From the Centre de Recherche en Rhumatologie et
Immunologie, (CHUL), Département de Médecine,
Université Laval, Quebec City, Quebec G1V 4G2, Canada and the
§ Laboratoire d'Immunologie Moléculaire,
Département de Microbiologie et Immunologie, Faculté de
Médecine, Université de Montréal, Montreal, Quebec
H3C 3J7, Canada
Received for publication, November 13, 2002, and in revised form, December 13, 2002
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ABSTRACT |
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Lipid rafts are plasma membrane microdomains that
are highly enriched in signaling molecules and that act as signal
transduction platforms for many immune receptors. The
involvement of these microdomains in HLA-DR-induced signaling is less
well defined. We examined the constitutive presence of HLA-DR molecules
in lipid rafts, their possible recruitment into these microdomains, and the role of these microdomains in HLA-DR-induced responses. We detected significant amounts of HLA-DR molecules in the lipid rafts of
EBV+ and EBV Although major histocompatibility complex
(MHC)1 class II molecules
do not possess any known signaling motifs in their cytoplasmic and
transmembrane domains, they act as signal transducers in addition to
playing a critical role in antigen presentation and autoimmune disease
susceptibility and severity (1, 2). Corley et al. (3)
reported that the recognition of MHC class II-peptide complexes by
specific T cell receptors (TCR) leads to the activation of both T
lymphocytes and antigen-presenting cells (APCs), suggesting that MHC
class II molecules can act as signal transducers. To confirm this
possibility, anti-MHC class II antibodies (Abs) and superantigens
(SAgs), which act as natural MHC class II ligands, were used to mimic
MHC class II-peptide complex recognition by specific TCRs. This
ligation led to various cellular events such as homotypic and
heterotypic cell-cell adhesion (4), B cell proliferation and
differentiation (5), cytokine production, and expression of
co-stimulatory molecules (6) and, under certain conditions,
cell death (7). Like other ligand-receptor interactions, some of these
events are dependent on MHC class II ligand dimerization (8, 9),
generate cAMP and intracellular calcium flux (1), and are mediated by
signaling pathways and secondary messengers including protein kinase C
(PKC), protein tyrosine kinase (PTK), cyclooxygenase 2, cytosolic
phospholipase A2, and phospholipase C.
A number of studies have addressed the structural basis by which MHC
class II molecules carry out its multiple functions. It has been shown
that a six amino acid region of the cytoplasmic domain of the The plasma membranes of eukaryotic cells contain microdomains enriched
in cholesterol and sphingolipids (16-18), commonly called lipid rafts
or glycolipid-enriched microdomain (GEM)s. These microdomains are
resistant to solubilization at low temperature by nonionic detergents
and can be separated and isolated from the rest of the plasma membrane
using sucrose density gradients (18, 19). They also harbor large
quantities of proteins such as G proteins, kinases, and adaptor
molecules that act as intermediate transducers for many receptors,
including TCR (20) and B cell receptor (BCR) (21). The relevance of
lipid rafts with regard to MHC class II-induced signal transduction
pathways and APC function is less clearly defined (22, 23). Anderson
et al. (22) reported that large numbers (20-50%) of MHC
class II molecules are located in the lipid rafts of murine and human B
cell lines. Such localization seems to be critical for T cell
activation when minimal numbers of relevant MHC class II-peptide
complexes are available on APCs. However, Huby et al. (23)
failed to detect MHC class II molecules in the lipid rafts of a human
IFN- In this study, we show that small but significant amounts of HLA-DR
molecules were constitutively present in the lipid rafts of normal
human B cells, human EBV Reagents--
The following antibodies were used: mAb L243
(mouse IgG2a, recognizes a conformational epitope of HLA-DR; ATCC,
Manassas, VA), mAb DA6.147 (mouse IgG1, recognizes the C-terminal
intracellular tail of the HLA-DR Tonsillar B Cells, Monocytes, and Cell Lines--
Resting human
tonsillar B cells and monocytes were purified as previously described
(25). The EBV-negative B lymphoma cell lines (BL28 and BJAB) and their
EBV-transformed counterparts were a gift from Dr. C. Pedro
(Hôtel-Dieu de Québec Hospital, Quebec City, QC) and Dr. J. Menezes (Sainte-Justine Hospital, Montreal, QC). The THP-1 monocytic
human cell line was purchased from ATCC. THP-1 cells (5 × 105 cells/ml) were treated with recombinant human IFN- Isolation of Lipid Rafts and Western Blotting--
Stimulated
and unstimulated cells (107) were washed twice in cold
serum-free RPMI 1640 and lysed in 400 µl of ice-cold TNE buffer (10 mM Tris, pH 7.5, 150 mM NaCl, 5 mM
EDTA) containing 1% Triton X-100, 2 mM
Na3VO4 and a mixture of protease inhibitors (Roche Molecular Biochemicals, Montreal, QC) for 30 min on ice. Cells
lysates were mixed with an equal volume of 85% sucrose in TNE and
deposited in SW60Ti centrifuge tubes. The samples were overlaid with
2.4 ml of 35% sucrose and 1 ml of 5% sucrose in TNE and centrifuged
for 16 h at 35,000 rpm at 4 °C. Eleven fractions (380 µl)
were collected beginning at the top of the tube. Aliquots of each
fraction were mixed with 6× Laemmli buffer containing 6%
2-mercaptoethanol and heated for 5 min at 95 °C. Samples were resolved by SDS-PAGE, and proteins were transferred onto polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA). After blocking with 5% skim milk and 0.05% Tween 20 in TBS, the membranes were incubated with DR Tyrosine Phosphorylation and Lyn and ERK1/2 Activation
by Immunoblotting--
THP-1 cells (106 cells/ml) were
incubated for 15 min at 37 °C in serum-free medium before
stimulation. Stimulations were carried out as indicated in the figure
legends and stopped by adding an equal volume of 2× modified Laemmli
sample buffer (29) preheated to 95 °C. Total cell lysates were
resolved by SDS-PAGE and transferred to polyvinylidene difluoride
membranes. After blocking with 2% gelatin and 0.15% Tween 20 in TBS,
membranes were immunoblotted with anti-phosphotyrosine mAb 4G10 and
subjected to chemiluminescent detection with HRP-conjugated anti-mouse
IgG Ab as described above. After stripping, the membranes were
sequentially reprobed with anti-phospho-ERK 1/2, anti-Erk1,
anti-phospho-Lyn, and anti-Lyn Abs.
