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RII with Low-Density Detergent-Resistant Membranes Is Important for Cross-Linking-Dependent Initiation of the Tyrosine Phosphorylation Pathway and Superoxide Generation1
*
Department of Physiology, Nihon University School of Dentistry, Matsudo, Japan;
Department of Hard Tissue Engineering, Biochemistry, Division of Bio-Matrix, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan;
Centre de Recherches Biomédicals des Cordeliers, Paris, France;
Department of Microbiology, Nihon University School of Medicine, Tokyo, Japan;
¶ Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan; and
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Brain Science Institute, Institute of Physical and Chemical Research, Wako, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Rs). In the present study, we
investigated role of lipid rafts, glycolipid- and cholesterol-rich
membrane microdomains, in the FcR-mediated responses. In retinoic
acid-differentiated HL-60 cells, cross-linking of FcRs resulted in a
marked increase in the tyrosine phosphorylation of FcRIIa,
p58lyn, and p120c-cbl,
which was inhibited by a specific inhibitor of Src family protein
tyrosine kinases. After cross-linking, FcRs and
tyrosine-phosphorylated proteins including
p120c-cbl were found in the low-density
detergent-resistant membrane (DRM) fractions isolated by
sucrose-density gradient ultracentrifugation. The association of
FcRs as well as p120c-cbl with DRMs did not
depend on the tyrosine phosphorylation. When endogenous cholesterol was
reduced with methyl-
-cyclodextrin, the cross-linking did not induce
the association of FcRs as well as p120c-cbl
with DRMs. In addition, although the physical association between
FcRIIa and p58lyn was not impaired, the
cross-linking did not induce the tyrosine phosphorylation. In human
neutrophils, superoxide generation induced by opsonized zymosan or
chemoattractant fMLP was not affected or increased, respectively, after
the methyl--cyclodextrin treatment, but the superoxide generation
induced by the insoluble immune complex via FcRII was markedly
reduced. Accordingly, we conclude that the cross-linking-dependent
association of FcRII to lipid rafts is important for the activation
of FcRII-associated Src family protein tyrosine kinases to initiate
the tyrosine phosphorylation cascade leading to superoxide
generation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Rs) are expressed on phagocytic
leukocytes and play a central role in immune responses following
interaction with Ab-Ag complexes. These responses include phagocytosis,
endocytosis, Ab-dependent cellular cytotoxicity, superoxide generation,
release of inflammatory mediators, as well as immune complex clearance
(for review, see Refs. 1, 2, 3, 4). FcR-mediated responses
are induced by multivalent Ag-Ab complexes that allow clustering of
FcRs, but not by monomeric IgG that binds to FcR with a 1:1
stoichiometry value (5, 6). Therefore, the molecular
mechanism by which clustering of FcRs triggers cell activation is
one basis for defense against IgG-opsonized pathogens.
FcRs have been classified into three classes (1, 2, 3, 4):
FcRI, FcRII (FcRIIa and FcRIIb), and FcRIII (FcRIIIa
and FcRIIIb). FcRI, FcRIIa, and FcRIII are individually
capable of inducing the phagocytosis of IgG-coated cells. FcRI have
high affinity for monomeric IgG, whereas FcRII and FcRIII have
low affinity for monomeric IgG and bind IgG-containing immune
complexes. Each class of FcRs is clustered by cross-linking of IgG
with multivalent Ag. Structurally, FcRI and FcRIIIa are composed
of oligomeric complexes along with - and 
-chain homo- and
heterodimers. The associated subunits contain the two YXXL boxes termed
immunoreceptor tyrosine-based activation motif
(ITAM).3 FcRIIIb is
a GPI-linked form of FcRIII. In contrast, receptors of the
FcRII class are monomeric transmembrane proteins and possess a
signal motif, similar but not identical to ITAM, within their
cytoplasmic region.
Although FcRs have no intrinsic tyrosine protein kinase (PTK)
activities, intracellular Src family protein tyrosine kinases
(Src-PTKs) are activated upon clustering of FcRs and phosphorylate
tyrosine residues in the ITAM (7, 8, 9). Phosphorylated ITAM
then serves as a docking site for the Src homology (SH) 2 domain
of cytosolic protein kinase p72syk
(10, 11, 12, 13). Such a tyrosine phosphorylation cascade is
required for FcR-mediated phagocytosis (14) and
superoxide generation (15). So far, physical and
functional association of Src-PTKs (Lyn and Hck) with FcRI
(9) and FcRIIa (7) have been reported.
Furthermore, the cross-linking-dependent activation of Lyn that is
constitutively associated with the cytoplasmic tail of the FcRIIa
has been suggested in neutrophils (16). However, the
molecular mechanism by which the aggregation of FcRs on the cell
surface induces the activation of intracellular Src-PTKs has not been
elucidated.
Recent advances in membrane biology suggest the glycolipid- and
cholesterol-rich membrane microdomains as lateral structural components
of the plasma membranes. These microdomains are called lipid rafts and
have been proposed to function as platforms for both signal
transduction and membrane trafficking (for review, see Ref.
