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*
Department of Cell and Molecular Biology, Karolinska Institute, Stockholm, Sweden; and
Department of Microbiology and Immunology, Medical College of Virginia, Richmond, VA 23298
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A limited number of observations of B cell spreading have been reported. Small numbers of spread cells are found in LPS plus IL-4-stimulated cultures of murine B cells, in addition to cell aggregates (5). Cambier and Lehmann have reported a high frequency of spread B cells, also cultured with LPS plus IL-4, on anti-MHC class II mAb-coated tissue culture plastic (6), while Santos-Argumedo et al. describe B cell spreading on surfaces coated with anti-CD44 (7). Finally, human B cells have been reported to spread on fibronectin in a very late Ag (VLA)3-4-dependent manner (8). However, the questions of what signals are involved, the molecular mechanism, or indeed the physiologic relevance of B cell spreading have hardly been addressed.
We have focused this investigation on the role of IL-4 in inducing cell spreading of primary B cells. IL-4 is a cytokine exerting many effects on different cell types. Perhaps the most profound effects are found on B lymphocytes, where it induces Ig class switching to IgE and IgG1 (mouse) or IgG4 (human), increased expression of MHC class II and CD23, and proliferation (9). Other reported activities of IL-4 on B cells, such as induction of cell adhesion (5) and motility (10, 11), suggest it can influence B cell morphology. IL-4 has also been reported to directly affect cell morphology and cytoskeletal arrangement in human endothelial cells (12), providing a clear precedent for its ability to influence cytoskeletal organization. We report the ability of IL-4 to induce cell spreading and microvilli in B lymphocytes and discuss the physiologic significance of these findings.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following Abs were produced in the laboratory of D.H.C.:
anti-mouse CD44 mAb RK3G9 (13), an anti-DNP mouse IgE mAb (14),
anti-mouse CD23 mAb B3B4 (15) and 4F8 (rat IgM), and polyclonal
rabbit anti-CD23 antiserum (IgG purified by protein G affinity
chromatography; Pharmacia, Uppsala, Sweden). An additional
anti-CD44 mAb KM201 was kindly provided by Southern Biotechnology
(Birmingham, AL) or purified from hybridoma supernatant. The hybridomas
KM201 and FD448.1 secreting anti-LFA-1 
L-subunit mAb were
purchased from the American Type Culture Collection (ATCC, Rockville,
MD). Goat anti-mouse IgM polyclonal Ig was purchased from Dakopatts
(Copenhagen, Denmark). Anti-IgM mAb Ak13 (16) was prepared by ammonium
sulfate precipitation from culture supernatants and, where indicated,
was used coupled to cyanogen bromide-activated Sepharose beads
(Pharmacia) according to the manufacturer’s instructions (kindly
prepared by Asta Miskiniené, Vilnius University, Vilnius,
Lithuania). Soluble Ak13 was used at 10 µg/ml and the
Sepharose-coupled Ab at 2% bead vol/vol. The hybridoma-secreting
anti-CD40 mAb, 1C10 (17), was generously provided by Maureen Howard
(DNAX, Paulo Alto, CA). 1C10 mAb supernatants were ammonium sulfate
precipitated and used at 10 µg/ml. The hybridoma producing a
neutralizing mAb 11B11, recognizing mouse IL-4, was kindly provided by
Dr. William Paul (Bethesda, MD). Goat anti-rat IgG was obtained
from Jackson ImmunoResearch (West Grove, PA). A biotinylated B220 mAb,
two mAbs recognizing the mouse 4 integrin subunit
(R1-2 and 9C10), and an anti-ICAM-1 mAb (3E2) were purchased from
PharMingen (Hamburg, Germany). A hybridoma (YN1/1.7.4) producing a mAb
recognizing ICAM-1 was obtained from ATCC. Purified anti-mouse CD4
mAb, L3T4, was kindly provided by Dr. Anders Örn, Stockholm,
Sweden. Streptavidin coupled to horseradish peroxidase or FITC were
purchased from Dakopatts. Recombinant murine IL-4 was produced from
IL-4 cDNA-transfected X63 cells (18), purified by passage through an
11B11 (anti-IL-4)-coupled column, and used at 5 IU/ml in all
experiments. The amount of IL-4 inducing a half-maximal response in
induction of DNA synthesis in Con A-stimulated T cells is defined as 1