Measurement of Fluorescence--
Cells were stimulated with
FITC-labeled L243 mAb or FITC-labeled isotype control mAb (8C12) for 5 min. Washed cells were lysed and fractionated on a sucrose gradient as
described above. Fractions 3, 4, and 5 (raft) and fractions 9, 10, and
11 (soluble) were pooled. FITC-labeled L243 and FITC-labeled 8C12 were
quantified in the two pools using a microplate fluorescence reader
(BIO-TEX, FL600). The results were normalized with respect to the
isotype control and expressed as percentages.
Disruption of Lipid Rafts--
To alter the cholesterol content
of the plasma membrane, cells were washed three times in serum-free
RPMI 1640 and resuspended at a concentration of 107
cells/ml in serum-free RPMI 1640 containing 10 mM M Cell-Cell Adhesion Assays--
CIITA-transfected THP-1 cells
were resuspended in RPMI 1640 containing 5% fetal bovine serum and
seeded in 96-well microtiter plates at 1 × 105
cells/well. Cells were stimulated at 37 °C for 1 h with mAb
W6/32, mAb L243, or the F(ab) fragment of L243 at 1 µg/ml. The cells were pretreated with erbstatin analog (5 µg/ml) or PP2 (10 µM) for 1 h at 37 °C or with 10 mM
M HLA-DR Molecules Are Constitutively Present in Lipid Rafts of Human
B Cells and Monocytic Cells--
To determine whether HLA-DR molecules
were present in lipid rafts in APCs and to study the role of their
presence in HLA-DR-induced responses, we first examined the baseline
distribution of these molecules in normal human B cells using sucrose
gradient fractions derived from 1% Triton X-100 cell lysates. While
HLA-DR molecules were mainly detected in soluble fractions (Fig.
1A), ~2% were found in the
lipid rafts of resting human tonsillar B cells as determined by
quantitative densitometry. GM1 ganglioside, a well-known constituent of
lipid rafts, was recovered in fractions 2-4, whereas CD45 phosphatase
was found exclusively in fractions 9-11, indicating that our
conditions were appropriate for separating raft and non-raft membrane
fractions. When Triton X-100 was titrated or other detergents were used
such as CHAPS, Brij 58, and Nonidet P-40, we found that 1% Triton is
the only condition that allow us to isolate GM1 without any detectable
levels of CD45 molecules (data not shown). Based on these results, 1%
Triton X-100 cell lysates were used in all the following
experiments.
Because it is well established that MHC class II molecules and BCR
signaling share numerous similarities (30), and the presence of LMP2A
in the lipid rafts of EBV-immortalized human B cell lines has recently
been shown to affect the distribution of BCR (31), we examined the
effect of the EBV transformation on the distribution of HLA-DR
molecules. To this end, we used two B cell lines (BL28 and BJAB) and
their EBV-transformed counterparts. Fig. 1, B and C shows that 4% (BJAB) to 5% (BL28) of HLA-DR molecules
were found in the fractions corresponding to lipid rafts, and the
presence of EBV gene products did not affect the partitioning of HLA-DR molecules.
We next analyzed the localization of HLA-DR molecules in normal human
monocytes and in a human THP-1 monocytic cell line. Monocytes were
purified from peripheral blood mononuclear cell by adherence to
plastic Petri dishes precoated with heat-inactivated autologous plasma.
The THP-1 cells, which are known to express very low levels of HLA-DR
molecules, were treated with IFN-
Our results indicate that all the cell types tested constitutively
express significant amounts of HLA-DR molecules in the lipid rafts,
that the proportion of these molecules does not show gross variations
among the cell types, and that the expression of EBV proteins in B cell
lines failed to affect the constitutive localization of the HLA-DR molecules.
Ligation of HLA-DR Molecules with a Bivalent Ligand Failed to
Induce Recruitment of HLA-DR Molecules into Lipid Rafts--
We next
looked at whether the distribution of HLA-DR molecules would change
following ligation with specific Abs, with SEA, or with MAM. SEA and
MAM are members of the SAg family that are known to bear two MHC class
II binding sites, the first of which interacts with the
Having determined that HLA-DR was constitutively present in the lipid
rafts of CIITA-transfected THP-1 cells and that anti-HLA-DR mAb failed
to induce further HLA-DR recruitment, we extended our analysis to
confirm the specificity of this localization. Cells were left untreated
or were treated with M M
To investigate this possibility, we stimulated CIITA-transfected THP-1
cells with isotype-matched control mAb, mAb L243, and the F(ab)
fragment of L243. Total cell lysates were resolved by SDS-PAGE and
analyzed by immunoblot using anti-phosphotyrosine mAb 4G10. We detected
tyrosine phosphorylation of several proteins ranging in molecular mass
from 50 to 150 kDa within 1 min in L243-stimulated samples (data not
shown). Maximum phosphorylation occurred at 5 min and persisted at
least for 15 min (Fig. 4A). In
contrast, stimulation with the F(ab) fragment of L243, the
isotype-matched control mAb, or the PMA induced no detectable tyrosine
phosphorylation. The detection of heavily phosphorylated proteins
(~53-55 kDa) migrating in samples stimulated with the anti-HLA-DR
mAb suggested that they might be members of the Src family of kinases,
in particular Lyn. To test this possibility, antibodies directed
against the phosphorylated and total forms of Lyn were used. Fig.