17). Lipid rafts are considered to be formed by tight
packing of long and mostly saturated acyl chains of glycolipids
interspaced by cholesterol (18). The components of lipid
rafts are biochemically separated as detergent-resistant membranes DRMs
or detergent-insoluble glycolipid-enriched domains based on their
insolubility in the detergent Triton X-100 in the cold (19, 20). Because of their high lipid content, DRMs can be isolated
in the low-density fraction after gradient centrifugation. DRMs
concentrate GPI-anchored proteins (19, 21, 22), some
transmembrane proteins including influenza hemagglutinin
(23), and also intracellular signaling proteins such as
dually acylated Src-PTKs Lck, Lyn, and Fyn (24) or
heterotrimeric GTP-binding proteins (25) and
phosphatidylinositol bisphosphate (26). Lipid rafts are
therefore likely to act as scaffolding for both extracellular proteins
and intracellular molecules. In the immune system, the requirement of
lipid rafts for tyrosine phosphorylation mediated via high-affinity FcR
for IgE (Fc
RI) (27, 28, 29) and TCR (30, 31, 32)
has been reported.
Among the various FcRs, FcRII is most widely distributed.
Kwiatkowska and Sobota (33) recently suggested the
recruitment of cross-linked FcRII to lipid rafts based on analysis
of the high molecular mass complex of clustered FcRII isolated by
gel filtration. In the present study, we report that lipid rafts are
crucial machinery in which clustering of FcRIIa induces the
activation of Src-PTKs. Moreover, it is shown that a lipid
raft-disrupting reagent (methyl--cyclodextrin (MCD)) inhibits
FcRII-mediated superoxide generation in human neutrophils, whereas
this reagent dose not impair the superoxide generation induced by
FcRIIIb/complement receptor 3 or formyl peptide receptor (G
protein-coupled receptor).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The human myeloid cell lines HL-60 was cultured in RPMI 1640 containing 10% heat-inactivated FCS and 200 µg/ml kanamycin at 37°C in 95% air and 5% CO2. HL-60 cells were treated with 1 µM retinoic acid (RA) for 2 days (RA-HL-60) as described previously (34). K562, a human erythroleukemia cell line, was obtained from The Institute of Physical and Chemical Research Cell Bank (Tsukuba, Japan) and maintained similarly.
Materials and Abs
Human IgG purified from serum and mouse IgG3 were purchased from
ICN Pharmaceuticals (Aurora, OH). Human IgG was
125I iodinated with chloramine-T. E-RDF medium
was purchased from Kyokuto Pharmaceutical (Tokyo, Japan). The medium
was completely serum free and contained insulin and transferrin.
4-Amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine
(PP1) was purchased from Biomol (Plymouth Meeting, PA). Anti-FcRIIs
mAb (IV.3, IgG2b) was purchased from Medarex (Annandale, NJ) and
125I iodinated with chloramine-T.
F(ab')2 of IV.3 was prepared by pepsin digestion
followed by protein A-Sepharose chromatography. Purity (>99%) was
assessed by SDS-PAGE and Coomassie brilliant blue staining.
Anti-FcRIIa polyclonal Ab (pAb 260) was raised against the
intracellular region of FcRIIa (35). Anti-Lyn mAb
(Lyn9) was purchased from Wako Pure Chemicals (Osaka, Japan).
Anti-Cbl, anti-Syk, and anti-SLP-76 polyclonal Abs were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Syk
polyclonal Ab was also purchased from Upstate Biotechnology (Lake
Placid, NY). HRP-conjugated anti-phosphotyrosine mAb (HRP-PY20) was
purchased from Amersham International (Buckinghamshire, U.K.).
Affinity-purified goat anti-human IgG (GAH)
F(ab')2 Ab was purchased from Rockland
(Gilbertsville, PA). Affinity-purified F(ab')2 of
goat anti-mouse IgG (GAM) Ab was purchased from Kirkegaard & Perry
Laboratories (Gaithersburg, MD). FITC-conjugated mouse anti-human
FcRII mAb (FITC-IV.3), FITC-conjugated mouse IgG3, and
anti-FcRI mAb were purchased from BD PharMingen (San Diego,
CA).
Cross-linking of FcRs
RA-HL-60 cells were washed with E-RDF three times and suspended
in E-RDF at a cell density of 2 x 108
cells/ml. The cells (200 µl) were incubated with 5 µg/ml
IgG/125I-labeled IgG for 25 min on ice. After
preincubation for 3 min at 37°C, they were treated with or without 80
µg/ml GAH for 1 min. The reaction was terminated on ice by adding the
same volume of a lysis buffer consisting of 40 mM Tris-HCl (pH 8.0),
300 mM NaCl, 2% Triton X-100, 20 mM iodoacetamide, 2 mM sodium
orthovanadate, 10 mM sodium fluoride, 10 mM sodium pyrophosphate, 20 mM
EDTA, 20 mM -glycerophosphate, 20 µg/ml leupeptin, 20 µg/ml
pepstatin, 2 mM PMSF, and 20 µg/ml aprotinin. The lysates were
directly subjected to sucrose density gradient centrifugation or
centrifuged at 14,000 x g for 20 min before
immunoprecipitation. For selective cross-linking of FcRI or
FcRII, the cells were incubated with the indicated amount of
anti-FcRI mAb or anti-FcRII mAb (either IgG or
F(ab')2), respectively, then with
F(ab')2 of GAM.