IU. IL-4 supernatant was used at the same unit activity in some
experiments in which larger culture volumes were used. LPS purified
from Escherichia coli 055:B5 was provided by the Department
of Microbiology and Tumour Biology at Karolinska Institute and used at
25 µg/ml. Recombinant IL-2 and IL-5 were produced from X63 cells
transfected with IL-2 and IL-5 cDNA, respectively (18). IL-2 and IL-5
activity was determined in [3H]thymidine incorporation
assays using CTLL or LyH7B13 cells, respectively, and were both used as
supernatants added at two times the concentration giving half-maximal
assay activity. Purified IL-10 was purchased from Genzyme, Cambridge,
MA and used at 250 ng/ml. IL-10 at this concentration was able to
inhibit IL-4-induced B cell polarization (an indication of cell
motility; 19 on freshly prepared B cells. Fibronectin was
purified from fresh frozen human plasma by affinity chromatography on
gelatin-Sepharose 4B (Pharmacia) and coated at a concentration of 10
µg/ml (a concentration used routinely to support attachment and
growth of adherent cell lines). BSA fraction V was purchased from
Boehringer Mannheim (Mannheim, Germany). Other reagents were purchased
from Sigma (St. Louis, MO).
Animals
C57BL/6 or (CBA/J x C57BL/6)F1 mice were used in all experiments. These were either bought from Bomholtgaard (Ry, Denmark), Charles River (Uppsala, Sweden), or bred and maintained in the animal facility at the Department of Cell and Molecular Biology, Karolinska Institute. CD23-deficient mice were kindly provided by Dr. Susanne Gustavsson (Uppsala, Sweden) (20). All experiments used age- and sex-matched animals.
Cell culture
Small resting B cells were prepared from splenic cell suspensions after T cell killing and Percoll gradient centrifugation as previously described (21). Cells were maintained in RPMI 1640 (Life Technologies, Paisley, Scotland) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 50 IU/ml penicillin, 50 µg/ml streptomycin (Life Technologies), 50 µM 2-ME, and 10% heat-inactivated FCS (Life Technologies) at 37°C in a humidified atmosphere containing 5% CO2.
Cell morphology assays
B cells were cultured at 5 x 105 per ml in the presence of various stimuli and for indicated times. Cells were resuspended by repeated pipetting and diluted to 105 per ml in medium containing the original activating stimulus, transferred to Ab-coated plates, and cultured for the indicated times. Ab coating of tissue culture plastic or glass cover slips was performed by adding 50 µg/ml of purified Ab in PBS including Mg2+ and Ca2+ and incubating for 1 h at room temperature. Plates were washed four times with Earle’s balanced salt solution (EBSS; Life Technologies). In initial experiments, 1% BSA was used to saturate protein binding sites. However, similar results were obtained when cells were added directly in FCS-containing medium, and so this step was generally omitted. Finally, the cells were fixed with an equal volume of 2.5% glutaraldehyde in PBS and observed under an inverted microscope. Cultures assayed without fixation were found to spread to the same extent, showing that the spread morphology was not an artifact of fixation (not shown). Spread cells (defined as having at least one dendritic process of at least one cell diameter) and round cells were enumerated and the percentage of spread cells calculated. A minimum of 300 cells were counted for each determination. Duplicate or triplicate determinations were used as indicated and mean values calculated from these. Most assays were performed in 96-well plates using 50-µl coating volumes and 150-µl cultures. For assays using various stimuli, 24-well plates and 500-µl culture volumes were employed.
Light microscopy
Cells were fixed with glutaraldehyde in RK3G9 (anti-CD44)-coated 24-well plates and photographed using an inverted microscope and a x40 objective. Alternatively, cells were fixed with glutaraldehyde after culturing in 24-well plates on RK3G9-coated cover slips, stained with a biotinylated anti-B220 (2 µg/ml) or an irrelevant biotinylated Ab followed by streptavidin FITC (1/3000 dilution), and photographed with a x100 oil immersion lens.