4B shows that only the bivalent anti-HLA-DR mAb triggered
the phosphorylation of Lyn, a response that was detected after 1 min
(data not shown) and persisted for at least 15 min.
Our findings suggest that tyrosine phosphorylation is probably the
first step in HLA-DR-induced signaling cascades and that the proximal
pTyr events associated with ligation of HLA-DR molecules are
critically dependent on their dimerization.
We conducted additional experiments to identify other signaling
pathways induced by the ligation of HLA-DR molecules. To that end, we
analyzed the phosphorylation status of two members of the MAPK family
(ERK1/2 and p38), each requiring phosphorylation at tyrosine and
threonine residues for activation (36). CIITA-transfected THP-1 cells
were stimulated as above, and cell lysates were sequentially immunoblotted with the phosphorylated ERK1/2 and ERK1 Abs. ERK1/2 activation was observed after 5 min of stimulation with mAb L243 and
persisted up to 15 min (Fig. 4C). Once again, dimerization of HLA-DR molecules was required to trigger the phosphorylation of
ERK1/2. We detected no activation of the p38 MAPK under our conditions
(data not shown). Stimulation with PMA-induced ERK1/2 activation (Fig.
4C), but had no effect on p38 (data not shown).
Having confirmed the requirement of HLA-DR dimerization for the
signaling events described above, we then investigated the role of raft
integrity in these responses. We stimulated M
To further confirm that the presence of HLA-DR molecules in lipid rafts
was crucial for their capacity to initiate tyrosine phosphorylation,
especially of Lyn, we pretreated CIITA-transfected THP-1 cells with
M
Taken together the above results indicate that although lipid rafts act
as a platform for HLA-DR-induced PTK and Lyn activation, they are not
involved in the activation of ERK pathway, which is a strong support
for the involvement of two separate HLA-DR signaling pathways in APCs.
To further confirm this hypothesis, and to demonstrate that ERK
activation is independent of Lyn function, the effects of PP2 and PP1
on HLA-DR-induced ERK activation was analyzed. Cells were pretreated
with PP2 or PP1 for 30 min prior to HLA-DR stimulation. Cells were then
lysated and analyzed by Western blot for PTK and ERK activation. Fig.
7 shows that although tyrosine
phosphorylation is completely blocked by treatment with both
inhibitors, activation of ERK is not affected by this treatment.
The Cytoplasmic Domain of Both HLA-DR-induced Homotypic Aggregation Requires Raft
Integrity--
The constitutive localization of HLA-DR molecules in
the lipid rafts of various cell types together with the dramatic effect of M
To further support our conclusion, we treated cells with the erbstatin
analog, a selective PTK inhibitor, and PP2, the Src family specific
kinase inhibitor. Fig. 10 shows that
PTK inhibitor and PP2 blocked HLA-DR-induced cell-cell adhesion whereas
PD98059, the selective inhibitor of the ERK1/2 pathway, did not. The
concentration of PD98059 used was sufficient to completely block
HLA-DR-induced ERK1/2 activation (data not shown). These results
confirm that HLA-DR-induced cell-cell adhesion involves PTK, especially
Src family members, and that the integrity of lipid rafts is essential for proper PTK signaling-induced cell-cell adhesion.
Cholesterol/sphingolipid-rich plasma membrane microdomains,
commonly called lipid rafts, are known to play a pivotal regulatory role in various cellular processes, including membrane trafficking and
signal transduction initiation pathways (17). Although their role in
BCR and TCR-signaling is well established, investigation of their
possible involvement in MHC class II-induced signal transduction pathways is just beginning. Anderson et al. (22) reported
that significant amounts of MHC class II molecules (20-50%) are
constitutively present in the lipid rafts of murine and human B cell
lines. In contrast, Huby et al. (23) failed to detect any
MHC class II molecules in the lipid rafts of a human IFN- The constitutive localization of HLA-DR in the lipid rafts is not
limited to B and monocytic cell lines. These molecules have also been
detected in the lipid rafts of both normal human tonsillar B cells and
monocytes. Although MHC class II molecules and BCR share numerous
similarities (30), and the presence of EBV viral proteins is reported
to block the translocation of BCR into rafts, the localization of
HLA-DR molecules in these domains is unaffected by the EBV
transformation (31). However, like BCR (21), HLA-DR localization in
rafts was unaffected by the deletion of the cytoplasmic tails of the
All the HLA-DR-induced events studied required HLA-DR dimerization on
the cell surface. None was dependent on the recruitment of additional
HLA-DR molecules, indicating that the small amount of HLA-DR
constitutively present in the lipid rafts of CIITA-transfected THP-1
cells was sufficient for various cellular events. This was strongly
supported by our results showing that ligation of HLA-DR with a
bivalent ligand failed to induce additional recruitment of HLA-DR
molecules into raft microdomains but induced rapid protein tyrosine
phosphorylation of many substrates, including Lyn, a member of the Src
family tyrosine kinase (16). However, we cannot rule out that
additional recruitment of HLA-DR molecules into lipid rafts can
increase the HLA-DR-induced response, and such recruitment can be
required if the studied cells express low levels of HLA-DR on their surfaces.
The slight but reproducible increase of HLA-DR recruitment observed
with SEA and MAM can be explained by the ability of these two SAgs to
oligomerize HLA-DR molecules on cell surface (33, 44). Upon binding to
HLA-DR molecules, these two SAgs are capable of inducing additional
recruitment of HLA-DR, which strongly supports the observation by Huby
et al. (23) of HLA-DR translocation using a combination of
primary and secondary antibodies.
