Microvilli structures on B lymphocytes: inducible functional domains?

Gediminas Greicius1, Lisa Westerberg1, Edward J. Davey2, Eva Buentke3, Annika Scheynius3, Johan Thyberg1 and Eva Severinson1

1 Department of Cell and Molecular Biology, Karolinska Institutet, 17177 Stockholm, Sweden 2 Department of Genetics and Pathology, Uppsala University, 75185 Uppsala, Sweden 3 Department of Medicine, Unit of Clinical Allergy research, Karolinska Institutet and Hospital, Stockholm, 17176 Stockholm, Sweden

Correspondence to: E. Severinson; E-mail: eva.severinson@ cmb.ki.se
Transmitting editor: P. W. Kincade


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Interactive contact between B lymphocytes and T cells is necessary for their expansion during an immune response. It has been shown that B lymphocytes receive signals from T cells, such as IL-4 and cross-linking of CD40, which are crucial for their differentiation. We previously found that these factors induce formation of microvilli on B cells and that this was correlated with increased homotypic adhesion of B lymphocytes. In this study we have investigated if IL-4 induce segregation of proteins to microvilli and lipid rafts. Using immuno-electron microscopy we analyzed cell-surface distribution of molecules involved in B–T cell co-activation. Recruitment to detergent-resistant membrane fractions was analyzed using sucrose gradient centrifugation. We found that microvilli were enriched in ICAM-1 and MHC class II molecules. In contrast, LFA-1 and CD40 were more abundant on the smooth cell surfaces, while B7-2 (CD86) was randomly distributed. We also discovered that depletion of cholesterol, using ß-methyl-cyclodextrin, lowered the number of microvilli, indicating that intact lipid rafts are required for their expression. Moreover, activation of B lymphocytes by lipopolysaccharide (LPS) induced increased expression of GM1, a marker for lipid rafts. However, although both surface and total levels of GM1 were similar in B lymphocytes stimulated with either LPS or LPS plus IL-4, GM1 was mainly expressed on microvilli in LPS plus IL-4-stimulated cells. Taken together, our results indicate that microvilli represent distinct inducible membrane domains that can regulate direct cell–cell interactions via grouping and three-dimensional presentation of cell-surface receptors.

Keywords: adhesion, B cell, IL-4, lipid raft, microvilli


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
It is becoming increasingly clear that lymphocytes have unique ways of organizing molecules on the cell surface that are likely to regulate their signaling and, ultimately, effector functions of the whole cell. The formations of distinct lymphocyte surface domains such as segregation into leading edge and uropod, lipid rafts and thin cytoplasmic extensions known as microvilli all offer an increased functionality in cellular motility, adhesion and signal transduction.

Presence of microvilli on the lymphocyte surface was first appreciated in early electron microscopy studies performed in the 1960s. These formations were later suggested as a diagnostic feature in acute lymphocyte leukemia and hairy cell leukemia (1). In the following years, microvilli received much attention as grouping sites for surface molecules. Thus, expression of selectins on the tips of microvilli is important for leukocyte binding to endothelial cells in flow conditions and for subsequent steps of extravasation (2). Likewise, a number of cell surface signaling molecules, including CD4, CCR5 and CXCR4 in T lymphocytes (3), and CD21 (4) and insulin receptor (5) in B lymphocytes, are all preferentially localized on microvilli.

Little is known about the mechanisms that regulate microvilli expression on the lymphocyte surface. As described in the study of Patarroyo et al. (6), microvilli-like membrane extensions are induced by phorbol ester in human peripheral blood leukocytes. A later study by Björck et al. (7) demonstrates that antibodies to CD40, a key molecule in T cell-dependent B cell activation, induce microvilli in human B lymphocytes. Our previous results (810) are in line with these studies and provide evidence that another key T cell-derived factor for B lymphocyte differentiation, IL-4, is a potent inducer of microvilli in activated mouse B lymphocytes (8). Decreased amounts of microvilli are observed in lymphocytes from Wiskott–Aldrich syndrome (WAS) patients (11). This disease, caused by mutations in WAS syndrome protein (WASP), affects hematopoietic cells and in severe cases leads to immunodeficiency. WAS patients are susceptible to pyogenic and viral infections, and suffer from eczema, autoimmune and lymphoproliferative diseases (12). The cause of the immunodeficiency is not clear, but it has been suggested to be associated with abnormal cell migration and altered geometry of the cell surface [for a review, see (13)].