Immunoprecipitation
Protein G-agarose beads (30 µl) were coated with 5 µg of IV.3 or mouse IgG3 and washed with washing buffer containing 20 mM Tris-HCl (pH 8) and 150 mM NaCl. The cell lysates (200 µl) were incubated with the beads for 2 h at 4°C. As negative controls, the immunoprecipitation was done using mouse IgG3 instead of IV.3 or in the absence of Ab. The beads were washed five times with lysis buffer containing 0.5% Triton X-100 and the immunoprecipitates were separated by SDS-PAGE.
Sucrose density gradient ultracentrifugation
The cell lysate was adjusted to 40% sucrose by the addition of the same volume of 80% sucrose in MES-buffered saline (MBS); 25 mM MES (pH 6.5), 150 mM NaCl, and 2 mM sodium orthovanadate and was placed at the bottom of an ultracentrifuge tube. A step gradient of 5–30% sucrose (5% steps, 400 µl each) in MBS was formed above the 40% homogenate. The mixture was centrifuged at 250,000 x g for 17 h at 4°C in a SW60Ti rotor (Beckman Instruments, Palo Alto, CA). Gradient columns were fractionated into 16 equal volumes (200 µl) taken from the top of the column. The radioactivity of 125I-labeled IgG present in each fraction was determined with a COBRA auto gamma counter (Packard Instrument, Downers Grove, IL).
Detection of GM1
After sucrose density gradient ultracentrifugation, fractions were dialyzed against distilled water and lyophilized. Lipids were extracted by chloroform/methanol (2:1, v/v) and developed on a precoated high performance TLC plate (Silica Gel 60; Merck, Darmstadt, Germany) with chloroform/methanol/12 mM CaCl2 (5:4:1, v/v/v). The immunoblotting assay on TLC plates with HRP-conjugated cholera toxin B subunit (List Biological Laboratories, Campbell, CA) was performed to detect GM1a as described previously (36).
Recovery of DRMs
After sucrose density gradient ultracentrifugation, low-density fractions (fractions 5–9) were collected and 5-fold diluted with MBS. DRMs were recovered by centrifugation at 250,000 x g for 45 min at 4°C in a SW60Ti rotor.
SDS-PAGE and immunoblotting
The proteins were separated on 10% SDS-PAGE (37). They were then transferred to nitrocellulose membranes (12 V, overnight) in transfer buffer (25 mM Tris, 192 mM glycine, 0.1% SDS, 20% methanol) as described elsewhere (38). Blots were blocked at room temperature for 1 h in 10% Block Ace (Yukijirushi-Nyugyo, Sapporo, Japan) and probed for 2 h with specific Ab in 1% Block Ace. The blots were washed three times with 1% Block Ace containing 0.05% Tween 20 and probed for 1 h with HRP-conjugated secondary Ab. Immunoreactivity was determined by means of the ECL reaction (Amersham International).
Cyclodextrin treatment
RA-HL-60 cells were washed three times with E-RDF and suspended
in E-RDF at a cell density of 2 x 107
cells/ml. The cells were incubated in the absence or presence of 10 mM
MCD (Sigma, St. Louis, MO) for 1 h at 37°C. The viability of
cells was >90% after treatment based on trypan blue staining. Based
on the colorimetric assay using MTT according to the manufacturer’s
instruction (Chemicon International, Temecula, CA), cell viability was
>75% after the treatment.
Cholesterol determination
RA-HL-60 cells were washed three times with PBS. Lipids were extracted by chloroform/methanol at sequential ratios of 2:1, 1:1, then 1:2, and the extracts were combined. Cholesterol was measured using a cholesterol oxidase-based assay kit (Boehringer Mannheim, Mannheim, Germany).
Flow cytometry analysis
RA-HL-60 cells and K562 cells were washed with PBS and treated
with FITC-conjugated anti-FcRIIs Ab or FITC-conjugated mouse
IgG3 for 30 min on ice (106 cells/20 µl
FITC-Ab/80 µl PBS). The cells were diluted with PBS and analyzed by
FACSCalibur (BD Biosciences, Mountain View, CA).
Isolation of neutrophils
Human neutrophils were isolated from heparinized venous blood from healthy adult donors with a discontinuous one-step Ficoll-Hypaque gradient as previously described (39). After isolation, neutrophils were suspended in HBSS containing 10 mM HEPES (pH 7.4) and 0.1% (w/v) low endotoxin BSA. The viability of cells was >95% after treatment based on trypan blue staining.
