 °C before addition of goat anti-mouse Ig
(F(ab`) fragment. After addition of the cross-linking
antibody, total tyrosine phosphorylation was never increased and a
decrease in phosphorylation of the band in the 40-kDa region was
reproducibly observed (Fig. 5).
Figure 5:
Cross-linking of L-selectin using
secondary Ab does not increase tyrosine phosphorylation. Neutrophils
were incubated with or without the anti-L-selectin mAb, DREG 56, for 20
min at 37 °C. After washing the cells were resuspended and treated
either with or without secondary, cross-linking goat anti-mouse (GAM) Ab. After 2 min the cells were sedimented, resuspended
in boiling sample buffer, and analyzed as before. Increased tyrosine
phosphorylation is seen after L-selectin ligation (lane 3)
which is not increased after cross-linking (lane 4). One
particular band at approximately 40 kDa was consistently
dephosphorylated after cross-linking.
Antibody-mediated
cross-linking of platelet integrins leads to rapid increases in
tyrosine phosphorylation of several cellular proteins (24). This
increase is inhibitable by pretreatment with cytochalasin, suggesting
that integrins transmit their signals via the actin cytoskeleton.
However, as illustrated in Fig. 6, cytochalasin D, at doses up to
5 µM, did not inhibit tyrosine phosphorylation induced by
binding of anti-L-selectin antibodies. These data suggest that, unlike
induction of tyrosine phosphorylation in platelets by integrin
cross-linking(25) , enhancement of tyrosine phosphorylation by
L-selectin does not require cytoskeletal reorganization in neutrophils.
Figure 6:
Tyrosine phosphorylation is not inhibited
by cytochalasin D. Neutrophils were incubated in the indicated
concentration of cytochalasin D (CYTO D) for 10 min prior to
addition of the anti-L-selectin mAb DREG 56. After incubation with or
without mAb, the cells were sedimented and resuspended in boiling
sample buffer. SDS-PAGE and immunoblotting was performed as above.
Increased tyrosine phosphorylation is observed after L-selectin
ligation (lane 2), which is not inhibited by either 2 or 5
µM cytochalasin D (lanes 3 and 4,
respectively).
As noted previously, prominent tyrosine phosphorylated bands were
seen in the 40-42 kDa range after treatment with either
anti-L-selectin antibodies or sulfatides. The MAP kinase pathway has
recently been shown to be activated by chemoattractants in human
neutrophils(26, 27) and MAP kinase itself is one of the
most prominent phosphorylated bands in chemoattractant-activated
neutrophils(28) . Because MAP kinase consists of 42- and 44-kDa
isoforms, consistent with the bands observed here, we examined whether
the MAP kinase was phosphorylated after ligation of L-selectin. After
treatment with anti-L-selectin antibodies (Fig. 7A) or
sulfatides (Fig. 7B), increased tyrosine phosphorylation
of 40-42 kDa protein species were observed. The nitrocellulose
membrane was stripped and reprobed with anti-MAP kinase mAb. Fig. 7illustrates that MAP kinase co-migrated with the
tyrosine-phosphorylated band at an estimated molecular mass of 42
kDa.(
)
Figure 7:
The prominent 42-kDa band co-migrates with
MAP kinase. Neutrophils were treated with either DREG antibodies (A) or sulfatides or cytochalasin B and fMLP (B) as
described previously. After SDS-PAGE and immunoblotting, the
nitrocellulose membranes were probed with anti-phosphotyrosine (lanes 1-4 in A, lanes 1-3 in B). The same membrane was then stripped and re-probed with
anti-MAP kinase (lanes 5-8 in A, lanes
4-6 in B). Both DREG 200 and sulfatides increase
tyrosine phosphorylation of several cellular proteins, including a
42-kDa band which appears to co-migrate with MAP
kinase.