Activation of B lymphocytes in the immune response is largely a result of their interaction with antigen-specific Th lymphocytes. It is believed that B lymphocytes can take up antigen directly via antigen receptors or as immuno- complexes via Fc receptors (14) and/or complement receptors (15,16). Thereafter, it is processed and presented to the antigen-specific T lymphocytes in the context of MHC class II molecules. When this interaction is further supported by recognition of B7 molecules that are up-regulated on activated B lymphocytes, Th lymphocytes receive a signal for proliferation and differentiation. In turn, expansion of antigen-specific B cells is triggered by CD40 that recognizes CD40 ligand on the surface of activated T cells (17) and/or IL-4-mediated co-stimulation of the BCR (18). Direct T cell interactions with antigen-presenting cells involve segregation of the surface molecules into lipid rafts—dynamic, cholesterol-ordered membrane domains that recruit and concentrate molecules involved in cellular signaling (19,20). As demonstrated by Anderson et al., the concentration of MHC class II molecules in lipid rafts facilitates antigen presentation (21).

In the present study we found that B lymphocytes are induced to express more and longer microvilli by lipopolysaccharide (LPS) plus IL4 or cross-linking of CD40, but not by LPS plus IL-2 or IL-5. We found that microvilli represent a membrane domain rich in ICAM-1, MHC class II and B7-2 (CD86), suggesting that they are involved in the regulation of adhesion and/or antigen presentation. Moreover, our studies indicate that microvilli are sensitive to cholesterol depletion, indicating a requirement for intact lipid rafts. Finally, lipid rafts appear to be concentrated to microvilli-rich regions.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
(CBA/J x C57BL/6)F1 mice purchased from Charles River (Uppsala, Sweden) were used unless otherwise stated and maintained in an animal facility at the Department of Cell and Molecular Biology, Karolinska Institutet. Mice deficient in WASP (WASP–/–) were kindly provided by Dr Scott Snapper and Dr Fred Alt (22). These mice were maintained and bred in the same animal facility. All experimental work with animals was performed according to ethical permissions N130/96, N73/99 and N84/02.

Cell culture
Splenic B cells were enriched as previously described, using a cocktail of anti-T cell antibodies and complement followed by size separation in Percoll gradients (8,23). Using flow cytometry, the cell population was tested for contamination by other cells in four different experiments, and found to consist of 80–93% B220+ cells (a B cell marker), 0.1–5% CD3+ cells (a T cell marker) and 2–8% Mac-1+ cells (a granulocyte/macrophage/monocyte marker). Cells (5 x 105/ml) were cultured in RPMI 1640 supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, penicillin 50 IU/ml, streptomycin 50 µg/ml (all from Gibco, Life Technologies, Paisley, UK), 50 µM 2-mercaptoethanol and 10% heat-inactivated FCS taken from selected batches (Gibco). Cultures of B lymphocytes were stimulated with agonistic rat anti-mouse CD40 mAb (1C10) (24) or LPS (extracted from Escherichia coli 055:B5; Sigma-Aldrich, Stockholm, Sweden). In some experiments, LPS was used in combination with hybridoma supernatants, containing recombinant murine IL-2, IL-4 or IL-5 as described previously (8,23,25). The cytokines were titrated in specific assays and used in saturating concentrations as described (8). Supernatants of 1C10 cells were ammonium sulfate precipitated and titrated in a proliferation assay with B cells from spleen, and used at saturating concentration. In four experiments the activated B cell population was tested and found to consist of 88–94% B220+ cells, 3–7% CD3+ cells and 2–5% Mac-1+ cells determined as stated above.

Electron microscopy preparation
Aggregates of stimulated B cells were fixed in suspension in 3% glutaraldehyde in 0.1 M sodium cacodylate–HCl buffer (pH 7.3) with 0.05 M sucrose (1 volume of cells in culture medium mixed with 3 volumes of fixative). After 1–2 h, the cells were sedimented by a two-step centrifugation procedure, first at 1000 r.p.m. for 5 min and thereafter in a smaller volume of fresh fixative at 10,000 r.p.m. for 15 min (Eppendorf centrifuge). The pellets so obtained were removed from the tubes and cut into small pieces. With this procedure, the shape and surface topography of the cells was preserved by the initial fixation in suspension, and no clear changes occurred during the subsequent centrifugations. After rinsing overnight in 0.1 M cacodylate buffer, the specimens were post-fixed in 1.5% osmium tetroxide in 0.1 M cacodylate buffer (pH 7.3) with 0.7% potassium ferrocyanate for 2 h at 4°C, dehydrated in ethanol (70, 95 and 100%), stained with 2% uranyl acetate in ethanol and embedded in Spurr low-viscosity epoxy resin.

