Regulation of Cell Morphology in B Lymphocytes by IL-4: Evidence for Induced Cytoskeletal Changes

Edward J. Davey, Johan Thyberg, Daniel H. Conrad and Eva Severinson
J Immunol June 1, 1998, 160 (11) 5366-5373;

Abstract

Lymphocyte activation is often accompanied by changes in cell morphology, for example, in cell adhesion or motility. IL-4 is a cytokine exerting many effects on B lymphocytes. In this study, we show that stimulation with LPS in combination with IL-4, but not LPS or IL-4 alone, results in a pronounced dendritic morphology of B cells. Using a culture system in which Abs directed to B cell surface markers are immobilized on the tissue culture plastic, we find that cell spreading can be mediated by a variety of Abs, including anti-CD44, -CD23, -LFA-1, -VLA-4, -ICAM-1, and -Ig. B cells stimulated with anti-Ig Abs plus IL-4, or anti-CD40 Abs in the presence or absence of IL-4, are also induced to spread, while IL-2, IL-5, or IL-10 in combination with LPS or alone fail to induce this. Spreading correlates with induction of tight cell aggregation. It is sensitive to cytochalasin B, indicating a requirement for intact actin cytoskeleton. CD44 is selectively detected in the detergent-insoluble fraction of cell lysates prepared from LPS plus IL-4-stimulated B cell cultures after Ab cross-linking of CD44, suggesting a membrane protein-cytoskeleton interaction. Interestingly, electron microscopy studies reveal induction of microvilli-like structures on LPS plus IL-4-stimulated blasts, suggesting that IL-4 can influence cell morphology on an ultra-structural level. In summary, our data show that stimulation with LPS plus IL-4 or ligation of CD40 is capable of inducing dramatic morphologic changes in murine B cells, which correlates with in vitro induction of strong cell adhesion.

Many cellular processes accompanying leukocyte activation lead to changes in cell morphology. Transendothelial migration involves spreading of activated leukocytes on endothelial cells before extravasation (1). Lymphocyte motility leading to a polarized cell morphology (2) is induced by many activating signals (3). Adhesion molecules, including ICAM-1, ICAM-3, and CD43, can accumulate in uropods of polarized lymphocytes, contributing to mechanisms for cell adhesion (4). Such processes reflect the importance of cell morphology in adhesion and motility in immune responses.

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.

Materials and Methods

Reagents and Abs

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 αL-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 α4 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 × 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 × 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 ×40 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 ×100 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 × 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 × 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 × 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 × 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 × 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 × 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 × 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.

Results

Cell spreading is induced in B cells stimulated with LPS plus IL-4

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. 1⇓b), 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. 1⇓c) not clearly seen with conventional phase contrast microscopy (Fig. 1⇓d). 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.

FIGURE 1.
FIGURE 1.

Cell spreading is induced in B cells stimulated with LPS plus IL-4. B cells stimulated with LPS plus IL-4 (a) or LPS (b) for 2 days were transferred to 24-well plates coated with RK3G9 (anti-CD44) and cultured for 20 h. The cells were fixed with glutaraldehyde and photographed directly in the wells using an inverted microscope. This is representative of more than 50 similar observations. Similarly cultured LPS plus IL-4-stimulated blasts were transferred to RK3G9-coated coverslips, cultured overnight, fixed, and stained with a biotinylated B220 mAb and streptavidin-FITC. Fluorescence and conventional phase contrast images are shown in c and d, respectively, and are representative of four similar experiments. Bars represent 20 μm.

IL-4 induction of dendritic morphology was dramatically reduced in the presence of anti-IL-4 mAb 11B11 (Fig. 2⇓). 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.

FIGURE 2.
FIGURE 2.

LPS plus IL-4 but not anti-CD40-induced cell spreading is inhibited by anti-IL-4 mAb 11B11. Day 2 LPS-stimulated B cell blasts were recultured in a 96-well plate coated with RK3G9 (anti-CD44) in the presence of LPS plus IL-4, LPS, or anti-CD40 Ab, in the presence or absence of anti-IL-4 mAb 11B11, respectively, for 20 h, fixed, and counted for cell spreading in triplicate. One representative experiment of two is presented. Error bars are SD.

