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IMMUNOBIOLOGY
From the Department of Cell and Molecular Biology,
Karolinska Institutet, Stockholm; the Ludwig Institute of Cancer
Research, Biomedical Center, Uppsala, Sweden; and the Gastrointestinal
Unit, Massachusetts General Hospital, Boston, MA.
Patients with the immunodeficiency disorder Wiskott-Aldrich
syndrome (WAS) have lymphocytes with aberrant microvilli, and their T
cells, macrophages, and dendritic cells are impaired in cytoskeletal-dependent processes. WAS is caused by a defective or a
missing WAS protein (WASP). Signal mediators interleukin-4 (IL-4) and
CD40 are important for actin-dependent morphology changes in B cells. A
possible function of WASP and its interacting partners, Cdc42 and Rac1,
was investigated for these changes. It was found that active Cdc42 and
Rac1 induced filopodia and lamellipodia, respectively, in activated B
cells. Evidence is given that IL-4 has a specific role in the regulated
cycling of Cdc42 because IL-4 partially and transiently depleted active
Cdc42 from detergent extract of activated B cells. WASP-deficient B
lymphocytes were impaired in IL-4- and CD40-dependent induction of
polarized and spread cells. Microvilli were expressed on WASP-deficient
B cells, but they appeared shorter and less dense in cell contacts than in wild-type cells. In conclusion, evidence is provided for the involvement of Cdc42, Rac1, and WASP in the cytoskeletal regulation of
B lymphocytes. Aberrations in WASP-deficient B lymphocytes, described
here, provide further evidence that WAS is a cytoskeletal disease of
hematopoietic cells.
(Blood. 2001;98:1086-1094) Activation of lymphocytes is often accompanied by
changes in cell morphology. Transendothelial migration requires
microvilli-dependent adhesion and spreading of
lymphocytes.1 The fine regulation of immune activation in
lymph nodes and spleen is dependent on migratory and adhesive
capacities of B and T cells.2,3 For migration and eventual
contact with other immune cells to occur, adhesion receptors are
essential and appear at the trailing uropod of a migrating cell. In
contrast, chemoattractant receptors are expressed at the leading
edge.4 Reorganization of microtubuli and dynamics of the
actomyosin filaments are crucial events during migration and
adhesion.5
T-cell-derived signals through interleukin-4 (IL-4) receptors or CD40
on B cells induce cytoskeletal rearrangements, altering B-cell
morphology. Thymus-independent stimulus lipopolysaccharide (LPS) from
gram-negative bacteria, and even more pronounced IL-4 and agonistic
anti-CD40 antibodies (anti-CD40 monoclonal antibody [mAb]), mimicking
the CD40 ligand, stimulate B lymphocytes to acquire a motile
shape.6-9 It has been shown that anti-CD40 or LPS plus
IL-4, but not LPS alone, induces the formation of dendritic protrusions
when B cells are cultured on immobilized antibodies directed to B-cell
surface determinants.10,11 LPS plus IL-412 or
anti-CD4013 induces homotypic B-cell aggregates that are tight and spherical, whereas LPS-induced aggregates are loose and
irregularly shaped.14 The induction of spread cells and tight aggregates are correlated with an increase in number and length
of villous structures on the B-cell surface.9
Wiskott-Aldrich syndrome (WAS) is a severe X-linked immunodeficiency
disorder caused by mutations in the gene encoding the WAS protein
(WASP). Lymphocytes from patients with WAS have aberrant formation of
microvilli.15-17 In addition, T cells16,17
and B cells18 have been reported to have abnormal
cytoarchitecture. WASP-deficient dendritic cells19 and
macrophages20,21 have aberrant polarization and are
impaired in stimulated migration. So far, WASP expression has only been
detected in cells of the hematopoietic lineage,22 whereas
its homologue N-WASP is more ubiquitously
expressed.23 WASP24 and N-WASP25
interact directly with the Arp2/3 complex, which is important for
polymerization from barbed ends and branching of actin
filaments.26 Similarly, WASP27,28 and
N-WASP23 colocalize with polymerized actin. Recently, mice
carrying a WASP null allele were described,29,30 and it
was found that T-cell function was impaired, whereas mutant B cells
proliferated normally to B-cell stimuli.