Immuno-electron microscopy
LPS plus IL-4 lymphocytes (15 x 106) were cultured for 48 h and then fixed by mixing with an equal volume of 4% phosphate-buffered formaldehyde for 10 min. Then cells were centrifuged at 1200 r.p.m. for 10 min and fresh phosphate-buffered 2% formaldehyde solution was added for 45 min. After fixation, cells were washed twice in PBS and non-specific binding was thereafter blocked in PBS containing 0.5% BSA (v/w) for 2 h. Following the blocking step, cell pellets were incubated with 350 µl PBS/BSA containing cocktails of rat mAb to ICAM-1 (YN1/7.4 Protein G-affinity purified from hybridoma supernatants in combination with either HB233, Protein G-affinity purified from hybridoma supernatants or 3E2; BD PharMingen, San Diego, CA; 20 µg/ml each, both cocktail formulations gave similar results), LFA-1 (50% ammonium sulfate-precipitated M17/5.2 hybridoma supernatant and 2D7; BD PharMingen; 40 µg/ml each), CD40 (50% ammonium sulfate precipitated hybridoma 1C10 supernatant and MCA1143; Serotec, Oxford, UK; 40 µg/ml each), CD86 (GL1; BD PharMingen and MCA1587; Serotec), MHC class II (M5/114.15.2, BD PharMingen) and isotype control antibodies (BD PharMingen) in respective concentrations for 2 h. Then cells were washed 3 times with PBS/BSA and incubated with biotinylated mouse anti-rat IgG (Jackson ImmunoResearch, West Grove, PA) diluted 1:20 in PBS/BSA. Following this incubation, cells were washed 3 times with PBS/BSA and incubated with streptavidin–gold particles (10 nm, Auroprobe; Amersham, Uppsala, Sweden) for 2 h. Thereafter, cell pellets were rinsed twice with PBS/BSA and once with 0.1 M cacodylate buffer, pH 7.3, followed by fixation in 3% cacodylate-buffered glutaraldehyde for 2 h and incubation in cacodylate buffer overnight. These staining steps were followed by standard electron microscopy procedures including post-fixation in osmium tetraoxide.

To study the distribution of GM1 on the surface of lymphocytes, cells were fixed and blocked as described above. Thereafter, they were incubated in PBS/BSA containing 20 µg/ml of biotinylated cholera toxin B (Sigma-Aldrich). After this step cells were fixed in 3% cacodylate-buffered glutaraldehyde, blocked in PBS/BSA and then incubated in PBS/BSA containing streptavidin–gold particles diluted 1:10. Thereafter, cells were washed in PBS/BSA and post-fixed in 3% cacodylate-buffered glutaraldehyde.

Thin sections were cut with a diamond knife on an LKB Ultrotome IV (Bromma, Sweden) or a Leica Ultracut (Vienna, Austria), picked up on carbon-coated Formvar films, stained with alkaline lead citrate, and examined in a JEOL 100CX electron microscope (JEOL, Tokyo, Japan) or a Philips CM120Twin electron microscope (Philips, Eindhoven, The Netherlands). For each experimental group, at least three or four specimens were sectioned and a large number of cells or cell aggregates were studied in the electron microscope.

Analysis of electron micrographs
We selected sections of 20 cells using the following criteria: non-polarized, intact cell morphology, central location and size of nucleus characteristic for sections located close to the middle of the cell body, and the presence of microvilli. Then membrane regions of these sections were photographed at x57,000 magnification to obtain five or six images per section for the determination of gold label distribution around the entire circumference of the cells. The latter analysis was performed after coding the samples (while measuring, the identity of the experimental groups was not known to the person doing the analysis). The aim was to investigate whether there are differences in the proportions of the specific antibody labeling on the microvilli between the five groups. The data were examined by analysis of covariance (ANCOVA) using Statistica software (StatSoft, Tulsa, OK). Microvilli surface and the total number of gold particles on the cell were used as covariates in the ANCOVA model. The assumptions of the ANCOVA model were tested and were found to be satisfactory fulfilled. In the dataset it was found that seven of the proportions were either 0 or 1. This may result in underestimated variances in some groups and in an underestimated P value, although the influence of these values was assessed to be rather small.