The kinetics of cell spreading

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 3⇓A 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. 3⇓B). This shows that IL-4 induces cell spreading with slow kinetics.

FIGURE 3.
FIGURE 3.

The kinetics of cell spreading. A, B cells were cultured in parallel RK3G9 (anti-CD44)-coated plates in the presence of stimuli as shown. At indicated time points, individual plates were fixed and spreading determined in duplicate. The data are representative of two experiments using RK3G9 coating and three experiments using B3B4 coating. B, Day 2 LPS blasts were washed, resuspended in medium, and transferred to parallel RK3G9-coated plates. The cells were allowed to settle for 2 h at 37°C, after which indicated stimuli were added. Individual plates were fixed and spreading determined in duplicate at indicated times. The data shown are the mean of two experiments and are representative of two more using B3B4-coated plates.

Abs to diverse B cell surface structures are capable of mediating cell spreading

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 (FcεRII 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.

FIGURE 4.
FIGURE 4.

B cell spreading on IgE-coated surfaces is mediated by CD23. Day 2 LPS plus IL-4-stimulated B cell blasts prepared from spleens from wild-type (+/+) or CD23 deficient (−/−) mice were transferred to wells coated with RK3G9 (anti-CD44), B3B4 (anti-CD23), IgE mAb, or L3T4 (anti-CD4), respectively. Wild-type cells were also transferred to Ab-coated surfaces after 30 min of preincubation with 20 μg/ml B3B4 and incubated in the continuous presence of the Ab (marked +/+ plus sB3B4 in graph key). The cells were fixed after 20 h of incubation and cell spreading counted in duplicate. The data are the mean of two experiments.

FIGURE 5.
FIGURE 5.

Fibronectin fails to support cell spreading in LPS plus IL-4-stimulated B cell blasts. B cells stimulated with LPS plus IL-4 for 2 days were transferred to 96-well plates coated with fibronectin, RK3G9 (anti-CD44), or BSA. Individual plates were fixed with glutaraldehyde and counted in duplicate for cell spreading after 2 and 20 h in culture. The data are the mean of three experiments. The error bars represent SD.

View this table:
Table I.

Ab mediating cell spreadinga

B cell spreading is induced by anti-CD40 and anti-Ig plus IL-4 stimulation

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.

View this table:
Table II.

A variety of stimuli are capable of inducing cell spreadinga

IL-2, IL-5, and IL-10 fail to induce cell spreading

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.

View this table:
Table III.

IL-2, IL-5 and IL-10 fail to induce cell spreadinga

Electron microscopy reveals microvilli-like structures on LPS plus IL-4-stimulated blasts

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.

FIGURE 6.
FIGURE 6.

Electron micrographs of B cell blasts from RK3G9-coated plates. Cells were stimulated with LPS plus IL-4 (a and b) or LPS alone (c and d). The scale bar represents 1 μm, N = nucleus, M = mitochondria, and G = Golgi complex. These observations are representative of two similar experiments. Induction of microvilli-like structures were observed consistently in at least four experiments using B cells cultured in suspension.

Interaction between CD44 and the cytoskeleton

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 8⇓A, 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. 8⇓B). 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).

FIGURE 7.
FIGURE 7.

Cell spreading is sensitive to cytochalasin B. B cells were stimulated for 2 days with LPS plus IL-4 or LPS alone, exposed to cytochalasin B at indicated concentrations for 2 h, transferred to RK3G9-coated wells, and incubated for an additional 2 h. The cells were then glutaraldehyde fixed and spreading was counted in duplicate determinations. Data are the mean of three experiments and error bars represent SD.

FIGURE 8.
FIGURE 8.

Detergent-insoluble localization of CD44 in LPS plus IL-4-stimulated B cells. LPS or LPS plus IL-4-stimulated B cells were treated with anti-CD44 and anti-rat Ig as indicated and solubilized with detergent. The soluble and insoluble material were separated as indicated in Materials and Methods. Nonidet P-40-insoluble pellets were solubilized in nonreducing sample buffer, separated with SDS/PAGE, and Western blotted (A). Detergent-soluble lysates (25 μg) were similarly blotted (B). Bands were detected with anti-CD44 mAb KM201. Similar observations of CD44 localization in the detergent-insoluble fraction were made in at least five experiments, three of which included blots of detergent lysates.