The induction of protein tyrosine phosphorylation following HLA-DR
dimerization was found to be critically dependent on raft integrity
since disruption by M In addition to the activation of PTK signaling pathways, the ligation
of HLA-DR molecules with bivalent mAb L243 resulted in the activation
of ERK 1/2, but not p38. This pathway was delayed compared with the PTK
pathway (maximum at 15 min versus 5 min). The
HLA-DR-mediated activation of ERK1/2 and p38 has been reported in human
monocytes (47) using solid-phase mAb L243, which is equivalent to a
high order of cross-linking. The ERK1/2 pathway was probably a parallel
signal transduction pathway in our model since raft disruption had no
impact on its activation. The dimerization of HLA-DR molecules by a
bivalent ligand was necessary and sufficient to initiate ERK1/2
activation despite the disruption of raft integrity, indicating that
the dimerization of non-lipid raft HLA-DR molecules might be sufficient
to trigger certain cellular events. These results point to two separate
HLA-DR signaling pathways in antigen presenting cells, as previously
proposed (12, 13). This possibility is strongly supported by the
results showing that the cytoplasmic domains of the Anderson et al. (22) reported that efficient antigen
presentation at low ligand densities is another important function of
the enrichment of MHC class II molecules in the lipid rafts of murine
and human B cell lines. Watts (48) used MHC class II molecules
immobilized on planar membranes to demonstrate that individual MHC
class II-peptide complexes must be less than 20-nm apart for proper T
cell activation. Both studies highlight the importance of the
localization and concentration of MHC class II molecules for antigen
presentation. Given the relatively small number of MHC-peptide
complexes required to activate T cells (49), our results with monocytes
suggest that it is not merely the number of MHC class II molecules in
individual rafts but rather their spatial organization that determines
T cell activation potential. Dimerization and/or oligomerization MHC
class II molecules at the cell surface might allow cross-activation,
thereby circumventing the need for large numbers of MHC molecules at
the site of the immunological synapse.
Since bivalent but not monovalent SAgs are able to induce cytokine gene
expression (8, 9), it will be interesting to determine the roles played
by lipid rafts in this event. If lipid rafts are indeed involved, it
will reinforce the notion that they are of immunological relevance for
APCs. Studies are currently underway to address this question and to
determine the regions as well as the HLA-DR residues involved in the
constitutive and/or additional recruitment of HLA-DR into these microdomains.
B cell lines, monocytic
cell lines, transfected HeLa cells, tonsillar B cells, and human
monocytes. Localization of HLA-DR in these microdomains was unaffected
by the deletion of the cytoplasmic domain of both the
and
chains. Ligation of HLA-DR with a bivalent, but not a monovalent,
ligand resulted in rapid tyrosine phosphorylation of many substrates,
especially Lyn, and activation of ERK1/2 MAP kinase. However, the
treatment failed to induce further recruitment of HLA-DR molecules into
lipid rafts. The HLA-DR-induced signaling events were accompanied by
the induction of cell-cell adhesion that could be inhibited by PTK and
Lyn but not ERK1/2 inhibitors. Disruption of lipid rafts by
methyl-
-cyclodextrin (M
CD) resulted in the loss of membrane raft
association with HLA-DR molecules, inhibition of HLA-DR-mediated
protein tyrosine phosphorylation and cell-cell adhesion. M
CD
did not affect the activation of ERK1/2, which was absent from lipid
rafts. These results indicate that although all the HLA-DR-induced
events studied are dependent on HLA-DR dimerization, some require the
presence of HLA-DR molecules in lipid rafts, whereas others do not.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
chain, in particular residues 225, 227, and 228, are required for
antigen presentation, cAMP accumulation, PKC
translocation, and the
expression of CD80 molecules in murine B cells (10-14). Other MHC
class II-induced biochemical events such as tyrosine phosphorylation,
phosphoinositide hydrolysis, and calcium mobilization, are
unaffected by the substitution or deletion of the cytoplasmic domains
of the
and
chains (12, 13). It has been proposed that both
transmembrane and cytoplasmic regions are involved in signaling via MHC
class II molecules using different pathways. Nevertheless, the two
pathways must be at least partially cooperative since disruption of
either leads to a loss of MHC class II-induced B cell differentiation
(13, 15).
-treated THP-1 monocytic cell line unless the molecules were
oligomerized by cross-linking with specific primary Abs followed by a
secondary Ab. Indeed, the translocation of MHC class II molecules into
lipid rafts seems to be a requirement for PTK activation.
, and EBV+ B cell
lines, monocytes, transfected HeLa cells, and monocytic THP-1 cells
treated with IFN-
or transfected with the MHC class II
transactivator CIITA. The localization of HLA-DR in the lipid rafts was
unaffected by the deletion of the cytoplasmic domains of the
and
chains. We further show that initiation of signal transduction such
as activation of PTK, in particular Lyn, and homotypic cell-cell
adhesion required the dimerization of MHC class II molecules in the
lipid rafts. In contrast, activation of ERK1/2 was critically dependent
on MHC class II dimerization, but did not require the presence of MHC
class II molecules in the rafts.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
chain; a generous gift from Dr. P. Cresswell, Yale University, New Haven, CT), mAb XD5 (mouse IgG1,
recognizes the HLA-DR
chain), mAb W6/32 (mouse IgG2a, anti-MHC
class I; ATCC, Manassas, VA), mAb 9.4 (mouse IgG2a, anti-CD45; ATCC,
Manassas, VA), 8C12 (mouse IgG2a, anti-SEB; produced in our
laboratory), anti-phosphotyrosine mAb 4G10 (mouse; Upstate Biochemical
Inc., Lake Placid, NY), rabbit anti-phospho-ERK 1/2 (New England
Biolabs, Beverly, MA), rabbit anti-phospho-Lyn, goat anti-Erk1, and
rabbit anti-Lyn (Santa Cruz Biotechnology Inc., Santa Cruz, CA). The F(ab) fragment of L243 was prepared using a commercially available kit
(Pierce). The secondary Abs included donkey anti-goat IgG coupled to
HRP, goat anti-mouse IgG coupled to HRP, and goat anti-rabbit IgG
coupled to HRP (Santa Cruz Biotechnology, Santa Cruz, CA). Recombinant
staphylococcal enterotoxin A (SEA) and Mycoplasma arthritidis-derived mitogen (MAM) were produced as previously described (8, 24). The following products were purchased from
commercial sources: erbstatin analog (Calbiochem, La Jolla, CA), PP1
(Biomol, Plymouth Meeting, PA), methyl-
-cyclodextrin (M
CD)
(Sigma), PMA (Sigma), and HRP-conjugated cholera toxin B subunit
(CTB-HRP) (Sigma).