Preparation of opsonized zymosan and immune complexes
Opsonized zymosan was prepared as previously described (40). Briefly, zymosan (20 mg of zymosan A; Sigma) was incubated with fresh human serum and finally suspended in 1 ml of PBS containing 1 mM CaCl2 (zymosan suspension). As for the nonopsonized zymosan, zymosan was similarly treated in the absence of serum. Insoluble immune complex (IIC) was prepared with BSA and rabbit anti-BSA (Cappel Oreganon Technica, Durham, NC) in a weight:weight ratio of 1:10 (molar ratio, 1:4) as previously described (41).
Measurement of superoxide generation
Superoxide generation was measured by the cytochrome
c reduction method as described previously
(42). Neutrophils (1 x 107
cells/ml, 100 µl) were incubated in HBSS with or without 10 mM MCD
at 37°C for 5 min. Then, cytochrome c (24.7 mg/ml, 5 µl)
and the stimulant as indicated were added and further incubated for 15
min. The reaction was terminated by adding 5 µl of 40 mM
N-ethylmaleimide. After centrifugation at 200 x
g for 5 min, the absorption spectrum (520–570 nm) was
measured by DU-640 (Beckman Instruments). Superoxide generation was
expressed by amount of cytochrome c reduction based on the
increase in absorbance at 550 nm.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Rs in RA-HL-60 cells
For cross-linking of FcRs, RA-HL-60 cells were incubated with
human IgG and IgG of GAH. The cross-linking of FcRs resulted in a
prominent increase in tyrosine phosphorylation of the 120-kDa protein
in the cell lysate as shown in Fig. 1
.
This protein was previously identified in HL-60 cells as the cytosolic
SH3 domain-binding protein p120c-cbl
(43).
|
RI and FcRII. RA-HL-60 cells do not express
FcRIII (44). As shown in Fig. 1
, Cbl was also the major
tyrosine phosphorylation substrate on independent and selective
stimulation of FcRI and FcRII using anti-FcRI (CD64) mAb
and F(ab')2 of anti-FcRII mAb IV.3,
respectively, along with F(ab')2 of GAM.
Association of FcRs with DRMs on cross-linking of FcRs
RA-HL-60 cells were incubated with
125I-labeled IgG, then further incubated with
GAH. The cells were then lysed with a buffer containing 1% Triton
X-100 and subjected to sucrose density gradient ultracentrifugation.
Distribution of 125I-labeled IgG was analyzed to
observe the surface localization of 125I-labeled
IgG-cross-linked FcRs. Most 125I-labeled IgG
(98%) was recovered in the bottom, heavy fractions (fractions 13–16)
in the absence and presence of GAH (data not shown). However, as shown
in Fig. 2, an appreciable increase of
125I-labeled IgG in the low-density fractions,
namely, DRMs (fractions 6–9) characterized by a localization of
ganglioside GM1a, was observed in the presence of GAH. It is therefore
considered that the increase in 125I-labeled IgG
is due to the presence of FcRs, which are cross-linked with
125I-labeled IgG and GAH on the cell surface,
in DRMs.
|
Rs in DRMs is actually due to
the association of FcRs with lipid rafts, we investigated the effect
of MCD. MCD has been used to reduce endogenous cholesterol
(45), a crucial component in the maintenance of lipid
rafts, and to impair raft-mediated processes (46, 47).
After RA-HL-60 cells were incubated with 10 mM MCD for 1 h at
37°C, endogenous cholesterol was decreased to 50% (3.4
nmol/106 cells to 1.7
nmol/106 cells). This decrease is closely similar
to that previously reported in RBL-2H3 cells (29). The
surface expression of FcRII was not significantly changed after
MCD treatment when cells were stained with FITC-IV.3 and analyzed by
flow cytometry (Fig. 3). However, as
shown in Fig. 2, the increase in 125I-labeled IgG
in DRMs by cross-linking of FcRs was appreciably diminished after
MCD treatment. Thus, cross-linked FcRs are considered to be
associated with lipid rafts.
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RII with DRMs
RA-HL-60 cells express both FcRI and FcRII. To investigate
an association of FcRII with DRMs, we examined a surface
localization of 125I-labeled IV.3-labeled
FcRII in K562 cells. K562 cells express FcRII but does not
express FcRI and FcRIII (48). As shown in Fig. 4C, stimulation of K562 cells
with IV.3 and F(ab')2 of GAM increased tyrosine
phosphorylation of p120c-cbl. After
sucrose density gradient ultracentrifugation, 9% of
125I-labeled IV.3-labeled FcRII was recovered
in DRMs (fractions 4–8) in unstimulated cells. The amount of FcRII
in DRMs was increased to 31% by cross-linking of FcRII (Fig. 4A). Such an increase was not observed when
F(ab')2 of GAM was added after cell lysis (data
not shown). Furthermore, the cross-linking-dependent increase of
FcRII in DRMs was reduced after MCD treatment (Fig. 4B), although the surface expression of FcRII was not
significantly changed based on a flow cytometry analysis (data not
shown). These results confirm a recruitment of FcRII into DRMs after
cross-linking.
|
Rs
The presence of Src-PTK Lyn in DRMs was previously reported by
Parolini et al. (49) in HL-60 cells. We also observed the
presence of
p60lyn/p58lyn
in DRMs in RA-HL-60 cells (Fig. 5).