WASP was identified as a binding partner for the small GTPase
Cdc42.27,31 Cdc42 and its relative Rac1 belong to the
family of Rho GTPases, known to operate as molecular switches, cycling between an active guanosine triphosphate (GTP)-bound and an inactive guanosine diphosphate (GDP)-bound state. This cycling is tightly regulated by guanosine nucleotide exchange factors (GEFs), which stimulate the exchange of GDP to GTP, and GTPase-activating proteins (GAPs), which increase the intrinsic rate of GTP
hydrolysis.32 Rho GTPases are involved in many cellular
processes in which the cell cytoskeleton is the final target, such as
migration,33,34 cytokinesis,35 and
phagocytosis.36,37 Effector proteins of Cdc42 and Rac1 are
numerous, and many of them, including WASP and N-WASP, have
Cdc42/Rac-interactive binding (CRIB) motifs that specifically recognize
the GTP-bound form.32
In this paper, we have investigated the intracellular pathways induced
by LPS, IL-4, and CD40 activation in B lymphocytes. We show that active
Cdc42 and Rac1 induced morphology changes in activated B cells. IL-4
partially and transiently depleted active Cdc42 from detergent extract
of B cells. Furthermore, we found that WASP-deficient B cells were
impaired in many morphologic processes. Interestingly, N-WASP was
clearly expressed in WASP-deficient B cells. The data suggest that
Cdc42, Rac1, and WASP are important for the induction of morphologic
changes leading to B-cell migration and adhesion.
Reagents and antibodies
Animals
Cell culture Small resting B cells were prepared from splenic cell suspensions after the removal of T cells and Percoll gradient centrifugation as previously described.14,42 Cells were cultured at 0.5 × 106 cells/mL in RPMI 1640 supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 50 U/mL penicillin, 50 µg/mL streptomycin, 5 µM 2-mercaptoethanol (all from Gibco BRL, Life Technology, Paisley, United Kingdom) and 5% to 10% of batch-selected FCS at 37°C in a humidified atmosphere containing 5% CO2. Cell lines secreting monoclonal antibodies were cultured in the same way but with 1% to 5% batch-selected FCS.Migration assay To exclude contaminants of chemoattractants, both the X63-derived IL-4 and the purified IL-4 (Peprotech) were tested in a migration assay using a 2-chamber system (polycarbonate filter plates, 5-µm pore size; Transwell, Costar, New York, NY). B cells (100 000) were loaded to the upper wells, and no stimulus (5% FCS in RPMI), X63 IL-4, purified IL-4, or SDF-1 (50 ng/mL; R&D Systems, Oxon, United Kingdom) was added onto the lower wells. B cells were allowed to migrate for 2 hours, and then cells in the lower wells were collected and enumerated in triplicate. FCS induced low migration (2400 ± 670 cells, background). X63 IL-4 and purified IL-4 induced a slightly higher number of migrating cells (3300 ± 370 and 3600 ± 350 cells, respectively). Compared to background levels, these changes were nonsignificant (P > .05) using a t test. SDF-1, a potent B-cell chemoattractant, induced the migration of 8300 ± 800 cells (P < .001 compared to background). Figures indicate the mean of triplicate results ± 1 SD. Thus, the X63 supernatant and purified IL-4 seem to lack classical chemokines inducing migration with fast kinetics.Transfections and stainings cDNAs of myc-tagged Cdc42L61, Cdc42N17, wild-type Cdc42, RacL61, and RacN17 in the pRK5 vector (Pharmingen, Stockholm, Sweden) was used. CB B lymphocytes were stimulated with LPS for 46 to 50 hours. Fourteen micrograms DNA was electroporated (960 µF, 350 V, and 200 ) into activated B cells (10 × 106), then
diluted in medium containing LPS or LPS plus IL-4. Cells were
transferred to anti-CD44-coated coverslip and cultured for 3 to 4 hours. They were thereafter fixed in either acetone-methanol (1:1) for
20 minutes at  20°C or 4% phosphate-buffered paraformaldehyde for
10 minutes at room temperature, followed by 0.1% Triton X-100 for 1 minute. Cells were first blocked in 10% mouse serum in
phosphate-buffered saline (PBS), stained with rabbit
anti-myc antibodies, biotinylated antirabbit antibodies, and
streptavidin-FITC. All solutions contained 10% mouse serum in PBS.