Aggregation assay
LPS or LPS plus IL-4-activated B lymphocytes were cultured for 48 h in 96-well plates. Then half of the medium was carefully removed and reconstituted with warm RPMI 1640 supplemented with either 0.5 or 0.25% ß-methyl-cyclodextrin (MCD; Sigma-Aldrich), 10% FCS and either LPS or LPS plus IL-4, respectively. Cell cultures were incubated for 30 min at 37°C in a humidified atmosphere containing 5% CO2. Levels of cellular aggregation were measured using a hemacytometer, counting at least 200 cells per group. Dead cells, stained with Trypan blue (Sigma-Aldrich), were not counted.

Flow cytometry
Staining with anti-ICAM-1
Activated or non-activated B cells were incubated sequentially with rat anti-ICAM-1 mAb (YN1/7.4, 10 µg/ml) and FITC-labeled anti-rat F(ab')2 (Jackson ImmunoResearch). Controls were stained with the second step alone.

Staining with cholera toxin B
Activated and non-activated B cells were fixed in 4% phosphate-buffered formaldehyde (pH 7.2) for 5 min and washed twice in Earle’s balanced salt solution (BSS; Gibco). Thereafter, samples used for intracellular staining were permeabilized by washing in BSS containing 0.1% Saponin (Sigma-Aldrich). All samples were then incubated with BSS containing 10 µg/ml of biotinylated cholera toxin B subunit (Sigma-Aldrich) at 20°C for 30 min. Following washing, samples were incubated in BSS containing streptavidin–FITC (Dakopatts, Älvsjö, Sweden). Staining of the permeabilized cells was performed in BSS containing 0.1% Saponin.

Cells (10,000 per sample) were analyzed using a FACScan (Becton Dickinson, San Jose, CA). Dead cells were excluded by staining with propidium iodide (Sigma-Aldrich). Single cells were gated and selected according to a characteristic forward scatter pattern.