To prove more definitively that MAP
kinase itself was tyrosine-phosphorylated, MAP kinase was
immunoprecipitated, resolved by SDS-PAGE, and transferred to a
nitrocellulose membrane which was probed with monoclonal
anti-phosphotyrosine antibody. Fig. 8illustrates that after
treatment of cells with sulfatides, MAP kinase was phosphorylated on
tyrosine. This phosphorylation was not seen if cells were incubated
with chymotrypsin, which cleaves L-selectin but not other surface
antigens such as FcRII, HLA class I, or CD18 (Fig. 2B)
from the cell surface(6, 22) , prior to treatment with
sulfatides (lane 3). Similar negative results were also
obtained using Type I and Type II galactocerebrosides as negative
controls (data not shown). To determine if MAP kinase was activated
following L-selectin ligation, gel renaturation kinase assays were
performed using both whole cell lysate and MAP kinase
immunoprecipitates. Cells were treated with control cerebrosides,
sulfatides, or cytochalasin B and fMLP before immunoprecipitation of
MAP kinase from the cell lysate. Samples of whole cell lysate or
immunoprecipitate were resolved on SDS-PAGE gels which incorporated the
MAP kinase substrate, myelin basic protein. After renaturation of the
protein within the gel, kinase activity was detected by incubating the
gel with [P]ATP. As Fig. 9A demonstrates, sulfatides, but not control cerebrosides, increased
the MAP kinase activity in both whole cell lysates and MAP kinase
immunoprecipitates. Using densitometric analysis, the kinase activity
induced by sulfatides was 20% of the response induced by cytochalasin B
and fMLP, consistent with the lower intensity of MAP kinase
phosphorylation seen in Fig. 8. Similarly, treatment of cells
with anti-L-selectin mAbs but not control mAbs resulted in activation
of MAP Kinase activity (Fig. 9B).
Figure 8:
Sulfatides increase tyrosine
phosphorylation of MAP kinase. Neutrophils were pretreated with DFP for
30 min, washed, and resuspended at 2 10
cells/ml in
HEPES-buffered RPMI. Cells were then treated with chymotrypsin 10
units/ml in PBS (lane 3) or PBS alone (lane 1, 2, and 4). After 10 min cells were treated with PBS alone, 400
µg/ml sulfatides, or cytochalasin B followed by fMLP. After 2 min
the cells were sedimented, lysed, and boiled and MAP kinase was
immunoprecipitated. The immunoprecipitate was resolved on SDS-PAGE and
immunoblotted with monoclonal anti-phosphotyrosine. Sulfatides caused
an increase in tyrosine phosphorylation of a 42-kDa band in the MAP
kinase immunoprecipitate (lane 2), which was not seen if
L-selectin was enzymatically cleaved by chymotrypsin pretreatment (lane 3). The response to cytochalasin B and fMLP is shown as
a positive control.
Figure 9:
Sulfatides or anti-L-selectin mAb cause
activation of MAP kinase activity. A, neutrophils were treated
with sulfatides or cytochalasin B and fMLP as described in the legend
to Fig. 8. After immunoprecipitation of MAP kinase, the
immunoprecipitate was resolved on 10% polyacrylamide gels containing
the MAP kinase substrate, myelin basic protein. The proteins within the
gel were then renatured and incubated with
[-
P]ATP, washed, and exposed to
autoradiographic film. Sulfatides caused an increase in the kinase
activity of the MAP kinase immunoprecipitate (lane 4), whereas
treatment with the control cerebrosides (lane 2 and 3) did not. The response of cells treated with cytochalasin B
and fMLP is shown as a positive control. B, neutrophils were
treated with mAb as in Fig. 1 or sulfatides (lane 6) as above.
Anti-L-selectin antibodies (lanes 2 and 3), but not
mAb against CD18 (IB4) or CD45 (4B2) stimulate MAP kinase
activity.
To determine if
enhanced tyrosine phosphorylation was necessary for any downstream
response to L-selectin ligation, cellular tyrosine kinases were
inhibited with the chemically unrelated tyrosine kinase inhibitors,
genistein, and herbimycin A. Both compounds inhibited the cytosolic
calcium transient in response to sulfatides (Fig. 10A and data not shown). Such transient increases in cytosolic calcium
have previously been shown to be required for potentiation of the
oxidative burst(5) . In addition, tyrosine kinase inhibition
with genistein decreased the amount of superoxide radical released from
neutrophils stimulated with sulfatides (Fig. 10B).
Genistein did not nonspecifically inhibit the NADPH oxidase, since the
oxidative burst induced by PMA was unaltered in its presence.
Figure 10:
Tyrosine phosphorylation is important and
the tyrosine kinase inhibitor, genistein, inhibits downstream events
induced by sulfatides. A, cells were incubated in 150
µM genistein or equivalent levels of the vehicle, DMSO,
simultaneously with Fura-2 AM for 30 min at 37 °C. After washing,
the cells were resuspended in Ca measuring buffer and
stimulated with 100 µg/ml sulfatides and subsequently fMLP
(10
M). Genistein selectively inhibits the
response to sulfatides but does not impair the calcium transient
induced by fMLP. B, neutrophils were incubated in genistein as
in A and resuspended in KRPD with cytochrome c.