Antibody coating of plastic culture vessels or coverslips was performed as follows: goat anti-rat IgG was added and incubated for 1 hour at 37°C, followed by addition of rat anti-mouse CD44 (RK3G9) antibodies, incubated overnight at room temperature. Coverslips were washed in balanced salt solution before cells were added. Glutathione-S-transferase pull-down assay Residues 201-321 (CRIB) of human WASP were cloned into the pGEX-2T (Pharmacia Biotech, Uppsala, Sweden). Production and purification of recombinant GST-CRIB was described elsewhere.43 Precipitation of Cdc42-GTP was performed as previously described.43 Briefly, CB B lymphocytes (25 or 50 × 106 cells per sample) were stimulated with LPS for 48 hours, and IL-4 or FCS-containing RPMI was added in the last half hour of incubation. Cells were centrifuged at 4°C and lysed for 10 minutes on ice in RIPA buffer (100 µL/106 cells) containing 2 µg/mL aprotinin and leupeptin, 1 mM phenylmethylsulfonyl fluoride, and 5 mM MgCl2. Cdc42-GTP was precipitated from lysates with glutathione-Sepharose beads (Pharmacia Biotech), preloaded with GST-CRIB, for 30 minutes at 4°C. The beads were washed, resuspended in reduced sample buffer (50 mM Tris, 50 mM dithiothreitol, 0.1% bromphenol blue, 10% glycerol, and 5% sodium dodecyl sulfate [SDS]), boiled, and loaded on a 15% polyacrylamide gel. Separated proteins were transferred to a nitrocellulose membrane by Western blotting.Cell polarization assays B lymphocytes, isolated from spleens of WASP or
WASP+ mice, were cultured in 24-well tissue culture plates
in the presence of LPS, IL-4 (derived from the X63 plasmacytoma or
purified from Peprotech), anti-CD40 mAb, or no stimulus for 20 to 24 hours. Cells were fixed by adding an equal volume of 2.5%
phosphate-buffered glutaraldehyde and were washed twice in PBS before
they were counted using an inverted light microscope. The percentage of
polarized cells cells that were not spherical but were tapered and had
uropodswas determined.
Cell spreading assay Cell spreading assays were performed in antibody-coated 96-well plates as previously described.11 Briefly, cells were stimulated with LPS, LPS plus IL-4 (derived from the X63 plasmacytoma or purified from Peprotech), or anti-CD40 mAb and were transferred to anti-CD44-coated plates for 17 to 19 additional hours of culture. Cells were thereafter fixed in 2.5% phosphate-buffered glutaraldehyde, and the percentage of spread cellsdefined as having at least one
dendritic process of at least one cell diameterwas determined using
an inverted light microscope.
Aggregation assays B lymphocytes, isolated from spleens of WASP or
WASP+ mice, were cultured for 46 to 50 hours. The
morphology of B-cell aggregates, from cultures stimulated with LPS, LPS
plus IL-4 (derived from the X63 plasmacytoma or purified from
Peprotech), anti-CD40 mAb or anti-CD40 plus IL-4, was photographed
directly using a camera connected to an inverted microscope. Percentage
cell aggregation was measured by adding 100 µL trypan blue to 200 µL/well cultures. Cells were then resuspended by gentle pipetting up
and down 6 times in a blunted disposable pipette and were transferred
to a hemocytometer. Aggregated cells (2 or more cells in association) and single cells were enumerated under an inverted light microscope. The percentage of aggregated cells was then calculated.