Enrichment of detergent-resistant membranes (DRM)
DRM were enriched according to the procedures described in Current Protocols in Immunology (26). In short, 1.2 x 108 cells were activated by LPS or LPS plus IL-4 for 2 days. After harvesting, cells were lysed in 1 ml of ice-cold TNE buffer (50 mM Tris, pH 7.6, 150 mM NaCl, 5 mM EDTA, 0.2 mM PMSF, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 10 mM NaF and 1 mM Na3VO4) containing 1% Triton X-100 for 30 min on ice. After lysis, cellular debris were removed by brief low-speed centrifugation (6000 r.p.m., 5 min, 4°C) and thereafter supernatants were mixed with an equal volume (1 ml) of ice-cold 80% sucrose solution in TNE buffer. The resulting mixtures of lysates in 40% sucrose were transferred to Beckman tubes and overlaid with TNE buffer containing 35% sucrose followed by TNE buffer containing 5% sucrose. All TNE buffer–sucrose mixtures contained the aforementioned inhibitors of proteases, kinases and phosphatases. Gradient centrifugation was performed at 35,000 r.p.m. using a SW 42Ti rotor for 18 h at 4°C. Fractions of 0.5 ml were collected from the top of the gradient. Close to the 5/35% sucrose interphase, fraction volume was reduced to 0.25 ml in order to get more concentrated protein samples. For western blot analysis, fractions were mixed with non-reducing 5 x sample buffer (Laemmli), boiled at 95°C for 5 min and loaded to wells of SDS–polyacrylamide gels. Western blotting was performed to detect MHC class II, B7-2 and ICAM-1. From the fractions containing soluble proteins in 40% sucrose, 25 µg was loaded per lane. From all other fractions, 30 µl were loaded per lane. We could not detect LFA-1 and CD40 in western blot with the antibodies used in the current study. In order to analyze distribution of these molecules in sucrose-gradient-fractionated samples, we performed dot-blots. Dot-blot analysis was done as follows. Small aliquots (1 µl) of each fraction were dried onto nitrocellulose membrane. Each fraction was dotted in duplicate. After blotting, membranes were blocked for 1 h in Tris-buffered saline (TBS) containing 5% non-fat milk and 0.05% Tween-20. Thereafter, membranes were incubated with the respective antibodies for 2 h at 20°C. MHC class II, ICAM and CD86 were detected with antibodies YN1/7.4 (affinity-purified from hybridoma supernatant), M5/114.15.2 (BD PharMingen) and MCA1587 (Serotec) respectively. LFA-1 was detected using equal parts of antibodies M17/5.2 precipitated from hybridoma supernatant and 2D7 (BD PharMingen). CD40 was detected using equal parts of 1C10 (ammonium sulfate-precipitated hybridoma supernatant) and MCA1143 (Serotec). Antibodies were used at 1 µg/ml in TBS containing 5% skimmed milk. Following this incubation, membranes were washed in TBS containing 0.05% Tween-20 and subsequently incubated with horseradish-peroxidase labeled rabbit anti-rat serum (Dako) diluted 1/1000 in TBS/milk/Tween-20 for 1 h. Membranes were washed and developed using SuperSignal (Pierce, Rockford, IL) reagent and analyzed either in a gel documentation system using Image Gauge software (version 3.41; Fuji Photo Film) or by exposure on films for autoradiography.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Induction of microvilli by IL-4 and anti-CD40
We previously showed that B cells activated with LPS plus IL-4, but not with LPS alone, express many long microvilli (8). Here we studied in more detail whether microvilli can be regulated by other T cell cytokines that also take part in B cell differentiation. B cells were activated for 2 days with LPS, LPS plus IL-4 or with agonistic anti-CD40 antibodies and analyzed by electron microscopy. In all electron microscopy experiments, the great majority of the cells had lymphoblast morphology. Since contamination by T cells was <5% (see Methods), we conclude that the majority of the cells we analyzed were B cell blasts. When focusing on cells in contact with each other, we noticed that LPS-activated cells had smooth surface contours and only few, short processes (Fig. 1A). In contrast, B cells activated with LPS plus IL-4 expressed many long microvilli. These were engaged in contacts with neighboring cells and each cell typically used several microvilli in the adhesion to other cells. It was also noted that the cells used microvilli to contact more than one other cell (Fig. 1B and C). B cells stimulated with anti-CD40 expressed even more microvilli. The stimulated B cells were in tight aggregates and the microvilli formed an elaborate system of cytoplasmic processes, often making it difficult to determine the contours of the individual cells (Fig. 2A). On the other hand, B cells stimulated with LPS plus either IL-2 or IL-5 did not express more microvilli than B cells stimulated with LPS alone. These cytokines stimulated neither aggregation nor dendritic formation [(8) and data not shown], but they are known to cause maturation, i.e. Ig secretion in B cells. Consequently, cells activated with LPS plus IL-2 or IL-5 expressed a prominent endoplasmic reticulum and Golgi complex (Fig. 2B and C). Thus, there was a strong correlation between the capacity to induce many long microvilli, increased levels of aggregation with round dense aggregates (23) and dendritic morphology (8).



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Fig. 1. Cell-surface morphology and cell–cell interactions in aggregates of mouse B lymphocytes cultured in the presence of LPS (A) or LPS plus IL-4 (B and C). In LPS-treated cultures (A), the cells had a relatively smooth contour and only few, short processes extended from the cell surface (arrowheads). In cultures treated with LPS plus IL-4 (B and C), an intricate system of microvilli-like processes was found on the cell surface. Via these processes, the individual cells established contact with one or more adjacent cells (arrows). M, mitochondria; N, nuclei. Bars = 1.0 µm.

 


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Fig. 2. Cell-surface morphology and cell–cell interactions in aggregates of mouse B lymphocytes cultured in the presence of anti-CD40 (A), LPS plus IL-5 (B) or LPS plus IL-2 (C). Stimulation with anti-CD40 (A) gave rise to large aggregates with closely opposed cells. An elaborate system of cytoplasmic processes connected the cells to each other (arrows). In the cultures treated with LPS plus IL-5 (B) or LPS plus IL-2 (C) the aggregates were smaller and the cells less densely packed. Only few and short processes appeared on the cell surface (arrowheads). In the cytoplasm, a prominent endoplasmic reticulum (ER) and Golgi complex (G) was typically found in these cells. M, mitochondria; N, nuclei. Bars = 1.0 µm.