(100 units/ml) for 48 h (Peprotech, Rocky Hill, NJ) to induce
HLA-DR expression. THP-1 cells were also transfected with the human
CIITA cDNA,2 obtained as
a generous gift from J. P. Ting, by electroporation (26). HeLa cells
expressing full-length HLA-DR
and
chains (27) or mutated
versions that are devoid of their
and
cytoplasmic tails (28),
were generously provided by Dr. Rafick Sékaly (Department of
Microbiology and Immunology, University of Montreal). All cell lines
were cultured in RPMI 1640 supplemented with 10% inactivated fetal
bovine serum, L-glutamine, 2-mercaptoethanol, penicillin, and streptomycin (WISENT, Saint-Bruno, QC).
mAb DA6.147, washed extensively, and subjected to
chemiluminescent detection with HRP-conjugated anti-mouse IgG Ab using
an ECL kit (PerkinElmer Life Sciences). The distribution of HLA-DR
molecules was determined by densitometry using a Bio Image Densitometer
and whole band analysis software (Millipore, MI). To ensure that the
results fell into the linear range, scans of multiple exposures were
obtained. Immunoblots with CD45 mAb were performed using the same
conditions. For some experiments, the membranes were stripped and
sequentially probed with anti-phosphotyrosine mAb 4G10,
anti-phospho-ERK 1/2, anti-Erk1, anti-phospho-Lyn, and anti-Lyn Abs. To
detect the GM1 raft marker, 10-µl aliquots of each fraction were
dotted on a polyvinylidene difluoride membrane, which was then
incubated with CTB-HRP and developed with chemiluminescent reagents as
described above.
CD
for 30 min at 37 °C. After extensive washing at room
temperature, the cells were stimulated with either isotype-matched
control mAb 8C12 or mAb L243 at a concentration of 0.5 µg/106 cells for 5 min at 37 °C. To reverse the effect
of cholesterol depletion, M
CD-treated cells were incubated at
37 °C for 2 h in RPMI 1640 containing 50 µg/ml of cholesterol
water-soluble.
CD for 30 min at 37 °C in serum-free RPMI 1640 when indicated.
Cell-cell adhesion was monitored using a light microscope, and
photographs were taken.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
HLA-DR molecules are constitutively present
in lipid rafts of human tonsillar B cells. The presence of EBV
proteins in human B cell lines does not interfere with constitutive
residency. A, human tonsillar B cells were lysed in 1%
Triton X-100 for 30 min on ice and then subjected to sucrose density
gradient ultracentrifugation. Fractions were collected beginning from
the top of the centrifuge tube. Samples from 8 × 106
(fractions 1-7) and 2 × 106 (fractions 8-11) cell
equivalents were resolved by SDS-PAGE and analyzed by Western blot
using anti-HLA-DR chain mAb DA6.147. The membrane was then stripped
and reprobed with a primary antibody to reveal CD45. Aliquots (10 µl)
of each fraction were dot-spotted, and the lipid raft marker GM1
ganglioside was detected using HRP-conjugated CTB. The relative
positions of the raft and soluble fractions are indicated based on the
distribution of GM1 ganglioside and CD45. B, human
EBV-negative and -positive BJAB cells, and C, human
EBV-negative and -positive BL28 cells were fractionated as described
above. Raft (fractions 2-4) and soluble protein (fractions 9-11)
samples from 2 × 106 and 5 × 105
cell equivalents, respectively, were resolved by SDS-PAGE and analyzed
by immunoblot using anti-HLA-DR
chain mAb DA6.147.
for 48 h to induce high
levels of HLA-DR expression and to allow them to respond to HLA-DR
ligation in a manner similar to that observed in human monocytes.
Western blot analyses (Fig. 2,
A and B) revealed the presence of HLA-DR
molecules in the lipid raft fractions of human monocytes (3%) and
IFN-
-treated THP-1 cells (3.5%). Although the purity of the
monocytes used exceeded 90% (OKM1-positive) and no CD20-positive cells
were observed in the preparation (data not shown), a possible
contribution to the response by contaminating B cells could not be
ruled out. Indeed, it could be argued that the presence of HLA-DR
molecules in lipid rafts might result from the priming effect of
IFN-
and/or cell activation that occurred during purification
process. To overcome this problem, we generated stable THP-1 cells
transfected with the human MHC class II transactivator gene
CIITA and tested the distribution of HLA-DR molecules.
Quantitative densitometry of the immunoblots (Fig. 2C)
revealed that significant amounts of HLA-DR molecules were
constitutively associated with the lipid rafts of these
CIITA-transfected cells (7.5%).
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Fig. 2.
HLA-DR molecules are constitutively present
in lipid rafts in purified human monocytes and human monocytic cell
lines. A, normal human monocytes; B,
IFN- -treated THP-1 cells or C, CIITA-transfected THP-1
cells were fractionated as described in the legend to Fig. 1. Samples
from 2 × 106 (fractions 1-7) and 5 × 105 (fractions 8-11) cell equivalents for THP-1 cells and
8 × 106 and 2 × 106 cell
equivalents for monocytes were resolved by SDS-PAGE and analyzed by
immunoblot using anti-HLA-DR
chain mAb DA6.147.
chain and
the second, with the
chain of another MHC class II molecule (32,
33). CIITA-transfected cells were stimulated for 5 min at 37 °C with
8C12 isotype-matched control mAb, anti-HLA-DR mAb L243, SEA, or MAM.