Electron microscopy B lymphocytes, isolated from WASP and
WASP+ mice, were stimulated with LPS or anti-CD40 mAb plus
IL-4 for 48 hours. Aggregates were gently pelleted to preserve
cell-cell adhesion and microvilli and then were fixed in
glutaraldehyde. Specimen preparation was described
previously.11 For examination, a Jeol 100CX electron microscope (Tokyo, Japan) was used at 80 kV.
Western blot analysis B lymphocytes, isolated from WASP and
WASP+ mice, were stimulated with LPS or LPS plus IL-4 for
46 to 50 hours. Cells were harvested, lysed in buffer (50 mM Tris, 150 mM NaCl, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and 2 µg/mL aprotinin and leupeptin) at 4°C for 15 minutes. Equal protein
concentrations of lysates were boiled in reduced sample buffer
(described above) containing 2% SDS and were thereafter loaded on a
10% polyacrylamide gel. Separated proteins were transferred to a
polyvinylidene difluoride membrane by Western blotting. Membranes were
blocked in Tris-buffered saline, 0.05% Tween 20, and 5% fat-free dry
milk (Blotto A) for 1 hour at 4°C and then incubated with primary
antibodies in Blotto A overnight at 4°C. Horseradish
peroxidase-conjugated secondary antibody in Blotto A was added and
incubated for 1.5 hours at 4°C. Between steps, membranes were
extensively washed in Tris-buffered saline with 0.05% Tween 20. Blots
were developed with enhanced chemiluminescence (Pierce, Rockford, IL).
Membranes were stained with Ponceau Red to confirm equal loading.
Cdc42 and Rac1 induce filopodia and lamellipodia in activated B lymphocytes We wanted to investigate a potential role of Cdc42 and Rac1 in the cytoskeletal rearrangements of B lymphocytes. These GTPases induced morphologic changes in fibroblasts after microinjection.44,45 Notably, active Cdc42 induced filopodia, a structure that seems similar to the protrusions found on spread B cells.10,11 We therefore considered Cdc42 and Rac1 as having a potential role in IL-4-induced spreading. To elucidate the role of Cdc42 and Rac1 in B lymphocytes, we used the fact that the Rho GTPases can be converted to constitutively active (CA) and dominant negative (DN) forms using single amino acid substitutions. CA mutants are locked in the GTP-bound form, thereby preventing intrinsic and GAP-induced GTP hydrolysis.46,47 DN mutants are locked in the GDP-bound form, thus forming a nonfunctional complex with cellular GEFs and competing with endogenous proteins for binding to GEFs.48 CA, DN, or wild-type (WT) myc-tagged constructs of Cdc42 or Rac1 were transiently electroporated into LPS-activated B lymphocytes. Cells were then cultured in the presence of LPS or LPS plus IL-4 on anti-CD44 antibody layers (B cells, in contrast to fibroblasts, can only spread in the presence of antibody layers). Cells were fixed, permeabilized, and stained with anti-myc antibodies. CA Cdc42 induced long filopodia in cells stimulated with LPS or LPS plus IL-4, whereas DN Cdc42 failed to induce these structures (Figure 1A). WT Cdc42 induced short filopodia structures or lamellipodia (Figure 1A). CA Rac1 mainly induced lamellipodia in cells stimulated with LPS or LPS plus IL-4 and, rarely, short filopodia (Figure 1B). DN Rac1 did not induce these membrane structures (Figure 1B). Morphologic changes induced by CA and WT Cdc42 or Rac1 in LPS-stimulated cells were indistinguishable from those in LPS plus IL-4 stimulated cells. Thus, in this system, the LPS stimulus alone is sufficient to induce membrane alterations. Induction of morphologic changes was dependent on coating with anti-CD44 antibodies because, in the absence of antibody layers, no morphologic changes were detected and cells were spherical (not shown).