Furthermore, immunoblotting analysis with HRP-conjugated
anti-phosphotyrosine mAb (HRP-PY20) revealed an increase in
tyrosine phosphorylation substrates at 120, 72, and 60 kDa to DRMs on
cross-linking of FcRs (Fig. 5). The 120- and 60-kDa proteins
correspond to p120c-cbl and
p60lyn, respectively, based on immunoblotting
with corresponding Abs. Identity of the 72-kDa protein is currently
under investigation. To date, we have been unable to detect Syk (with
two different Abs) and SLP-76 in DRMs by immunoblotting (data not
shown).
|
CD treatment, the cross-linking-dependent increase in
tyrosine-phosphorylated proteins in DRMs was completely suppressed
(Fig. 5). We also observed an almost complete loss of Lyn that is
constitutively associated with DRMs. These results suggest recruitment
of the phosphorylation substrates with lipid rafts on FcR
cross-linking. The association of FcRs as well as the intracellular
phosphorylation substrates with DRMs on FcR cross-linking further
suggests the involvement of lipid rafts in the FcR-mediated tyrosine
phosphorylation process.
The recruitment of p120c-cbl to DRMs was also
observed when FcRII was selectively stimulated in RA-HL-60 cells
with 1 µg/ml IV.3 and 22 µg/ml F(ab')2 of
GAM. Furthermore, recruitment was suppressed by MCD treatment (data
not shown), suggesting a role of lipid rafts in the FcRII
signaling.
Lack of effect of Src-PTK inhibitor on association of FcRs and
p120c-cbl to DRMs
To investigate whether tyrosine phosphorylation is required for
the association of FcRs and p120c-cbl with
DRMs or whether the association is needed for tyrosine phosphorylation,
we examined the effect of PP1, a specific inhibitor of Src-PTKs
(50). In RA-HL-60 cells, phosphorylation of
p120c-cbl in lysates on FcR cross-linking was
largely inhibited in the presence of PP1 (Fig. 6A). In addition, as shown in
Fig. 6B, tyrosine phosphorylation of FcRII (40 kDa) and
the 58-kDa protein (identified as p58lyn in Fig. 9) in the IV.3 immunoprecipitates was also suppressed in the presence
of PP1. Thus, Src-PTKs play a crucial role in phosphorylation on FcR
cross-linking.
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A, however, PP1
had no effect on the recruitment of FcRs to DRMs after
cross-linking. Furthermore, although the tyrosine phosphorylation of
p120c-cbl in DRMs was decreased by PP1
treatment, the amount of p120c-cbl was unchanged
(Fig. 7B). These results indicate that the recruitment of
FcRs as well as p120c-cbl to the DRMs is
independent of their tyrosine phosphorylation.
|
CD on FcR-mediated tyrosine
phosphorylation
We investigated the effect of MCD on tyrosine phosphorylation
on FcR cross-linking to investigate whether initiation of the
tyrosine phosphorylation pathway is mediated via lipid rafts. As shown
in Figs. 4 and 8, the increase in
tyrosine phosphorylation of p120c-cbl was no
longer observed in MCD-treated cell lysate on either FcR (Fig. 8A) or FcRII cross-linking (Figs. 4C and
8B). These results thus suggest the crucial involvement of
lipid rafts in tyrosine phosphorylation. Since the surface expression
of FcRII was not appreciably reduced (Fig. 3), the decrease in
tyrosine phosphorylation is probably due to dysfunction of
FcRII-linked molecular machinery.
|
R cross-linking is considered
to be the activation of Src-PTKs associated with the FcRs (2, 7, 16). A marked increase in tyrosine phosphorylation was
observed at 40-kDa (FcRII) and 58-kDa bands in the IV.3
immunoprecipitates on FcRII cross-linking (Fig. 9A), which was inhibited by a
Src-PTK inhibitor, PP1 (Fig. 6B). The 58-kDa
protein was identified as p58lyn based on
immunoblotting (Fig. 9C). It should be noted that
p58lyn/p60lyn doublet was
present in the DRMs (Fig. 5), whereas
p58lyn was present in the IV.3
immunoprecipitates (Fig. 9C) in their
tyrosine-phosphorylated forms. In support of this observation, it has
been reported that the lower molecular mass isoform of Lyn is
selectively phosphorylated following association to FcRII in THP-1
cells (7). As shown in Fig. 9A, the tyrosine
phosphorylation of FcRII and p58lyn was
almost completely inhibited when RA-HL-60 cells were treated with
MCD before FcRII cross-linking. In contrast, the amount of
FcRIIa (Fig. 9B) and p58lyn (Fig. 9C) in the IV.3 immunoprecipitates was not decreased after
MCD treatment. The decreased immunoreactivity at the 40-kDa band
from the stimulated cells (Fig. 9B) is probably due to
anti-FcRIIa polyclonal Ab (pAb 260) reacting with
nonphosphorylated FcRIIa more strongly than with the phosphorylated
one (51). These results strongly suggest that MCD
treatment impaired the activation of Src-PTKs responsible for the
tyrosine phosphorylation of FcRIIa, p58lyn,
and p120c-cbl. Along with the inhibitory effect
of MCD on the association of FcRII to DRMs, it is considered that
the association of FcRs with lipid rafts is a prerequisite for the
activation of Src-PTKs in initiating the subsequent tyrosine
phosphorylation processes on clustering of FcRII.