 
We considered the possibility that microvilli were induced as a consequence of the increased level of adhesion between B cells. However, when analyzing B cells stimulated by LPS plus IL-4 in the presence of anti-LFA-1 antibodies, cells showed a similar amount of microvilli, although adhesion was almost entirely inhibited (data not shown). Thus, we conclude that microvilli can be formed even though B cells are not engaged in adhesive contacts with other cells.

Adhesion and co-stimulatory molecules are expressed on microvilli
As shown above, stimulation of B cells with LPS plus IL-4 or anti-CD40 led to the formation of two different cell-surface domains: a more ‘rigid’, smooth cell body and microvilli that are fine, long and flexible membrane extensions. Since these two domains offer different physical properties for direct cell–cell interactions, we investigated how adhesion and co-stimulatory molecules were distributed on microvilli and smooth areas of B cells. Initially, we performed confocal microscopy studies for localization of ICAM-1. We detected staining on relatively large membrane protrusions. However, this method did not allow measurement of the fine microvilli structures (data not shown). To study the B cell surface in more detail, we used immuno-electron microscopy. B lymphocytes activated in the presence of LPS plus IL-4 were labeled with antibodies to ICAM-1, LFA-1, CD40, MHC class II and B7-2 (CD86) or respective isotype controls, and then stained with streptavidin–gold particles. Thereafter, random central sections of B cells expressing microvilli were acquired and the distribution of gold particles in each group was assessed in a blind manner. The microvilli surface was not significantly different among the analyzed groups (see legend to Fig. 4). An example of immuno-electron microscopy micrographs of anti-ICAM-1-labeled B lymphocytes is presented in Fig. 3. We found that on the surface of LPS-activated B cells lacking microvilli, ICAM-1 labeling was distributed in a dispersed pattern (Fig. 3A and B). However, ICAM-1 was preferentially localized to microvilli on cells stimulated with LPS plus IL-4 (Fig. 3C–E). A summary of the data on the distribution of specific labeling for ICAM-1 and other molecules on microvilli is presented in Fig. 4. We found that gold particles were present on both microvilli and smooth surfaces of cells in all the groups. The groups stained with isotype-matched control antibodies contained significantly lower (5- to 10-fold) amounts of gold particles than other groups. Using ANCOVA, we distinguished two significantly (P < 0.05) different types of labeling (Fig. 4). The first group included staining with ICAM-1 and MHC class II-specific antibodies with immuno-gold particles predominantly distributed on microvilli surfaces. In contrast, gold labeling in a second group consisting of LFA-1 and CD40 was predominantly localized on smooth areas of the cell surface. The co-stimulatory molecule B7-2 was randomly dispersed on both smooth and microvilli surfaces. We conclude that there is no stringent sorting mechanism between localization on microvilli and smooth cell surfaces. However, a relative increase in MHC class II and ICAM-1 on microvilli indicates that this morphological domain possesses a distinct composition of molecules involved in contact mediated co-stimulation.



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Fig. 4. Expression of adhesion and co-stimulatory molecules on the microvilli of B lymphocytes. B cells activated with LPS plus IL-4 were fixed and labeled with antibodies and streptavidin-coated gold particles as indicated in Methods. Surface distribution of gold particles was assessed on 20 randomly selected cell sections per group. The percentage of microvilli surface was similar in all groups (53 ± 3.9% of the total surface). Bars represent the mean percentage of gold particles found on microvilli. Error bars represent 0.95 confidence interval.

 


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Fig. 3. Immuno-electron microscopy demonstration of ICAM-1 on the surface of mouse B lymphocytes cultured in the presence of LPS (A and B) or LPS plus IL-4 (C–E). Pre-embedding staining was done with a mixture of two mAb against ICAM-1 (YN1/7.4 and 3E2) followed by biotinylated anti-rat IgG and streptavidin–10 nm gold (for further details, see Methods). In LPS-treated cells (A and B), a fine dispersed staining was seen on the cell surface (arrowheads). In cells exposed to LPS plus IL-4 (C–E), the staining was concentrated to microvilli-like processes (arrows), but some reactivity also appeared on smooth parts of the cell surface (arrowheads). M, mitochondria; N, nuclei. Bars = 0.5 µm.

 
It appeared from the pictures shown in Fig. 3 that ICAM-1 was expressed at a higher level on LPS plus IL-4- as compared to LPS-activated B cells. Indeed, as shown in Fig. 5 using flow cytometry, the former population had a slightly higher level of staining than the latter.