Short pulses of IL-4 partially and transiently deplete active Cdc42 from the B-cell cytosol Many proteins, including specific GAPs and GEFs, regulate the cycling of Cdc42. IL-4 may have a potential role in this process, thereby inducing cytoskeletal changes in activated B cells. We have used a GST-CRIB (from WASP) fusion protein to precipitate the cytosolic fraction of active Cdc42 from detergent lysates of LPS-stimulated B lymphocytes given short pulses of IL-4. In each experiment, extract from cells stimulated with LPS alone was used as a standard for the detected level of Cdc42. A clear decrease in the level of cytosolic Cdc42-GTP was detected after IL-4 stimulation for 5 minutes (Figure 2, lane 4) compared to the LPS standard (lane 1). No reduction was detected after IL-4 stimulation for 30 minutes, 10 minutes (lanes 2 and 3, respectively), or 1 minute (not shown). By adding FCS-containing medium alone, we controlled for the reduction being caused by IL-4 stimulation (not shown). Similar results were obtained in 4 experiments, suggesting that IL-4 stimulation of B cells leads to a transient depletion of active Cdc42 from the cytosol. In some other experiments, however, no difference between the groups was obtained. It is unlikely that these data were obtained by chance or the amount of active Cdc42 in the different experimental groups would have been totally variable, which was not the case. Instead, we believe this reflects some of the difficulties when working with rapidly cycling proteins.49
GTP Induction of B-cell polarization is impaired in the absence of WASP We have investigated whether the defects in polarization and migration of WAS dendritic cells and macrophages also apply to B cells from WASP-deficient mice. Lymphocytes undergoing locomotion characteristically have a polarized or prolonged cell body. Thus, assessment of cells with a motile cell shape can be used to determine locomotion capacity of lymphocytes.6,7,50 In WASP+ B cells (Figure 3, open bars), IL-4, anti-CD40 mAb, and LPS all induced a higher frequency of motile-shaped cells than noninduced cells, the 2 former stimuli giving higher responses than the latter. In WASP B cells (Figure
3, black bars), LPS stimuli induced a frequency of motile-shaped cells
similar to that in WASP+ cells. However, IL-4 and anti-CD40
induction of motile-shaped WASP B cells was impaired in
comparison to WASP+ cells. This suggests that WASP has an
important role for IL-4- and CD40-induced motility but that
LPS-induced signaling can bypass the requirement for WASP or use
completely different pathways.
Impaired spreading in the absence of WASP We also investigated the capacity of B-cell spreading in the absence of WASP. LPS plus IL-4 or anti-CD40 mAb induced a high number of spread WASP+ B cells (Figure 4, open bars). However, WASP B cells were impaired in spreading in response to
the corresponding stimuli (Figure 4, black bars). Only occasional
spread cells were seen in LPS cultures. Still, in the absence of WASP,
spreading was not completely abolished because the percentage of
WASP spread cells was higher in the presence of
suboptimal concentrations of IL-4 (Figure 4) or anti-CD40 (not shown)
than with LPS alone. Spreading did not occur in the absence of
immobilized antibodies or on surfaces coated with irrelevant antibodies
(not shown).
Aggregation is mildly impaired in the absence of WASP IL-4 and CD40 are known to induce B-cell homotypic adhesion, resulting in the formation of tight, spherical aggregates.12-14 We investigated whether WASP was important for this process. LPS-stimulated WASP+ B cells formed loose, irregularly shaped aggregates, and these were indistinguishable from those of WASP B cells (Figure
5A). LPS plus IL-4 induced tight,
spherical aggregates to a similar extent in WASP+ and
WASP B cells. However, anti-CD40 mAb- or anti-CD40 plus
IL-4-induced WASP aggregates were smaller, though still
spherical, and less dense than WASP+ aggregates. Another
method to estimate cellular aggregation is to gently resuspend
aggregate cultures and then enumerate single and aggregated cells.