Electron microscopy
B cells were stimulated with LPS plus IL-4 or LPS for 2 days, transferred to anti-CD44-coated 9-cm tissue culture dishes at 2 x 105 cells per ml, and cultured overnight. The medium was carefully removed from the plates and a primary fixative of 3% glutaraldehyde in 0.1 M sodium cacodylate-HCl buffer (pH 7.3) containing 0.05 M sucrose was applied. The cells were then removed by scraping and collected by centrifugation. Between 1 and 2 x 106 cells were used per specimen. Postfixation was done for 2 h at 4°C in 2% osmium tetroxide in 0.1 M cacodylate buffer (pH 7.3) containing 0.5% potassium ferrocyanate. The specimens were then dehydrated in graded ethanol (70–100%), stained with 2% uranyl acetate (in ethanol), and embedded in Spurr low viscosity epoxy resin. Thin sections were cut on an LKB Instruments (Gaithersburg, MD) ultratome IV and examined in a JEOL (Tokyo, Japan) 100CX electron microscope at 60 kV.
Western blotting
B cells stimulated with LPS plus IL-4 or LPS alone for 3 days were harvested and resuspended in original medium at a cell density of 2 x 106 cells per ml. These cells were then incubated for 2 h in the presence of anti-CD44 mAb RK3G9 (5 µg/106 cells) or goat anti-rat IgG at 5 µg/ml (added for the final 90 min), or a combination of the two, or no Ab. Each determination used 10 to 40 x 106 cells. After washing four times with EBSS containing 100 µM Na3VO4, cells were solubilized for 5 to 10 min at 4°C in a lysis buffer containing 0.5% Nonidet P-40, 150 mM NaCl, 10 mM Tris (pH 7.6), 100 µM Na3VO4, 10 mM Na4P2O7, 10 mM NaF, 1 mM PMSF, and 1 µg/ml respectively, of aprotinin and leupeptin. Lysates were centrifuged at 10,000 x g for 15 min. The cleared lysates were retained and the detergent-insoluble pellet was washed twice with lysis buffer and once in TBS. The detergent-insoluble pellets or 25 µg of detergent lysate were solubilized in nonreducing SDS-PAGE sample buffer and resolved on 7% SDS-PAGE gels. Western transfer to nitrocellulose (BA 83; Schleicher & Schüll, Dassel, Germany) was performed in 25 mM Tris, 192 mM glycine, and 20% methanol using a miniblot transfer chamber (Bio-Rad, Hercules, CA). The transfer conditions were 100 V (constant voltage) for 3 h at 4°C. The filters were reversibly stained with 0.3% Ponceau S in 0.3% TCA (Serva, Heidelberg, Germany) to control for even transfer. Nonspecific protein binding sites were blocked by incubation for 20 min with blotting buffer containing 5% skimmed milk powder and 0.05% Tween-20 in 1 x TBS. The filters were stained with biotin-conjugated anti-CD44 mAb KM201 (1 µg/ml in blotting buffer for 1 h at 4°C) and washed four times with 0.05% Tween-20 in 1 x TBS. Streptavidin horseradish peroxidase conjugate was applied at a 1/3000 dilution in blotting buffer for 30 min and washed as above. Detection was achieved by enhanced chemiluminescence using standard procedures and exposure to Fuji film.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Murine B cells cultured in the presence of LPS plus IL-4 for 2 to
3 days were previously reported to form large regular round cell
aggregates. A low frequency of spread cells was consistently observed
in such cultures. However, when cells were stimulated with LPS alone,
neither spread cells nor regular large round aggregates were observed
(5). We initially observed a high frequency of spread cells showing
pronounced dendritic morphology in LPS plus IL-4-stimulated cultures
containing anti-CD23 Abs and found this to be mediated by Abs bound
to the tissue culture plastic (E.J.D. and E.S., unpublished
observations). We have since made similar observations using a number
of Abs deliberately immobilized to the tissue culture plastic. We used
anti-CD44 mAb RK3G9 extensively throughout this paper. Many cells
bearing long and often branched dendritic projections were observed
when B cells prestimulated with LPS plus IL-4 (Fig. 1
a), but not LPS alone
(Fig. 1b), were transferred to RK3G9-coated plates.