Inhibitory effect of MCD on FcRII-mediated superoxide
generation
Finally, we investigated the effect of MCD on superoxide
generation in human neutrophils to evaluate functional relevance of
lipid rafts to immune responses. Resting human neutrophils express both
FcRII and the GPI-linked form of FcRIII (FcRIIIb).
Superoxide generation can be induced by various stimuli such as
chemoattractants, opsonized microorganisms, or immune complexes. As
shown in Fig. 10A,
chemoattractant fMLP-induced superoxide generation was enhanced in
MCD-treated cells. On the other hand, the superoxide generation
induced by opsonized zymosan (opsonized with both IgG and complement
C3) was not affected in MCD-treated cells (Fig. 10B).
Complement receptor 3 (CR3, Mac-1,
M2, CD11b/CD18) and
FcRIIIb are considered to cooperate in this respiratory burst
(52). In contrast to these responses, the superoxide
generation by IIC was appreciably suppressed after the MCD treatment
(Fig. 10C). The effect of IIC is mainly mediated via
FcRII (53). Accordingly, among the typical three types
of stimulation in neutrophils, the inhibitory effect of MCD was
specifically observed in the case of the FcRII-mediated pathway. The
results strongly suggest that lipid rafts are important in the
FcRII-mediated superoxide generation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Rs together with
tyrosine-phosphorylated proteins, including
p120c-cbl to DRMs. Furthermore, our results
strongly suggest that the association of FcRII to DRMs is necessary
for the tyrosine phosphorylation and superoxide generation
induced by clustering of FcRII. The cholesterol-dependent
specialized membrane structures, lipid rafts, are considered to be
crucial for the FcRII-mediated response induced by
IgG-opsonized Ags.
Association of cross-linked FcRs to lipid rafts
The distribution of 125I-labeled IgG on
sucrose density gradient centrifugation (Fig. 2) does not lead us to
conclude directly that uncross-linked FcRs are not present in DRMs,
because the final concentration of 125I-labeled
IgG (5 x 10-8 M) was lower than the
dissociation constant between FcRIIs and IgG
(>10-7 M), and thus FcRIIs were not bound to
monomeric 125I-labeled IgG (3).
However, we showed the cross-linking-dependent recruitment of FcRII
into DRMs using K562 cells and 125I-labeled IV.3
(Fig. 4). Accordingly, our data strongly suggest that FcRIIs are
recruited to DRMs after cross-linking.
We suggest here that the association of cross-linked FcRs to DRMs
does not require the tyrosine phosphorylation mediated by Src-PTKs.
Previously, Field et al. (28) showed that the recruitment
of FcRI to DRMs on receptor cross-linking precedes tyrosine
phosphorylation. Therefore, it is suggested that lipid rafts are
essential at the initial stage of the response induced by clustering of
FcRs for both IgE and IgG. In contrast, the recruitment of TCR/CD3 to
DRMs induced by CD3 ligation is inhibited by a Src-PTK inhibitor, PP1,
suggesting that tyrosine phosphorylation is required for the
recruitment (30). It is proposed that lipid rafts are
essential structures that ensure the efficient colocalization of
TCR/CD3 with CD4/Lck (31) and are stabilized during
immunological synapse formation (54) due to actin filament
organization (55). Accordingly, FcRs and TCRs may have
distinct requirement for lipid rafts; they are probably required as the
initial trigger of FcR signaling on receptor clustering, and in the
course of the TCR activation through successive interaction between T
cells and APCs.
The fact that FcRs might pre-exist in an oligomeric state on the
cell surface, as suggested by recent crystallographic studies
(56, 57), is compatible with the lack of requirement of
Src-PTK-dependent tyrosine phosphorylation for the association of
FcRs with rafts. According to the present results, FcR oligomers
may have a tendency to associate with lipid rafts. Previously, Harder
et al. (58) proposed that some membrane proteins
(GPI-anchored proteins and influenza hemagglutinin) tend to be
surrounded by raft lipids and thus they have an affinity for lipid
rafts. If low-affinity FcRs also have such lipids, the surrounding
lipids would form an extended edge around the parallel oligomer, which
intrinsically favors association with lipid rafts.
Interestingly, the association of FcRI with DRMs is only observed
when cells are lysed with a low concentrations (0.05%) of Triton X-100
(27, 28, 29), which coincides with the condition for the
coimmunoprecipitation of Src-PTK activity with FcRI
(59). FcRI is a multisubunit receptor composed of a
high-affinity IgE-binding subunit () and two ITAM-containing
subunits ( and ). In contrast, it has been noted that the kinase
activity is coimmunoprecipitated with FcRII in a 1% Triton X-100
cell lysate (7). Consistent with this, we showed the
association of FcRs with DRMs using 1% Triton X-100 lysates. Thus,
the physical property of the lipid rafts involved in the signaling of
monomeric FcRII may be different from that of oligomeric
FcRI.