Again, using this method, no differences in percentage of aggregated
cells were detected in LPS or LPS plus IL-4 cultures (Figure 5B). In
anti-CD40 or anti-CD40 plus IL-4 cultures, a mild defect was detected
in WASP B cells (black bars) compared to
WASP+ B cells (open bars). Enumeration and morphologic
characterization of aggregates suggest that WASP can be a component in
the induction of homotypic B-cell adhesion, resulting in the formation
of tight aggregates. A mild defect in aggregation can be a secondary
cause of impaired polarization of WASP B cells because
motility most likely is required for the formation of big
aggregates.
Activated WASP B
lymphocytes. LPS-stimulated WASP or WASP+ B
cells expressed few and short microvillilike structures (Figure 6). WASP B cells stimulated
with anti-CD40 mAb and IL-4 expressed more and longer microvilli on
their surfaces, but they were shorter than on similarly activated
WASP+ B cells. Higher magnification of cell contacts is
shown in Figure 7. WASP+ B
cells had extensive connections through microvilli in cell contacts
(Figure 7A, white arrows). The microvilli connections in cell contacts
of WASP B cells was less dense or absent, leading to more
frequent close contact through flat surfaces (Figure 7B, white arrows).
In addition, WASP B cells had smooth surfaces, whereas
WASP+ B cells were more irregularly shaped. In summary,
microvilli were expressed on activated WASP B cells, but
they appeared shorter and less dense in cell contacts than in
wild-type cells.
N-WASP is expressed in activated murine B lymphocytes We have described partial morphologic defects in B cells lacking expression of WASP. The WASP-deficient mice used here, like some patients with WAS, seem to have a less severe phenotype.18,51 A likely possibility is that another protein can substitute for some of the function of WASP. We investigated the protein levels of N-WASP, a likely substitute candidate, in activated murine B cells. For this purpose, we used specific antibodies directed to either the CRIB domain of WASP or to a peptide of N-WASP. The anti-WASP antibody detected WASP in detergent lysates of WASP+ B cells, whereas WASP was completely absent in WASP cells (Figure
8A). On the other hand, N-WASP was
clearly detected in both WASP+ and WASP B
cells (Figure 8B), in similar concentrations. Thus, N-WASP was
expressed in activated B lymphocytes, but it does not appear that in
the absence of WASP, the level of N-WASP was increased.
Cdc42, Rac1, and WASP are all implicated as important for the reorganization of the actin cytoskeleton. WASP and N-WASP regulate actin polymerization through direct interaction with the Arp2/3 complex. We investigated morphologic changes in B lymphocytes induced by IL-4 or CD40 activation and found that Cdc42 and Rac1, when overexpressed in activated B cells, induced filopodia and lamellipodia, respectively. WASP-deficient B cells were impaired in polarization, spreading, homotypic aggregation, and microvilli formation in response to IL-4- and CD40-dependent stimuli. Partial morphologic deficiencies can be explained by functional redundancy of a WASP homologous protein. In agreement with this, we found that the WASP homologue, N-WASP, was expressed in B lymphocytes at a level similar to those in WASP knockout and wild-type cells. In the early 1990s, Hall and coworkers44,45 performed
crucial experiments to explain the function of the Rho GTPases. When active Cdc42 and Rac1 were micro-injected into the Swiss 3T3 fibroblast cell line, specific membrane structures, filopodia, and lamellipodia, respectively, were induced. When we transiently expressed these proteins in activated B cells, we could induce similar phenotypes, indicating that Cdc42 and Rac1 also induce these specific structures in
B cells. This activation was dependent on the immobilization of B cells
through antibodies to cell membrane molecules. It was clear from our
results that LPS alone was sufficient to induce these events, because
LPS- and LPS plus IL-4-stimulated cells were indistinguishable in both
the morphologic phenotypes and the kinetics of induction (not shown).