Cell lysates were prepared and fractionated as described above for the
analysis of HLA-DR partitioning by immunoblot. Under these conditions, there was no additional recruitment of HLA-DR molecules into the raft
compartment when CIITA-transfected THP-1 cells were stimulated with
anti-HLA-DR (Fig. 3A) (7.5%
for isotype control versus 8% for L243). In contrast,
stimulation with SEA or MAM (Fig. 3B) led to a slight but
reproducible increase in HLA-DR in the lipid rafts (10% for SEA and
11% for MAM). Similar results were obtained with IFN-
-treated THP-1
and B cell lines (data not shown).
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Fig. 3.
Ligation of HLA-DR molecules with a bivalent
mAb L243 failed to induce additional recruitment of HLA-DR molecules
into rafts. A, CIITA-transfected THP-1 cells were
stimulated for 5 min at 37 °C with: (i) 8C12 isotype-control mAb or
anti-HLA-DR mAb L243 (0.5 µg/106 cells), or (ii) SEA or
MAM at a concentration of 1 µg/106 cells. Cells were then
lysed in 1% Triton X-100 and fractionated as described in the legend
to Fig. 1. Raft (fractions 2-4) and soluble protein (fractions 9-11)
samples from 2 × 106 and 5 × 105
cell equivalents, respectively, were resolved by SDS-PAGE, and analyzed
by immunoblot using anti-HLA-DR chain mAb DA6.147. C,
disruption of lipid rafts by M
CD treatment abolished the association
of HLA-DR molecules with membrane rafts. Cells pretreated or not with
10 mM M
CD for 30 min at 37 °C were stimulated as
above. After lysis in 1% Triton X-100 and fractionation, samples of
pooled raft and pooled soluble fractions from 2 × 106
and 5 × 105 cell equivalents, respectively, were
subjected to SDS-PAGE and immunoblot analysis.
CD, a drug that disrupts rafts by extracting
cholesterol (34). Washed cells were then stimulated for 5 min at
37 °C with isotype-matched control mAb 8C12 or anti-HLA-DR mAb L243.
Cell lysates were fractionated, pooled as indicated in the figure
legend, and analyzed for HLA-DR partitioning by immunoblotting.
Treatment with M
CD did not affect the interaction of HLA-DR with the
specific Abs as determined by fluorescence-activated cell sorting
(FACS) analysis, and cell viability was not compromised as measured by
trypan blue exclusion (data not shown). Fig. 3C shows that
M
CD treatment led to the complete disappearance of HLA-DR molecules
from these microdomains of both stimulated and unstimulated cells.
CD Inhibits HLA-DR Dimerization-mediated Proximal Tyrosine
Phosphorylation Events but Not ERK Activation--
As mentioned above,
Huby et al. (23) reported that oligomerization of HLA-DR
molecules by cross-linking with primary and secondary Abs leads to
HLA-DR recruitment into the lipid rafts of human IFN-
-treated THP-1
monocytic cells, an event that seems to be required for PTK activation.
On the other hand, Mehindate et al. (8, 9, 35) demonstrated
that dimerization of HLA-DR molecules by SAgs is sufficient for various
HLA-DR-induced cellular events to occur, particularly
PTK-dependent cytokine expression. We therefore
hypothesized that while most HLA-DR-induced events require HLA-DR
dimerization, some are raft-dependent while others are
raft-independent, and none require an additional recruitment of HLA-DR
molecules into lipid rafts.
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Fig. 4.
Ligation of HLA-DR molecules by the bivalent
mAb L243 but not by the F(ab) fragment of L243 induces rapid protein
tyrosine phosphorylation, phosphorylation of Lyn, and ERK1/2 activation
in a CIITA-transfected THP-1 monocytic cell line. After 5 and 15 min of stimulation at 37 °C with 8C12 isotype-control mAb, anti-DR
mAb L243, and the F(ab) fragment of L243, CIITA-transfected THP-1 cells
were lysed, and total cell lysates from 5 × 104
(A and C) and 2 × 105
(B) cell equivalents were subjected to Western blot analysis
with Abs specific for either: A, pTyr; B, p-Lyn
and Lyn; or C, p-ERK1/2 and ERK1.
CD-treated and
untreated CIITA-transfected THP-1 cells for 5 min at 37 °C with 8C12
control mAb and mAb L243. Fig.
5A shows that treatment with
M
CD led to a significant inhibition of HLA-DR-induced tyrosine phosphorylation in total cell lysates. Because Lyn is enriched in lipid
rafts (16, 19) and Lyn phosphorylation is triggered by the anti-HLA-DR
mAb, we then examined the phosphorylation status of Lyn. The increased
phosphorylation of Lyn resulting from the stimulation by the
anti-HLA-DR mAb was also inhibited following pretreatment of cells with
M
CD (Fig. 5B). In contrast, HLA-DR-mediated activation of
pERK1/2 was unaffected by M
CD (Fig. 5C).
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Fig. 5.
Raft disruption by
M CD inhibits HLA-DR-mediated protein tyrosine
phosphorylation and the phosphorylation of Lyn but not ERK1/2
activation in a CIITA-transfected THP-1 monocytic cell line.
CIITA-transfected THP-1 cells were pretreated or not with 10 mM M
CD for 30 min at 37 °C then stimulated with the
isotype control mAb or the anti-DR mAb for 5 min at 37 °C. Cells
were lysed, and total cell lysates from 5 × 104
(A and C) or 2 × 105
(B) cell equivalents were subjected to Western blot analysis
with Abs specific for either: A, pTyr; B, p-Lyn
and Lyn; and C, p-ERK1/2 and ERK1.
CD before stimulation and fractionation on a sucrose gradient.
Although most tyrosine-phosphorylated proteins following L243 mAb
stimulation were recovered in the soluble fractions, significant
tyrosine phosphorylation was also observed in the lipid raft fractions
(Fig. 6A). Tyrosine
phosphorylation events in rafts were undetectable after the M
CD
treatment, whereas phosphorylation in soluble fractions was markedly
inhibited. As expected, the phosphorylation of Lyn in rafts was
abolished following raft disruption (Fig. 6B). However,
ERK1/2 were not present in the lipid rafts and their activation by mAb
L243, as measured by immunoblotting of their phosphorylated forms, was
not affected by the M
CD treatment (Fig. 6C).