Production of O
was measured as described
under ``Experimental Procedures.'' Sulfatides induce a
measurable oxidative burst which return to control levels in cells
pretreated with genistein. Genistein does not significantly affect the
response to PMA. Note broken y axis to accommodate the much
larger response to PMA.
, Me
SO;
,
genistein.
fragments of DREG 56 were tested for
their ability to induce tyrosine phosphorylation. While the response
was of lower magnitude, these fragments were capable of inducing
increases in tyrosine phosphorylation. Similar supporting evidence was
also obtained using Fab fragments of DREG 200. In addition, blocking
neutrophil Fc receptors with combined treatment with IV.3 and 3G8, mAbs
against FcRII and FcRIII, respectively, did not inhibit the response to
anti-L-selectin mAbs. Furthermore, chymotrypsin did not remove FcRII
from the cell surface but did inhibit the response to sulfatides,
supporting our conclusion that L-selectin, and not FcRII, is the actual
signal-transducing molecule.
transients or potentiate the oxidative
burst(5, 6) , whereas in the present study, tyrosine
phosphorylation occurred after binding of whole anti-L-selectin mAb
alone. One possibility was that bivalent antibodies were cross-linking
two adjacent L-selectin molecules which might have been sufficient to
transduce intracellular signals. However, this did not appear to be
required, since Fab fragments of anti-L-selectin antibodies induced
similar responses, implying that univalent ligation of L-selectin was
capable of induction of tyrosine phosphorylation. We speculate that
L-selectin-mediated signaling may be a two-step process. Certain
intracellular signals are transmitted by univalent ligation, including
but perhaps not limited to tyrosine phosphorylation, whereas other
signals, including Ca
transients, require subsequent
cross-linking of L-selectin. Surprisingly, deliberate cross-linking
with secondary anti-mouse antibodies did not cause an increase in
phosphorylation. In fact, such treatment appeared to cause
dephosphorylation, at least of the 40-kDa band. This may have been due
to proteolytic cleavage of L-selectin, resulting in termination of the
signal, as antibody-mediated cross-linking has been reported to cause
shedding of L-selectin from the surface of the neutrophil(32) .
More specifically, protein tyrosine kinases regulated by L-selectin
might be deactivated by cleavage and shedding of L-selectin with
termination of the presumed ``on'' signal. However, it was
not possible to test this hypothesis, since the cleavage of L-selectin
cannot be inhibited by any known protease inhibitors(12) .
Alternatively, cross-linking may induce activation of a protein
tyrosine phosphatase. It is not currently known if this
dephosphorylation has any physiological consequences.
chain
co-immunoprecipitates with L-selectin in lymphocytes (35) and
P-selectin co-immunoprecipitates with
pp60
in platelets(36) .
(42, 43) , which
has been suggested to play an important role in neutrophil priming and
other responses. The pathways upstream of MAP kinase have recently been
characterized and can be activated by receptor tyrosine kinases (44) or through G-protein coupled receptors(27) .
Activation of MAP kinase by integrins has been recently reported (45) but activation by any selectin appears to be a novel
finding.
which is found
in neutrophils (46) while Ca
mobilization
after fMLP stimulation is mediated by phospholipase C
and is
therefore unaffected by tyrosine kinase inhibition. The level of
intracellular calcium in neutrophils is an important regulator of a
variety of neutrophil processes, such as motility and phagocytosis,
exocytosis, priming, and the respiratory burst. We have previously
shown that prevention of such calcium transients prevents the
potentiation of the oxidative burst seen after L-selectin
cross-linking(5) . Treatment with sulfatides but not
antibody-mediated L-selectin ligation is sufficient to activate the
respiratory burst. Crockett-Torabi et al. (7) have recently
reported that cross-linking L-selectin using antibodies adherent to a
planar surface does, in fact, activate NADPH oxidase. Sulfatides are
poorly soluble in aqueous buffer and form micelles. The detectable
oxidative burst after sulfatides might be due to large micelles acting
as a planar surfaces or ligand mimcry as discussed earlier. These minor
discrepancies notwithstanding, the important observation is that the
oxidative burst can be inhibited by genistein. Thus, for L-selectin
signaling it appears that tyrosine phosphorylation is necessary for
Ca
signaling, and both are required for the
downstream events, in this case, the oxidative burst.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.