These results were surprising given that LPS failed to induce spreading
in nontransfected B lymphocytes. One reason for this difference may be
that the induction of filopodia and lamellipodia is a process separate
from the induction of spreading. Another possibility is that these
processes may, in part, contain common intracellular pathways but that
spreading requires additional factors. However, yet another important
difference is that in the transfection experiments, active Cdc42 or
Rac1 were in much higher concentrations than their endogenous levels. This may influence downstream effectors and perhaps bypass the tight
regulation of signaling by the GTPases. Recently, WASP was shown to
have an auto-inhibitory contact between the GTPase binding domain
(CRIB) and the C-terminal part, suggesting that WASP activation is also
tightly regulated.52 The same mechanism most likely occurs
in N-WASP.25 High levels of active Cdc42 or Rac1 can probably shift the GTPase-WASP equilibrium to the favor of GTPase binding to WASP, keeping the latter in an open, active state. Therefore, in this "simplified system," LPS and LPS plus IL-4 can
induce morphologic changes. Interestingly, when active constructs were
expressed in WASP Our results show that IL-4 induces a transient decrease in the level of
active cytosolic Cdc42. This observation can be explained by an altered
endogenous localization of Cdc42. In the cytoplasm, RhoGDI (guanosine
nucleotide dissociation inhibitor) seems to have a major role in
sequestering the GDP-bound GTPases, thereby inhibiting the spontaneous
exchange of GDP to GTP.32,54 By way of a
posttranslationally added geranylgeranyl lipid modification, GTP-bound
proteins are able to translocate to membranes.55 For example, phorbol myristate acetate (PMA)-triggered activation of NADPH
in human neutrophils correlates with the translocation of Rac2-GTP to
the plasma membrane.56 In this system, the translocation is rapid, occurring after a 5-minute stimulation. In a cell-free system, using isolated neutrophil membranes and purified proteins, Rac2
and Cdc42 were rapidly translocated to membranes after GTP We also investigated a function for WASP in IL-4- and CD40-induced
slow processes, such as the induction of motile and spread cells.
WASP As judged by scanning electron microscopy, nonactivated human blood
lymphocytes have fine villous projections. WAS blood lymphocytes have
fewer microvilli, and those expressed are usually abnormally shaped in
that they are short and blunted.15-17 In our experiments using transmission electron microscopy, microvilli were clearly induced
on anti-CD40 mAb plus IL-4-activated murine WASP In some patients with WAS of less severe phenotype, truncated WASP
transcripts and proteins have been detected.18,51 We could
confirm that shorter WASP transcripts were completely absent in B cells
from WASP-deficient mice (data not shown).29 Thus, partial
morphologic deficiencies in murine WASP Increased surface area through filopodia, lamellipodia, and microvilli is a prerequisite for many cells that make extensive cell-to-cell or cell-to-matrix contacts. For example, in the immunologic synapse,5 a T cell forms filopodia and lamellipodia and clusters membrane molecules on these to sense and contact an antigen-presenting cell. During an immune response, B and T cells encounter the antigen in the spleen and lymph nodes. There, the collaboration between migratory and adhesive capacities is instrumental for B-cell activation to take place.2,3 We suggest that T-cell-derived signals by IL-4 and CD40 can induce morphologic alterations necessary for B-cell migration and interaction with T cells, follicular dendritic cells, and other B cells in B-cell foci and germinal centers (GCs). The importance of CD40-CD40 ligand interactions in GCs was revealed in CD40-deficient mice, which exhibit impaired GC formation.61 Earlier reports, in which electron microscopy was used to examine antigen-specific interaction between B and T cells, revealed extensive membrane contacts between cells.62 Later on, polarized expression of IL-4 in contact areas was shown to enhance conjugate formation.63,64 Impaired migration and contact between immune cells from patients with WAS, because of reduced surface plasticity, can most likely explain some of the severity of the disease. In addition, the antigen presentation capacity of WAS B cells may be impaired because antigen presentation on B cells seems to be dependent on CD40-CD40 ligand interaction, whereas other cells can present antigen in a CD40-independent fashion.65 We are investigating how LPS, IL-4, and anti-CD40 activation influences the antigen-presenting capacity of B cells. It will be interesting to include WASP-deficient B cells and to examine whether WASP-dependent morphology changes are important for antigen presentation by B cells. We have described impairment in the cytoskeletal regulation of WASP-deficient B cells. This is, to our knowledge, the first time a defect in murine B-cell function is shown. Facchetti et al18 showed that Epstein-Barr virus-transformed B-cell lines from patients with WAS are impaired in F-actin regulation. Together, these reports suggest that impaired B-lymphocyte responses may contribute to the immunodeficiency of WAS.