IgG immune complexes are also strongly implicated in the pathogenesis
of hematologic and rheumatic autoimmune disorders (1, 2, 3, 4).
Our results raise the possibility that IgG-mediated events are affected
by perturbation of lipid rafts by autoantibodies that react with raft
component, for example, anti-ganglioside Ab in
Landry-Guillain-Barré syndrome (60).
Involvement of lipid rafts in FcR signaling will be a new aspect to
understand the mechanism of humoral immunity as well as autoimmune
diseases.
Role of lipid rafts in activation of Lyn associated with FcRIIa
The inhibitory effect of MCD on the
cross-linking-dependenttyrosine phosphorylation suggests the crucial
involvement of lipid rafts in the activation of Src-PTKs in
FcR signaling. It has been proposed that Src-PTKs are activated in
part by tyrosine phosphorylation of the stimulatory site in the kinase
domain and/or the dephosphorylation of the inhibitory site in the
COOH-terminal tail (61).
The immunoprecipitation experiment suggests a constitutive association
of p58lyn with FcRIIa in both the presence
and absence of FcR clustering (Fig. 9). This finding is supported by
the previous report that Lyn is associated with a recombinant
GST-fusion protein of the cytoplasmic tail of FcRIIa in both resting
and activated human neutrophils (16). On the other hand,
activation of Lyn is controlled by phosphorylation of positive
regulatory tyrosine residue of Lyn in immature B cells
(62). Therefore, although we have not yet confirmed
whether Lyn is a dominant Src-PTK in the FcR signaling, the
cross-linking-dependent tyrosine phosphorylation of
p58lyn in IV.3 immunoprecipitates suggests the
activation of Lyn associated with FcRIIa.
The tyrosine phosphorylation of p58lyn was
inhibited by MCD treatment, whereas this treatment did not disrupt
the physical association between FcRIIa and
p58lyn (Fig. 9). This excludes the possibility
that tyrosine phosphorylation of FcRIIa-associated
p58lyn occurs simply via a transphosphorylation
mechanism upon FcR clustering. Rather, the involvement of lipid
rafts is strongly suggested. It is considered that FcR cross-linking
causes the association of the FcRIIa-p58lyn
complex to DRMs and then induces the tyrosine phosphorylation of
p58lyn. Recent papers have proposed several
mechanisms to explain the increase in phosphorylation or decrease in
dephosphorylation of Src-PTKs in DRMs. In FcRI, phosphorylation of
Lyn bound to FcRI is promoted by compartmentation within DRMs that
are rich in Src-PTKs (27, 28, 29). In TCR, protection of Lck
in DRMs from attack by a tyrosine phosphatase such as CD45 has been
reported (30, 31, 32). Moreover, activation of the intrinsic
kinase activity of Src-PTK Yes by Shiga toxin binding to globotriaosyl
ceramide in DRMs (63) or that of Lck and Fyn in the
vicinity of glycolipids or GPI-anchored proteins in DRMs
(64) has been also suggested.
Recruitment of p120c-cbl with DRMs on FcR
cross-linking
In contrast to a constitutive localization of
p120c-cbl with DRMs in Jurkat T cells
(31), the association of p120c-cbl
with DRMs in RA-HL-60 cells depends on FcR cross-linking.
Furthermore, the recruitment does not require tyrosine phosphorylation.
The direct interaction between p120c-cbl and Lyn
mediated by the SH3 domain of Lyn has been previously reported
(43). As Lyn is coimmunoprecipitated with FcRIIa (Fig. 8), two types of recruitment of p120c-cbl are
possible: one is that some population of
p120c-cbl is associated with the cytoplasmic
region of FcRIIa constitutively via Lyn and is thus recruited to the
inner leaflet of DRMs on cross-linking; the other is that
p120c-cbl preferentially binds to Lyn in
the FcRIIa-Lyn complex within DRMs. As
p120c-cbl does not seem to associate with the
Lyn resident in DRMs, the FcRIIa-associated Lyn in DRMs may act as
an efficient scaffold for p120c-cbl. The
tyrosine phosphorylation of p120c-cbl probably
occurs after recruitment to DRMs by the FcRIIa-associated Src-PTKs
activated within the DRMs.
p120c-cbl possesses a long proline-rich region
(65), which could mediate its interactions with SH3
domain-containing proteins such as Grb2, SLP-76 (66), or
Nck (15). p120c-cbl also has
several tyrosine residues and upon phosphorylation could interact with
the SH2 domains of Shc (15), phosphatidylinositol
3-kinases (67), or Vav (68). The tyrosine
phosphorylation of Vav and SLP-76 on FcRII cross-linking has been
reported in HL-60 cells (50). Vav is a guanine nucleotide
exchange factor specific for Rac1, one of the Rho family small
molecular mass GTP-binding proteins (69). The activation
of Rac is required for the actin cytoskeleton reorganization which
leads to phagocytosis mediated by FcRs (70). On the other
hand, activated Rac can stimulate Nck-associated Pak1 kinase activity,
which may contribute to the oxidative burst through NADPH oxidase
activation (71). Accordingly, the recruitment of
p120c-cbl with DRMs and its subsequent tyrosine
phosphorylation on cross-linking has the potential significance of
causing the accumulate of a large number of signaling proteins, in turn
triggering FcR-mediated phagocytosis or superoxide generation at the
contact sites of IgG immune complexes.