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Fig. 6.
M CD abolishes the
association of HLA-DR molecules with membrane rafts, which results in
the inhibition of their ability to induce protein tyrosine
phosphorylation and Lyn phosphorylation in a CIITA-transfected THP-1
monocytic cell line. Cells were pretreated or not with 10 mM M
CD for 30 min at 37 °C, then stimulated with the
isotype-control mAb or the anti-DR mAb for 5 min at 37 °C. After
lysis in 1% Triton X-100 and fractionation, samples of pooled raft and
pooled soluble fractions from 5 × 105 and 1 × 105 cell equivalents (A and B) or
2 × 106 and 5 × 105 cell
equivalents (C), respectively, were subjected to sequential
Western blot analysis as described in the legend to Fig. 5.
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Fig. 7.
The Src family kinase inhibitors PP1 and PP2
inhibit HLA-DR-induced tyrosine phosphorylation but fail to affect
activation of ERK1/2 in CIITA-transfected THP-1 monocytic cell
line. Cells were either untreated or pretreated with 10 µM PP1 or PP2 for 30 min at 37 °C, and then stimulated
for 15 min at 37 °C with 8C12 isotype control mAb or anti-DR mAb
L243. Total cell lysates from 5 × 104 cell
equivalents were subjected to Western blot analysis with Abs specific
for either: A, pTyr; B, p-ERK1/2 and ERK1.
and
Chains Is Not Required
for HLA-DR Association with Lipid Rafts--
The results presented
above indicate that HLA-DR-mediated tyrosine phosphorylation requires
raft integrity while ERK1/2 activation does not. Based on these
observations and studies showing that the deletion of the cytoplasmic
domains of both the
and
chains of HLA-DR does not affect
HLA-DR-induced tyrosine phosphorylation (37), we hypothesized that the
cytoplasmic domains are not involved in the localization of HLA-DR
molecules in the lipid rafts. To verify this hypothesis, HeLa cells
transfected with wild-type HLA-DR or truncated HLA-DR (cytoplasmic
domains of both the
and
chains deleted) and that expressed
comparable levels of these molecules at the cell surface were used
(Fig. 8A). Fractions of Triton
X-100 cell lysates were isolated by sucrose gradient and analyzed by
Western blot as described above. Our analysis (Fig. 8B)
indicated that there were small but significant amounts of HLA-DR in
the lipid rafts of both cell types (4.4% for wild-type HLA-DR
versus 2.5% for truncated HLA-DR). Since, it is well
established that large quantity of HLA-DR is retained in the
intracellular compartments of cells transfected with truncated HLA-DR
(37), we conducted a fluorometric analysis to determine the percentage of raft versus non-raft HLA-DR molecules on the cell surface
(38). To this end, cells were treated with FITC-conjugated 8C12 isotype control or FITC-conjugated L243. Cells were washed, lysed in Triton X-100, fractionated, and analyzed using a Microplate Fluorescence Reader. Our results indicated that both cell types expressed
significant amounts of HLA-DR constitutively localized in lipid rafts
(6.6% for wild-type HLA-DR versus 5.8% for truncated
HLA-DR). These results indicate that the cytoplasmic domains of the
and
chains of HLA-DR are not required for HLA-DR localization in
lipid rafts.
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Fig. 8.
The cytoplasmic domains of the
and
chains of HLA-DR are
not required for HLA-DR association with lipid rafts.
A, analysis of the expression of HLA-DR in transfected HeLa
cells by flow cytometry. B, transfected-HeLa cells were
lysed in 1% Triton X-100 for 30 min on ice, subjected to sucrose
density gradient ultracentrifugation and fractionated as above. Samples
from 7 × 106 (fractions 2-4) and 2 × 106 (fractions 9-11) cell equivalents were resolved by
SDS-PAGE and analyzed by Western blot using anti-HLA-DR
chain mAb
DA6.147 or XD5 for the HLA-DR
chain.
CD on early signaling events suggest that the presence of these
molecules in lipid rafts is of biological relevance. Based on this
observation, and since MHC class II-induced cell-cell adhesion of human
B lymphocytes and B cell lines (4, 39, 40) is known to involve PKC
and/or tyrosine kinase activation, we hypothesized that HLA-DR-induced
adhesion is critically dependent on lipid raft integrity. To test our
hypothesis, M
CD-treated and untreated CIITA-transfected THP-1 cells
were stimulated with the control anti-class I mAb W6/32, anti-HLA-DR
mAb L243, and the F(ab) fragment of L243. As previously reported in
human B cells (41), stimulation of CIITA-transfected THP-1 cells with bivalent anti-HLA-DR mAb for 1 h led to cell-cell adhesion (Fig. 9). However, the F(ab) fragment of L243
and anti-MHC class I mAb failed to induce any detectable response.
Pretreatment of CIITA-transfected THP-1 cells with M
CD abolished
HLA-DR-induced cell-cell adhesion, indicating that lipid raft integrity
and HLA-DR dimerization is essential for the HLA-DR-induced response.
We then attempted to reverse the inhibitory effect by adding
cholesterol. M
CD-treated cells were incubated in RPMI 1640 containing 50 µg/ml of cholesterol for 2 h and then stimulated
with L243 or W6/32. Our results clearly demonstrate that such a
treatment reverses the inhibitory effect induced by M
CD
treatment.
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Fig. 9.