We thank Dr Johan Thyberg for excellent guidance with the electron microscopy and Karin Blomgren for assistance in specimen preparation. We thank Dr Fredrick Alt for providing the WASP knockout mice. We also thank Dr Edward J Davey for stimulating and helpful discussions.
Submitted December 28, 2000; accepted April 5, 2001.
Supported by grants from the Swedish Natural Science Research Council, the Swedish Cancer Foundation, the Network for Inflammation Research funded by the Swedish Foundation for Strategic Research, the Fifth Framework Programme of the European Commission, the Åke Wiberg Foundation, and the Karolinska Institutet.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Lisa Westerberg, Department of Cell and Molecular Biology, Karolinska Institutet, von Eulers väg 3, 171 77 Sockholm, Sweden; e-mail: lisa.westerberg{at}cmb.ki.se.
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A. Meyer-Bahlburg, S. Becker-Herman, S. Humblet-Baron, S. Khim, M. Weber, G. Bouma, A. J. Thrasher, F. D. Batista, and D. J. Rawlings Wiskott-Aldrich syndrome protein deficiency in B cells results in impaired peripheral homeostasis Blood, November 15, 2008; 112(10): 4158 - 4169. [Abstract] [Full Text] [PDF]
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J. Magdalena, T. H. Millard, S. Etienne-Manneville, S. Launay, H. K. Warwick, and L. M. Machesky Involvement of the Arp2/3 Complex and Scar2 in Golgi Polarity in Scratch Wound Models Mol. Biol. Cell, February 1, 2003; 14(2): 670 - 684. [Abstract] [Full Text] [PDF]
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C. W. Dawson, G. Tramountanis, A. G. Eliopoulos, and L. S. Young Epstein-Barr Virus Latent Membrane Protein 1 (LMP1) Activates the Phosphatidylinositol 3-Kinase/Akt Pathway to Promote Cell Survival and Induce Actin Filament Remodeling J. Biol. Chem., January 31, 2003; 278(6): 3694 - 3704. [Abstract] [Full Text] [PDF]
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D. Cai, K. N. Felekkis, R. I. Near, G. M. O'Neill, J. M. van Seventer, E. A. Golemis, and A. Lerner The GDP Exchange Factor AND-34 Is Expressed in B Cells, Associates With HEF1, and Activates Cdc42 J. Immunol., January 15, 2003; 170(2): 969 - 978. [Abstract] [Full Text] [PDF]
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H. Falet, K. M. Hoffmeister, R. Neujahr, and J. H. Hartwig Normal Arp2/3 complex activation in platelets lacking WASp Blood, August 28, 2002; 100(6): 2113 - 2122. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
S are shown in lane 5. All lanes were
on the same membrane, but the exposure time was shorter for lane 5 because the signal was strong. Data are representative of 4 experiments.
) and WASP+ (
) mice
were cultured with indicated stimuli for 24 hours, after which they
were fixed. Polarized cells were defined as having a tapered cell body
and uropods, whereas nonpolarized cells were spherical. Polarized cell
frequency was determined. Results are presented as mean values of
triplicate experiments, error bars represent 1 SD, and brackets
indicate P < .005 using a 2-tailed t test. At
least 300 cells were counted in each group, and the data are
representative of at least 4 experiments.