Taken together, the cytochalasin sensitivity of cell spreading and detergent-insoluble localization of CD44 suggest that cross-linking of CD44 induces attachment to the cytoskeleton in LPS plus IL-4 but not LPS-stimulated cells. We cannot exclude, however, that CD44 could instead associate with other molecules after IL-4 stimulation to produce a detergent-insoluble complex.

Discussion

Induction of an adherent or a motile cell phenotype frequently plays an important role in lymphocyte activation and is accompanied by changes in lymphocyte morphology. We describe here a novel function of IL-4 on murine B cells, in which it synergizes with LPS or anti-Ig reagents to induce cell spreading on adhesive surfaces to produce a striking dendritic morphology. A similar response is seen when B cells are stimulated with anti-CD40. While the physiologic significance of these observations still is unclear, they suggest a possible means by which IL-4 and CD40 may modulate immune responses modifying interactions of B cells with other cells or substrates.

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 β-1 integrin (as found in VLA-4) (39), ICAM-1 is reported to associate with α-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.

Acknowledgments

We thank Karin Blomgren for excellent assistance in preparing specimens for electron microscopy.

Footnotes

  • ↵1 This work was supported by grants from the Swedish Natural Research Council, the Swedish Medical Research Council, and the Swedish Heart Lung Foundation, and by Grant AI 18697 from the U.S. Public Health Service.

  • ↵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 March 27, 1997.
  • Accepted February 5, 1998.