Role of lipid rafts in superoxide generation
Superoxide generation is an important host defense mechanism against infectious substances and on the other hand causes the pathogenesis of various inflammatory diseases. The assembly of NADPH oxidase on the membranes, which is necessary to generate oxygen radicals, requires translocation of cytosolic components of NADPH oxidase, Rac and p47phox-p67phox complex, to the membranes (72). Here, we examined the role of lipid rafts on the superoxide generation induced by three typical stimuli; IIC, opsonized zymosan, and chemoattractant. Each stimulus activates its distinct signaling pathway and is differently affected by cytochalasin B, namely, not affected (52), inhibited (52), and stimulated (73), respectively.
IIC can cross-link both FcRII and FcRIIIb in neutrophils.
However, the effect of IIC is mainly mediated via FcRII
(53). Consequently, the inhibitory effect of MCD (Fig. 10C) suggests that lipid rafts are required for the
FcRII-mediated superoxide generation. Since tyrosine phosphorylation
is necessary for the FcRII-mediated superoxide generation
(15), the requirement for initiation of tyrosine
phosphorylation probably underlies the crucial role of lipid rafts in
FcRII-induced superoxide generation. In addition, the recruitment of
p120c-cbl with DRMs and its subsequent tyrosine
phosphorylation also support the dependency of the FcRII-mediated
superoxide generation on lipid rafts.
When neutrophils are stimulated with opsonized zymosan through
cooperation of FcRIIIb and CR3, both the actin cytoskeleton and
tyrosine phosphorylation are required for the superoxide generation
(52). In contrast to IIC, the MCD treatment had little
effect on opsonized zymosan-induced superoxide generation (Fig. 10B). Although an involvement of lipid rafts in
FcRIIIb/CR3 signaling has not been demonstrated, it is considered
that FcRIIIb, a GPI-anchored protein, is constitutively localized
with DRMs. Since stabilization of CD44-containing lipid rafts by actin
cytoskeleton has been reported in EpH4 cells (74), the
MCD resistance of opsonized zymosan-stimulated superoxide generation
implies that the FcRIIIb/CR3-containing rafts are stabilized via
actin cytoskeleton.
Cellular responses to the fMLP receptor in neutrophils, including
phospholipase C stimulation, Ca2+ mobilization,
superoxide generation, and enzyme release, are mediated by pertussis
toxin-sensitive Gi-type G proteins (75). Based on the
increase of fMLP-induced superoxide generation in the presence of
MCD (Fig. 10C), it is possible to speculate that lipid
rafts are negatively involved in fMLP-induced superoxide generation. As
the localization of heterotrimeric GTP-binding proteins in the
cytoplasmic face of lipid rafts has been reported (25), a
disruption of lipid rafts could release Gi from DRMs and promote their
access to the fMLP receptor, especially when the fMLP receptor is
present outside DRMs. It is necessary to examine whether the fMLP
receptor is localized with DRMs or not.
In conclusion, the present study provides evidence that lipid rafts are
required for the initiation of tyrosine phosphorylation and important
for subsequent superoxide generation upon FcRII cross-linking. Our
results also suggest that the contribution of lipid rafts varies among
several stimuli. Further work focusing how lipid rafts define
intracellular signaling pathways via localization or recruitment of
distinct proteins will be necessary to understand the highly regulated
NADPH oxidase assembly process.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Miki Hara-Yokoyama, Department of Hard Tissue Engineering, Biochemistry, Division of Bio-Matrix, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan. E-mail address: m.yokoyama.bch{at}tmd.ac.jp
3 Abbreviations used in this paper: ITAM, immunoreceptor tyrosine-based activation motif; Src-PTK, Src family protein tyrosine kinase; DRM, detergent-resistant membrane; RA, retinoic acid differentiated; PP1, 4-amino-5-(4-metylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; GAH, goat anti-human IgG; GAM, goat anti-mouse IgG; MCD, methyl--cyclodextrin; IIC, insoluble immune complex; SH, Src homology; MBS, MES-buffered saline; CR3, complement receptor 3.
Received for publication September 11, 2000. Accepted for publication September 18, 2001.
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) and with (
) 80 µg/ml GAH. The
cells were then lysed in 1% Triton X-100 lysis buffer and fractionated
by sucrose density gradient ultracentrifugation as described in
Materials and Methods. The distribution of
125I-labeled IgG-Fc