LPS plus IL-4-stimulated B cells were also found to spread on
coverslips coated with RK3G9. Fluorescence staining with an
anti-B220 mAb revealed finely branched processes (Fig. 1c) not clearly seen with conventional phase contrast
microscopy (Fig. 1d). Greater than 95% of spread
cells were routinely found to be surface positive for B220, confirming
that they were B cells and not a contaminant cell population.
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). When LPS blasts were recultured in
the presence of LPS plus IL-4 on RK3G9-coated surfaces, 36% of cells
were found to spread after 20 h. This was reduced to 7.6% when
cells were similarly recultured in the presence of 10% 11B11
supernatant.
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We investigated the kinetics of cell spreading to determine
whether this correlated with other known B cell phenomena involving
cell morphology. Early events in IL-4 signal transduction such as
specific tyrosine phosphorylations occur within minutes of receptor
engagement (22). Induction of cell polarization (an indication of cell
motility) is clearly detectable 2 h after addition of IL-4 to
primary B cells and maximal after 24 h (11), whereas homotypic B
cell adhesion seen in LPS plus IL-4-stimulated cultures is maximal
after 3 to 4 days (5). To study the kinetics of the spreading response,
we plated freshly prepared B cells into RK3G9-coated wells and measured
spreading at different time points. Figure 3A shows a response obtained
within 20 h and that reached a maximum after 60 h of LPS plus
IL-4 stimulation. Spreading was rarely observed in cultures before
6 h and fell after 3 days as cell death in cultures increased (not
shown). B cells stimulated with either LPS or IL-4 alone or cultured in
medium alone showed little tendency to spread. Thus, cell spreading
appeared to correlate with blast formation in LPS plus IL-4-stimulated
cultures. We were therefore interested in investigating whether late
additions of IL-4 could induce spreading in LPS blasts and, if so, how
rapidly this occurred. Day 2 LPS blasts were washed, resuspended in
medium, and allowed to settle on RK3G9 wells in the presence of LPS,
IL-4, combinations of the two, or no stimulus. Blasts restimulated with
LPS plus IL-4 or IL-4 alone did not give a response that was different
from that of LPS at 1 or 2 h time points, but did give a good
response after 20 h (Fig. 3B). This shows that
IL-4 induces cell spreading with slow kinetics.
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We were interested in investigating whether Abs to different cell
surface determinants or other substrates could mediate a cell-spreading
response. In Table I we show that Abs to
diverse surface structures, including CD23 (a C-type lectin and type II
glycoprotein), CD44 (a proteoglycan), LFA-1, VLA-4 (both integrins),
ICAM-1 (an Ig superfamily member), and membrane Ig, all supported cell
spreading in the presence of LPS plus IL-4 (18–55% spread cells),
while LPS blasts plated on the same Abs showed little tendency to
spread (4.4% or less). We have yet to find an Ab that stains B cell
blasts brightly in FACS analysis and fails to mediate cell spreading. A
rat IgM anti-CD23 mAb (4F8) and a mouse IgE both supported
spreading, suggesting that Fc receptor interactions are not essential.
The surface expression of CD23 is greatly up-regulated on LPS plus
IL-4-stimulated B cell blasts (13), possibly explaining the difference
in the ability of LPS plus IL-4 and LPS blasts to spread. However, CD44
expression is increased after LPS stimulation but not further
up-regulated in the presence of IL-4 (13), demonstrating that high
surface expression is not a sufficient requirement for spreading. The
IgE-mediated cell spreading was further investigated to determine
whether it was mediated by CD23 (Fc
RII or the low affinity IgE
receptor), the only known receptor for IgE on B cells. In Figure 4, we show that cells prepared from
CD23-deficient mice were incapable of spreading on either B3B4 or
IgE-coated surfaces while spreading normally on RK3G9-coated surfaces.