References

  1. ↵
    Springer, T. A.. 1994. Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell 76: 301
    OpenUrlCrossRefPubMed
  2. ↵
    Newman, I., P. C. Wilkinson. 1992. Methods for phenotyping polarized and locomotor human lymphocytes. J. Immunol. Methods 147: 43
    OpenUrlCrossRefPubMed
  3. ↵
    Komai-Koma, M., F. Y. Liew, P. C. Wilkinson. 1995. Interactions between IL-4, anti-CD40, and anti-immunoglobulin as activators of locomotion of human B cells. J. Immunol. 155: 1110
    OpenUrlAbstract/FREE Full Text
  4. ↵
    Del Pozo, M. A., P. Sánchez-Mateos, F. Sánchez-Madrid. 1996. Cellular polarization induced by chemokines: a mechanism for leukocyte recruitment. Immunol. Today 17: 127
    OpenUrlCrossRefPubMed
  5. ↵
    Elenström, C., E. Severinson. 1989. Interleukin 4 induces cellular adhesion among B lymphocytes. Growth Factors 2: 73
    OpenUrlPubMed
  6. ↵
    Cambier, J. C., K. R. Lehmann. 1989. Ia-mediated signal transduction leads to proliferation of primed B lymphocytes. J. Exp. Med. 170: 877
    OpenUrlAbstract/FREE Full Text
  7. ↵
    Santos-Argumedo, L., P. W. Kincade, S. Partida-Sanchez, R. M. E. Parkhouse. 1997. CD44-stimulated dendrite formation (spreading) in activated B cells. Immunology 90: 147
    OpenUrlCrossRefPubMed
  8. ↵
    Postigo, A. A., R. Pulido, M. R. Campanero, A. Acevedo, A. García-Pardo, A. L. Corbi, F. Sánchez-Madrid, M. O. De Landazuri. 1991. Differential expression of VLA-4 integrin by resident and peripheral blood B lymphocytes: acquisition of functionally active α4 β1-fibronectin receptors upon B cell activation. Eur. J. Immunol. 21: 2437
    OpenUrlPubMed
  9. ↵
    Paul, W. E.. 1991. Interleukin-4: a prototypic immunoregulatory lymphokine. Blood 77: 1859
    OpenUrlFREE Full Text
  10. ↵
    Wilkinson, P. C., L. N. Islam. 1989. Recombinant IL-4 and IFN-gamma activate locomotor capacity in human B lymphocytes. Immunology 67: 237
    OpenUrlPubMed
  11. ↵
    Clinchy, B., C. Elenström, E. Severinson, G. Möller. 1991. T and B cell collaboration: induction of motility in small, resting B cells by interleukin 4. Eur. J. Immunol. 21: 1445
    OpenUrlPubMed
  12. ↵
    Klein, N. J., K. P. Rigley, R. E. Callard. 1993. IL-4 regulates the morphology, cytoskeleton, and proliferation of human umbilical vein endothelial cells: relationship between vimentin and CD23. Int. Immunol. 5: 293
    OpenUrlAbstract/FREE Full Text
  13. ↵
    Rao, M., R. Knox, D. H. Conrad. 1991. Characterization of Pgp-1 antigen on murine B lymphocytes using a new anti-Pgp-1 monoclonal antibody. Hybridoma 10: 281
    OpenUrlPubMed
  14. ↵
    Liu, F. T., J. W. Bohn, E. L. Ferry, H. Yamamoto, C. A. Molinaro, L. A. Sherman, N. R. Klinman, D. H. Katz. 1980. Monoclonal dinitrophenyl-specific murine IgE antibody: preparation, isolation, and characterization. J. Immunol. 124: 2728
    OpenUrlPubMed
  15. ↵
    Rao, M., W. T. Lee, D. H. Conrad. 1987. Characterization of a monoclonal antibody directed against the murine B lymphocyte receptor for IgE. J. Immunol. 138: 1845
    OpenUrlAbstract/FREE Full Text
  16. ↵
    Leptin, M., M. J. Potash, R. Grützmann, C. Heusser, M. Shulman, G. Köhler, F. Melchers. 1984. Monoclonal antibodies specific for murine IgM. I. Characterization of antigenic determinants on the four constant domains of the μ heavy chain. Eur. J. Immunol. 14: 534
    OpenUrlCrossRefPubMed
  17. ↵
    Heath, A. W., W. W. Wu, M. C. Howard. 1994. Monoclonal antibodies to murine CD40 define two distinct functional epitopes. Eur. J. Immunol. 24: 1828
    OpenUrlCrossRefPubMed
  18. ↵
    Karasuyama, H., F. Melchers. 1988. Establishment of mouse cell lines which constitutively secrete large quantities of interleukin 2, 3, 4 or 5, using modified cDNA expression vectors. Eur. J. Immunol. 18: 97
    OpenUrlCrossRefPubMed
  19. ↵