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Fig. 5. Expression of ICAM-1 on activated B cells. Freshly prepared B cells (A) or B cells cultured in the presence of LPS (B) or LPS + IL-4 (C). Cells were thereafter stained with anti-ICAM-1 as detailed in Methods and analyzed using a FACScan. Negative controls (dashed line) were stained with the second step alone. Aggregated and dead cells were excluded by forward scatter gating. Histograms represent FITC fluorescence intensity (x-axis) versus cell counts (y-axis). Numbers represent mean fluorescence intensity.

 
Microvilli and detergent-insoluble lipid domains
Since cell-surface signaling events involve lipid rafts (19), we investigated whether stimuli-induced microvilli and cellular adhesion were sensitive to disruption of these membrane domains. To this end, we measured cellular aggregation and microvilli formation in cultures incubated with MCD. This compound is known to extract cholesterol from lipid bilayers and thereby destabilizes lipid rafts. We found that homotypic B cell aggregation was significantly reduced following exposure to MCD for 30 min. As shown in Fig. 6(A), cellular aggregation was reduced up to 10-fold by treatment with 0.25% MCD, while cellular viability was not affected (see legend).



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Fig. 6. Intact lipid raft structure is important for homotypic adhesion and microvilli expression. (A and B) B lymphocytes were cultured with either LPS alone or LPS plus IL-4 for 2 days and thereafter incubated in the presence of 0.25% MCD or without MCD for 30 min, as indicated. (A) Aggregation was counted using a hemacytometer. The viability, as judged by Trypan blue exclusion, was similar in MCD-treated and untreated groups (>90%). Aggregation of LPS- and LPS plus IL-4-stimulated cells was significantly reduced in the presence of MCD (P < 0.01). A representative experiment from two independent ones is shown. (B) Activated cells were fixed and analyzed using electron microscopy. Membrane extensions >500 nm were defined as microvilli. Microvilli and smooth surface areas were measured in 20 cells (approximate mid-sections) per group. Microvilli surface on LPS plus IL-4-activated cells was significantly increased when compared to other groups (*P < 0.05). In contrast, following incubation with MCD, the microvilli surface on LPS plus IL-4-stimulated cells was reduced and not significantly different from LPS-activated cells in the presence or absence of MCD. (A and B) The results are presented as the mean of triplicates, error bars represent SD. (C) FACS analysis of LPS plus IL-4-activated B lymphocytes, incubated in the absence or presence of MCD and thereafter stained with cholera toxin B, as indicated in Methods. Solid line indicates staining of LPS plus IL-4-stimulated cells in the absence of MCD. Staining of LPS plus IL-4-stimulated cells treated with 0.5% MCD for 30 min is shown by the dotted line. Negative control staining is indicated by the dashed line. The marked region indicates the gate for mean fluorescence values (MCD non-treated: 628; MCD treated: 564). The experiment shown is representative of two independent experiments. (D) GM1 distribution on the surface of LPS plus IL-4-stimulated cells. Lymphocytes were stained with cholera toxin and labeled with immuno-gold as described in Methods. Thereafter, the distribution of gold particles was analyzed on sections of 20 cells. Beads localized to very basal parts of microvilli are defined as ambiguous.

 
Using the same experimental set-up and electron microscopy, we measured the proportion of microvilli surface in LPS- and LPS plus IL-4-activated B cells in the presence or absence of MCD (Fig. 6B). The measurements were performed on micrographs of randomly selected cell sections, which were localized in the proximity to the central plane of the cell body. As indicated in Fig. 6, we found that amounts of microvilli surface on LPS-activated cells did not change significantly after treatment with MCD. In contrast, the number of long (>500 nm in length), LPS plus IL-4-induced microvilli was significantly diminished following exposure to MCD and the cell morphology became similar to that of LPS-activated B cells (Fig. 6B). Apart from an absence of microvilli structures, the cells remained intact after MCD treatment (data not shown). Thus, we conclude that membrane cholesterol is important for sustained expression of long microvilli. In Fig. 6(C) we measured the levels of ganglioside GM1, a marker of lipid rafts, in MCD-treated or untreated LPS plus IL-4-activated B cells. Cells were stained with fluorescent-labeled cholera toxin B that binds to GM1 and analyzed by flow cytometry. As shown, MCD treatment somewhat reduces GM1 levels on the membrane, but the majority of label is still intact. Thus, MCD treatment does not solubilize GM1 from the cell surface.