Furthermore, when added to the medium at 20 µg/ml, B3B4 inhibited
spreading of wild-type cells on B3B4 and IgE-coated surfaces, while not
affecting that of RK3G9 coating. We conclude that B cell spreading on
IgE-coated surfaces is indeed mediated by CD23. We wished to determine
whether LPS plus IL-4-stimulated B cells are capable of spreading on a
natural substrate. To do so, we chose an extracellular matrix
component, fibronectin, which has previously been shown to support
spreading of human B cells (8). When transferred to plates coated with
fibronectin, B cells prestimulated for 2 days with LPS plus IL-4 failed
to show any significant degree of spreading when compared with those
transferred to BSA-coated control wells, either at 2 or 20 h after
transfer, while a high response was observed from cells transferred to
anti-CD44-coated plastic (Fig. 5).
This suggests that fibronectin is not capable of supporting this
response and, as cells show little tendency to bind to the bottom of
the wells, we assume that this is due to a low affinity for
fibronectin.
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We next investigated whether other stimuli could induce cell
spreading. Cells were stimulated for 2 days with anti-IgM reagents
in the presence or absence of IL-4 and transferred to RK3G9-coated
plates. In Table II, we see that such
cells readily spread in the presence of IL-4 (17 and 22%,
respectively, for anti-IgM-coupled Sepharose and soluble Ab),
albeit at a lower level than for LPS plus IL-4-stimulated cells (48%).
No spreading was observed in the absence of IL-4. Anti-CD40-stimulated
B cells have previously been shown to form tight round cell aggregates
(23, 24) reminiscent of those seen in LPS plus IL-4-activated cells
(5). A high proportion of anti-CD40-stimulated cells spread in both
the presence and absence of IL-4 (74 and 82%, respectively, as shown
in Table II) and anti-CD40-induced cell spreading was not inhibited
by a neutralizing anti-IL-4 mAb (11B11, Fig. 2
), indicating that
this phenomenon is not unique to IL-4-stimulated cells.
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IL-2, IL-5, and IL-10 all fail to induce cell spreading used alone
or in combination with LPS (Table III).
IL-10 has previously been reported to inhibit IL-4-induced B cell
motility, and as this also involves alterations in cell morphology, we
attempted to inhibit cell spreading with IL-10. However, IL-10 did not
inhibit spreading induced by LPS plus IL-4.
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B cells cultured on RK3G9-coated surfaces after prestimulation
with LPS plus IL-4 (Fig. 6, a
and b) and LPS (Fig. 6, c and
d) were examined by electron microscopy for
structural features. Both groups displayed a ribosome-rich cytoplasm
consistent with rapidly dividing blasts. Long cytoplasmic extensions
generally devoid of membranous organelles were specifically found in
the LPS plus IL-4-stimulated samples (not shown). Interestingly,
microvilli-like structures were found frequently on LPS plus
IL-4-stimulated cells (Fig. 6, a and b)
and, while not being absent on LPS blasts, they are shorter and far
less abundant in these cells (Fig. 6, of c and
d). Thus, IL-4 appears to influence B cell morphology
at both a macroscopic and ultrastructural level.