    Clinchy, B., P. Björk, S. Paulie, G. Möller. 1994. Interleukin-10 inhibits motility in murine and human B lymphocytes. Immunology 82: 376
    OpenUrlPubMed
  20. ↵
    Fujiwara, H., H. Kikutani, S. Suematsu, T. Naka, K. Yoshida, T. Tanaka, M. Suemura, N. Matsumoto, S. Kojima, T. Kishimoto, N. Yoshida. 1994. The absence of IgE antibody-mediated augmentation of immune responses in CD23 deficient mice. Proc. Natl. Acad. Sci. USA 91: 6835
    OpenUrlAbstract/FREE Full Text
  21. ↵
    Davey, E. J., W. C. Bartlett, H. Kikutani, H. Fujiwara, T. Kishimoto, D. H. Conrad, E. Severinson. 1995. Homotypic aggregation of murine B lymphocytes is independent of CD23. Eur. J. Immunol. 25: 1224
    OpenUrlCrossRefPubMed
  22. ↵
    Wang, L. M., A. D. Keegan, W. E. Paul, M. A. Heidaran, J. S. Gutkind, J. H. Pierce. 1992. IL-4 activates a distinct signal transduction cascade from IL-3 in factor-dependent myeloid cells. EMBO J. 11: 4899
    OpenUrlPubMed
  23. ↵
    Björck, P., S. Paulie. 1993. Inhibition of LFA-1-dependent human B-cell aggregation induced by CD40 antibodies and interleukin-4 leads to decreased IgE synthesis. Immunology 78: 218
    OpenUrlPubMed
  24. ↵
    Klaus, G. G., M. Holman, J. Hasbold. 1994. Properties of mouse CD40: the role of homotypic adhesion in the activation of B cells via CD40. Eur. J. Immunol. 24: 2714
    OpenUrlPubMed
  25. ↵
    Tsukita, S., K. Oishi, N. Sato, J. Sagara, A. Kawai, S. Tsukita. 1994. ERM family members as molecular linkers between the cell surface glycoprotein CD44 and actin-based cytoskeletons. J. Cell Biol. 126: 391
    OpenUrlAbstract/FREE Full Text
  26. ↵
    Lokeshwar, V. B., L. Y. Bourguignon. 1991. Post-translational protein modification and expression of ankyrin-binding site(s) in GP85 (Pgp-1/CD44) and its biosynthetic precursors during T-lymphoma membrane biosynthesis. J. Biol. Chem. 266: 17983
    OpenUrlAbstract/FREE Full Text
  27. ↵
    Elenström-Magnusson, C., W. C. Chen, B. Clinchy, B. Öbrink, E. Severinson. 1995. Il-4-induced B cell migration involves transient interactions between β-1 integrins and extracellular matrix components. Int. Immunol. 7: 567
    OpenUrlAbstract/FREE Full Text
  28. ↵
    Hamann, A., D. Jablonski-Westrich, H. G. Thiele. 1986. Contact interaction between lymphocytes is a general event following activation and is mediated by LFA-1. Eur. J. Immunol. 16: 847
    OpenUrlCrossRefPubMed
  29. ↵
    Rothlein, R., T. A. Springer. 1986. The requirement for lymphocyte function-associated antigen 1 in homotypic leukocyte adhesion stimulated by phorbol ester. J. Exp. Med. 163: 1132
    OpenUrlAbstract/FREE Full Text
  30. ↵
    Björck, P., S. Paulie, B. Axelsson. 1992. Interleukin-4-mediated aggregation of anti-IgM-stimulated human B cells: inhibition of aggregation but enhancement of proliferation by antibodies to LFA-1. Immunology 75: 122
    OpenUrlPubMed
  31. ↵
    Hou, J., U. Schindler, W. J. Henzel, T. C. Ho, M. Brasseur, S. L. McKnight. 1994. An interleukin-4-induced transcription factor: IL-4 Stat. Science 265: 1701
    OpenUrlAbstract/FREE Full Text
  32. ↵
    Wang, L. M., A. D. Keegan, W. Li, G. E. Lienhard, S. Pacini, J. S. Gutkind, M. J. Myers, X. J. Sun, M. F. White, S. A. Aaronson, W. E. Paul, J. H. Pierce. 1993. Common elements in interleukin 4 and insulin signaling pathways in factor-dependent hematopoietic cells. Proc. Natl. Acad. Sci. USA 90: 4032
    OpenUrlAbstract/FREE Full Text
  33. ↵
    Kaplan, M. H., U. Schindler, S. T. Smiley, M. J. Grusby. 1996. Stat6 is required for mediating responses to IL-4 and for development of Th2 cells. Immunity 4: 313
    OpenUrlCrossRefPubMed
  34. ↵
    Shimoda, K., J. van Deursen, M. Y. Sangster, S. R. Sarawar, R. T. Carson, R. A. Tripp, C. Chu, F. W. Quelle, T. Nosaka, D. A. Vignali, P. C. Doherty, G. Grosveld, W. E. Paul, J. N. Ihle. 1996. Lack of IL-4-induced Th2 response and IgE class switching in mice with disrupted Stat6 gene. Nature 380: 630