HLA-DR-induced cell-cell adhesion in
CIITA-transfected THP-1 cells is blocked by
M CD treatment, an effect that can be reversed
by cholesterol treatment. Cells were pretreated or not with 10 mM M
CD for 30 min at 37 °C in serum-free RPMI 1640. After extensive washing, the cells were resuspended in RPMI 1640 containing 5% fetal bovine serum and seeded in 96-well microtiter
plates at 1 × 105 cells/well. The cells were then
stimulated at 37 °C for 1 h with mAb W6/32, mAb L243, or the
F(ab) fragment of L243 at 1 µg/ml. To restore the integrity of rafts,
M
CD-treated cells were resuspended in RPMI 1640 containing 50 µg/ml of cholesterol water-soluble (chol.) and incubated
for 2 h at 37 °C. Cells were then stimulated at 37 °C for
1 h with mAb W6/32, mAb L243, or the F(ab) fragment of L243 at 1 µg/ml as described. Cell aggregation was monitored using a light
microscope, and photographs were taken with a camera coupled to
the microscope.
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Fig. 10.
Specific inhibitors of PTK and Src family
kinases but not of the ERK1/2 pathway inhibit HLA-DR-induced homotypic
aggregation in a CIITA-transfected THP-1 monocytic cell line.
CIITA-transfected THP-1 cells were pretreated or not with erbstatin
analog (5 µg/ml), PP2 (10 µM), or PD98059 (20 µM) for 1 h at 37 °C in RPMI 1640. The cells were
then seeded in 96-well microtiter plates at 1 × 105
cells/well and were left unstimulated or were stimulated at 37 °C
for 1h with the anti-DR mAb L243 at 1 µg/ml. Cell aggregation was
monitored using a light microscope, and photographs were taken using a
camera coupled to the microscope.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-treated
THP-1 monocytic cell line. Data obtained in the course of the present
investigation showed that small but significant amounts (3-8%) of
HLA-DR molecules were constitutively present in the lipid rafts of
various cell types. This is consistent with the results reported by
Kropshofer et al. (42), who showed that ~3% of HLA-DR
molecules are constitutively located in the lipid rafts of human B cell
lines and dentritic cells. The variability in the amount of HLA-DR
molecules in the lipid rafts reported in these various studies is
likely to be due to the cholesterol and/or sphingolipid content of the
cell culture medium as reported in another cell system (43), and to
different detergents and/or concentration of the used detergents.
and
chains.
CD resulted in the loss of HLA-DR molecules
from the raft compartment and the complete abrogation of
phosphorylation of pTyr and Lyn. One of the consequences of pTyr and
Lyn activation was the induction of cell-cell adhesion, an event that
was critically dependent on the dimerization of HLA-DR molecules and
their presence in lipid rafts. These results indicate that membrane
rafts provide a microenvironment crucial for proximal HLA-DR-mediated
pTyr events. In fact, these events, particularly Lyn activation, must
occur in membrane rafts to allow the proper association of HLA-DR
molecules with components that are essential for transducing signals
initiated by HLA-DR given that these molecules are devoid of classic
signaling motifs (15). What then is the mechanism involved in the
induction of PTK and Lyn activation? It is possible that the
dimerization of HLA-DR molecules promotes the formation of an immune
signaling complex involving HLA-DR, co-receptors, adaptor molecules,
and Src family tyrosine kinases. This complex would then be able to
transactivate raft-associated tyrosine kinases, thereby initiating
signal transduction cascades. The high degree of trans-species
conservation of the transmembrane sequences of the
/
chains of
MHC class II molecules and the dependence on intact transmembrane
domains for proper MHC class II-mediated tyrosine phosphorylation (12,
13, 15, 37) point to interactions between HLA-DR and partner molecules. This is strongly supported by studies demonstrating, at least in B
cells, the existence of a physical association between MHC class II
molecules and cell surface receptors, including CD20, CD40, CD19,
CD79a/CD79b, and several members of the tetraspanin family such as CD81
and 82. The role of the CD79a/CD79b association in MHC class II-induced
signaling has been elegantly demonstrated in murine B cells (30), and
the involvement of CD19 and CD20 has been demonstrated in
HLA-DR-induced PTK activation in human B cells (45, 46). Since none of
these molecules is expressed in monocytes and monocytic cell lines,
studies are currently underway to determine whether HLA-DR molecules in
human monocytes exist in association with other cell surface molecules
and whether HLA-DR molecules are directly or indirectly associated with
signaling partners.
and
chains
are not required for HLA-DR localization in lipid rafts.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. C. Pedro
(Hôtel-Dieu-de-Québec Hospital, Quebec City, QC) and Dr. J. Menezes (Sainte-Justine Hospital, Montreal, QC) for providing the B
cell lines and Dr. Rafick Sékaly (University of Montreal) for
providing the HLA-DR and
chains devoid of their cytoplasmic tails.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from CIHR, the Arthritis Society of Canada, and the Canadian Arthritis Network.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Recipient of a Formation Chercheurs & Aide Recherche studentship.
Recipient of a Canadian Institutes for Health Research scholarship.
** Recipient of a scientist award from the Arthritis Society of Canada. To whom correspondence should be addressed: Centre de Recherche en Rhumatologie et Immunologie, CHUL, 2705 boulevard Laurier, T1-49, Quebec City, Quebec G1V 4G2, Canada. Tel.: 418-654-2772; Fax: 418-654-2765; E-mail: Walid.Mourad@crchul.ulaval.ca.
Published, JBC Papers in Press, December 22, 2002, DOI 10.1074/jbc.M211566200
2 H. Khalil, W. Mourad, J. Thibodeau, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
MHC, major
histocompatibility complex;
PTK, protein tyrosine kinase;
ECL, enhanced
chemiluminescence;
MAPK, mitogen-activated protein kinase;
ERK, extracellular signal-regulated kinase;
MCD, methyl-
-cyclodextrin;
Ab, antibody;
FITC, fluorescein isothiocyanate;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
HRP, horseradish peroxidase;
SAgs, superantigens;
TCR, T cell receptor;
CIITA, class II transactivator;
APC, antigen-presenting cells;
BCR, B
cell receptor;
SEA, staphylococcal enterotoxin A;
MAM, Mycoplasma
arthritidis-derived mitogen.
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