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The dramatic changes in B cell morphology induced by LPS plus IL-4
stimulation suggest that a reorganization in the cytoskeleton takes
place. The formation of dendritic morphology was found to be partially
inhibited at 1 µM and totally inhibited at 10 µM cytochalasin B
(Fig. 7), showing that it is dependent on
intact actin cytoskeleton. Spread cells on Ab-coated surfaces were
observed using video time-lapse photography. Some cells were anchored
to the tissue culture plastic via the ends of dendritic processes,
while the main body of cytoplasm moved back and forth between these
points (not shown). These observations are consistent with a molecular
mechanism of cell spreading including a direct linkage between the
cytoskeleton and the membrane-attached protein. This would predict that
surface molecules in LPS plus IL-4-stimulated B cells be anchored to
the cytoskeleton. To address this issue, we looked for localization of
CD44 in the insoluble fraction (containing the cytoskeleton) of
detergent cell lysates. CD44 was initially chosen as it has been
reported to interact with the cytoskeletal proteins (25, 26) and it is
expressed equally on LPS plus IL-4 and LPS-stimulated B cell blasts
(13). LPS or LPS plus IL-4-activated B cells were cross-linked with rat
anti-mouse CD44 mAb and goat anti-rat Ig, solubilized with
detergent, and the insoluble and soluble material were separated. As
shown in Figure 8A, the
detergent-insoluble fraction of the LPS plus IL-4-activated cells
revealed a strong band of about 90 kDa (the m.w. of the common form of
CD44). Significantly, weaker or no bands were detected in the
case of LPS plus IL-4-stimulated cells treated with single or
no Abs or similarly treated LPS blasts. In contrast, CD44 was always
detected in the detergent-soluble fractions of cells treated in
different ways, although it appears that LPS plus IL-4-activated
cells express higher levels than did LPS-activated cells
(Fig. 8B). Additionally, we have examined LPS plus
IL-4-stimulated B cells spread on anti-CD44 (RK3G9)-coated
coverslips after paraformaldhyde fixation for localization or
capping of CD44, as this would give a further indication of CD44
interaction with the cytoskeleton. However, while strong staining for
CD44 was found over the entire cell, no specific localization was seen
(not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-4 has previously been reported to induce cell motility (11, 27) and cell adhesion in B lymphocytes (5). It is therefore interesting to ask if the spreading phenomenon represents either of these activities. Many activating stimuli, including LPS (11) and CD40 ligation (3), in addition to IL-4 are capable of inducing cell motility in B cells. We have observed, using time-lapse photography, 2-day-stimulated LPS plus IL-4 and LPS B cell blasts (which differ greatly in their ability to spread) for motile behavior when cultured on thin collagen gels. Both blasts appear highly motile, exhibiting cytoplasmic movement and locomotion over the substrate, suggesting that cell spreading does not reflect differences in motile capacity.
Mitogen-induced homotypic B cell aggregation in vitro has been well documented (5, 24, 28, 29). Aggregation resulting in tight spherical cell clusters has been seen when cells are stimulated with LPS plus IL-4 (5), anti-Ig plus IL-4 (30), and anti-CD40 (which is often enhanced in the presence of IL-4) (24). We find that the same stimuli also induce cell spreading, whereas LPS-stimulated B cells form large loose cell aggregates and fail to spread. Cell spreading has similar slow kinetics as has the formation of tight aggregation, with both increasing slowly over 3 days. Taken together, this suggests that spreading and tight aggregation have mechanistic similarities.
Much progress has recently been made in understanding IL-4-mediated signal transduction. The IL-4 receptor can activate two signaling pathways, namely via the STAT 6 protein (31) and the insulin receptor substrate (IRS)-1 and -2 proteins (32). STAT 6 induces Ig class switching to IgG1 and IgE, and CD23 expression (33, 34, 35), while IRS-1 and -2 are heavily phosphorylated molecules that are capable of docking various signaling molecules via SH2 interactions and mediate an IL-4-dependent proliferative signal (36). We have shown that IL-2, IL-5, and IL-10 fail to substitute for IL-4 in our assay system, suggesting that an additional proliferative signal is not sufficient to induce this phenomenon. At present it is not known to what extent the STAT 6 and IRS-1 or -2 pathways contribute to morphology-related phenomena such as cell adhesion or cell motility. Interestingly, phosphatidylinositol-3 kinase has been implicated in platelet-derived growth factor-induced cytoskeletal rearrangements (37) and in IRS-1 signaling (22). Whether IL-4 can influence cell morphology by a phosphatidylinositol-3 kinase-dependent mechanism has yet to be determined.