    OpenUrlCrossRefPubMed
  35. ↵
    Takeda, K., T. Tanaka, W. Shi, M. Matsumoto, M. Minami, S. Kashiwamura, K. Nakanishi, N. Yoshida, T. Kishimoto, S. Akira. 1996. Essential role of Stat6 in IL-4 signalling. Nature 380: 627
    OpenUrlCrossRefPubMed
  36. ↵
    Sun, X. J., L. M. Wang, Y. Zhang, L. Yenush, M. J. Myers, E. Glasheen, W. S. Lane, J. H. Pierce, M. F. White. 1995. Role of IRS-2 in insulin and cytokine signalling. Nature 377: 173
    OpenUrlCrossRefPubMed
  37. ↵
    Wennström, S., A. Siegbahn, K. Yokote, A. K. Arvidsson, C. H. Heldin, S. Mori, L. Claesson-Welsh. 1994. Membrane ruffling and chemotaxis transduced by the PDGF β-receptor require the binding site for phosphatidylinositol 3′ kinase. Oncogene 9: 651
    OpenUrlPubMed
  38. ↵
    Kupfer, A., P. Burn, S. J. Singer. 1990. The PMA-induced specific association of LFA-1 and talin in intact cloned T helper cells. J. Mol. Cell. Immunol. 4: 317
    OpenUrlPubMed
  39. ↵
    Arroyo, A. G., P. Sanchez-Mateos, M. R. Campanero, I. Martin-Padura, E. Dejana, F. Sanchez-Madrid. 1992. Regulation of the VLA integrin-ligand interactions through the beta 1 subunit. J. Cell Biol. 117: 695
    OpenUrl
  40. ↵
    Carpen, O., P. Pallai, D. E. Staunten, T. A. Springer. 1992. Association of intercellular molecule-1 (ICAM-1) with actin containing cytoskeleton and alpha-actinin. J. Cell Biol. 118: 1223
    OpenUrlAbstract/FREE Full Text
  41. ↵
    Braun, J., P. S. Hochman, E. R. Unanue. 1982. Ligand-induced association of surface immunoglobulin with the detergent-insoluble cytoskeletal matrix of the B lymphocyte. J. Immunol. 128: 1198
    OpenUrlAbstract/FREE Full Text
  42. ↵
    Takeuchi, K., N. Sato, H. Kasahara, N. Funayama, A. Nagafuchi, S. Yonemura, S. Tsukita, S. Tsukita. 1994. Perturbation of cell adhesion and microvilli formation by antisense oligonucleotides to ERM family members. J. Cell Biol. 125: 1371
    OpenUrlAbstract/FREE Full Text
  43. ↵
    Berryman, M., R. Gary, A. Bretscher. 1995. Ezrin oligomers are major cytoskeletal components of placental microvilli: a proposal for their involvement in cortical morphogenesis. J. Cell Biol. 131: 1231
    OpenUrlAbstract/FREE Full Text
  44. ↵
    von Andrian, U. H., S. R. Hasslen, R. D. Nelson, S. L. Erlandsen, E. C. Butcher. 1995. A central role for microvillous receptor presentation in leukocyte adhesion under flow. Cell 82: 989
    OpenUrlCrossRefPubMed
  45. ↵
    Sanders, V. M., J. M. Snyder, J. W. Uhr, E. S. Vitetta. 1986. Characterization of the physical interaction between antigen-specific B and T cells. J. Immunol. 137: 2395
    OpenUrlAbstract
  46. ↵
    Sanders, V. M., R. Botran-Fernandez, J. W. Uhr, E. S. Vitetta. 1987. Interleukin 4 enhances the ability of antigen-specific B cells to form conjugates with T cells. J. Immunol. 139: 2349
    OpenUrlAbstract
  47. ↵
    Kupfer, A., T. R. Mosmann, H. Kupfer. 1991. Polarized expression of cytokines in cell conjugates of helper T cells and splenic B cells. Proc. Natl. Acad. Sci. USA 88: 775
    OpenUrlAbstract/FREE Full Text
  48. ↵
    Kupfer, H., C. R. Monks, A. Kupfer. 1994. Small splenic B cells that bind to antigen-specific T helper (Th) cells and face the site of cytokine production in the Th cells selectively proliferate: immunofluorescence microscopic studies of Th-B antigen-presenting cell interactions. J. Exp. Med. 179: 1507
    OpenUrlAbstract/FREE Full Text
  49. ↵
    Noelle, R. J., M. Roy, D. M. Shepherd, I. Stamenkovic, J. A. Ledbetter, A. Aruffo. 1992. A 39-kDa protein on activated helper T cells binds CD40 and transduces the signal for cognate activation of B cells. Proc. Natl. Acad. Sci. USA 89: 6550
    OpenUrlAbstract/FREE Full Text
Back to top

In this issue

The Journal of Immunology
Vol. 160, Issue 11
1 Jun 1998
Print
Download PDF
Article Alerts
Email Article
Citation Tools

Jump to section