The altered cell morphology described in this paper strongly suggests
reorganization of the cytoskeleton. Furthermore, cell spreading
mediated by immobilized Abs to plasma membrane molecules implicates an
interaction between these molecules and the cytoskeleton. There are an
increasing number of reports of interactions between plasma membrane
proteins and the cytoskeleton. Interestingly, CD44 is reported to
interact with ankyrin (26) and the ERM family members ezrin, radixin,
and moesin (25). Talin has been shown to colocalize with LFA-1 (38) and
the ß-1 integrin (as found in VLA-4) (39), ICAM-1 is reported to
associate with -actinin (40), surface Ig receptor is reported to
localize in detergent-insoluble fractions (41), while there are no
reports of CD23-cytoskeleton interaction. The regulation of such events
is likely to be of importance in modulating events such as cell
adhesion or signal transduction. Our observation that IL-4 can
influence the detergent-insoluble localization of CD44 suggests that
IL-4 may have such a regulatory role. We have also found that higher
numbers of microvilli-like structures are induced on LPS plus
IL-4-stimulated B cell blasts, compared with LPS blasts. The induction
of microvilli is a complex process involving organization of
microfilaments and their association with actin binding proteins such
as ERM family members (42, 43) and provides further evidence that IL-4
can influence cytoskeleton organization. The microvilli-like structures
have also been observed in LPS plus IL-4-induced aggregates (not
shown), where they often make cell-cell contact, suggesting a role in
cell adhesion. Involvement of microvilli in lymphocyte adhesion is not
unprecedented, as microvilli have been reported on lymphocytes
undergoing rolling in an L-selectin-dependent manner (44). The
molecular nature of an IL-4-induced cytoskeleton-CD44 interaction and
formation of microvilli are currently being investigated in our
laboratory.
Cell spreading on immobilized Abs, while being useful for dissecting an interesting cellular response, is obviously an artificial system and raises the question of what these observations represent in vivo. The ability of two T cell-derived signals, namely IL-4 and CD40 ligation, to induce cell spreading strongly suggests the involvement of T cells. An interesting possibility is that the cell spreading we observe represents mechanisms involved in T cell-B cell collaboration. Early reports in which electron microscopy was used to examine Ag-specific interactions between B and T cells revealed extended areas of membrane contact between the two cell types and some attachments made via B cell microvilli (45). IL-4 has been observed to enhance the ability of Ag-specific B cells to form conjugates with T cells (46), suggesting a role for it in this process. More recent reports have shown that IL-4 is concentrated in T cells close to the B cell-contacting surface in an Ag-specific conjugate cell pair (47, 48). This provides a model for how a soluble factor like IL-4 can act in a cell-specific manner and enhance interaction between T and B cells. CD40 ligation, in addition to the spreading we observe, has been reported to induce strong homotypic B cell adhesion (23, 24). It is possible that signaling by CD40 ligand (gp39) expressed on activated T cells to B cells (49) results in enhanced Ag-specific interaction between the two cell types. We suggest that cytoskeletal changes induced by IL-4 and CD40 ligation make B cells bind more strongly to T cells, follicular dendritic cells, and/or other B cells within germinal centers.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Eva Severinson, Department of Cell and Molecular Biology, Karolinska Institute, S-171 77 Stockholm, Sweden.
3 Abbreviations used in this paper: VLA, very late antigen; IRS, insulin receptor substrate.
Received for publication March 27, 1997. Accepted for publication February 5, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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4 ß1-fibronectin receptors upon B cell activation. Eur. J. Immunol. 21:2437.[Medline]
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Q. Vos, C. M. Snapper, and J. J. Mond Th1 versus Th2 cytokine profile determines the modulation of in vitro T cell-independent type 2 responses by IL-4 Int. Immunol., September 1, 2000; 12(9): 1337 - 1345. [Abstract] [Full Text] [PDF]
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E. J. Davey, G. Greicius, J. Thyberg, and E. Severinson STAT6 is required for the regulation of IL-4-induced cytoskeletal events in B cells Int. Immunol., July 1, 2000; 12(7): 995 - 1003. [Abstract] [Full Text] [PDF]
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 Y. Yamashita, K. Oritani, E. K. Miyoshi, R. Wall, M. Bernfield, and P. W. Kincade Syndecan-4 Is Expressed by B Lineage Lymphocytes and Can Transmit a Signal for Formation of Dendritic Processes J. Immunol., May 15, 1999; 162(10): 5940 - 5948. [Abstract] [Full Text] [PDF]
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 E. U. Rudge, A. J. Cutler, N. R. Pritchard, and K. G.C. Smith Interleukin 4 Reduces Expression of Inhibitory Receptors on B Cells and Abolishes CD22 and Fc{gamma}RII-mediated B Cell Suppression J. Exp. Med., April 15, 2002; 195(8): 1079 - 1085. [Abstract] [Full Text] [PDF]
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