Next, we wanted to measure whether IL-4 regulated the level of GM1. This lipid is found on the cell surface as well as in intracellular compartments (27). As shown in Fig. 7, the surface levels of GM1 were up-regulated upon lymphocyte activation, but not different in LPS- or LPS plus IL-4-activated cells. We found that the total level of GM1, measured in saponin-permeabilized cells, also was higher after stimulation. Again, there was no significant difference between LPS- and LPS plus IL-4-activated cells. From these studies we conclude that IL-4 is not involved in regulation of GM1 levels. We also assessed levels of GM1 in WASP-deficient B cells activated by LPS and LPS plus IL-4, since they have been observed to be deficient in microvilli formation (10). As indicated in Fig. 7, we could not detect any significant difference between wild-type and WASP-deficient B cells in the level of GM1 on the cell surface or in the intracellular pools.



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Fig. 7. GM1 levels in resting and stimulated wild-type and WASP B cells. B lymphocytes from wild-type and WASP-deficient mice were either stained fresh or after activation for 48 h in the presence of LPS or LPS plus IL-4, as indicated. Following fixation, B lymphocytes were either stained on the surface or permeabilized with saponin (indicated as total), and then stained with biotinylated cholera toxin B subunit and streptavidin–FITC, as described in Methods. Staining with streptavidin–FITC alone is indicated by the dashed line and cholera toxin B-specific staining is marked by the solid line. Figures indicate mean fluorescence intensity of the marked population. The experiment shown is representative of three independent experiments.

 
It was of interest to determine the localization of lipid rafts on microvilli-expressing B cells. To this end we analyzed LPS plus IL-4-activated cells stained with cholera toxin B by electron microscopy, as described in Methods. As shown in Fig. 6(D), there is a relative enrichment of cholera toxin B staining on microvilli. All together, the above data indicate that lipid rafts are necessary for the formation and maintenance of microvilli.

We further investigated whether molecules found on microvilli would localize to DRM in LPS plus IL-4-activated cells. Cells were activated by LPS in the presence or absence of IL-4 for 48 h, harvested and lysed on ice. Thereafter, we used discontinuous sucrose gradient centrifugation to separate DRM from soluble membrane proteins. As indicated in Fig. 8(A), we found that a small fraction of MHC class II molecules was localized in the DRM fraction following LPS plus IL-4-induced activation. MHC class II molecules were translocated to lipid rafts to a lesser extent when cells were stimulated by LPS alone.



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Fig. 8. MHC class II is recruited to the DRM fraction in LPS plus IL-4-activated B cells. B lymphocytes (1 x 108) were activated with either LPS or LPS plus IL-4, harvested, lysed and loaded on sucrose gradients as described in Methods. (A) Western blots were performed using anti-MHC class II, -B7-2 and -ICAM-1 antibodies, as explained in Methods. Soluble fractions (40 and 35% sucrose) are indicated with S. Fractions collected from the 35/5% sucrose interphase represent DRM. These fractions were loaded sequentially, in order of high to low sucrose concentration. The experiment shown is representative of three independent experiments. (B) Measurement of staining with cholera toxin B, anti-LFA-1 and anti-CD40 in soluble and insoluble fractions using dot-blot analysis, as detailed in Methods. The results are presented as percentage of total amount of protein detected in all fractions. The figures are distributed horizontally to match respective fractions in western blots. An asterisk indicates <0.1% of protein detected. The experiment shown is representative of two independent experiments.

 
In contrast to MHC class II molecules, ICAM-1, CD86 (B7-2), ICAM-1, LFA-1 and CD40 were all localized to Triton X-100-soluble fractions (Fig. 8A and B). These results suggest that recruitment to microvilli and to DRM may be two different IL-4-induced events on the B cell surface. However, microvilli require intact MCD-sensitive lipid rafts to maintain integrity and adhesive functions.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The present study extends our observations (810) that IL-4 significantly increases the plasticity of the B lymphocyte surface. Here we describe microvilli as a feature of LPS plus IL-4- or anti-CD40-induced B cell activation that is not observed when cells are activated by either LPS alone or in combination with IL-2 or IL-5. It is interesting that the latter two cytokines, which induce Ig secretion in B cells, fail to induce microvilli. Similarly, the agents that induce microvilli also lead to formation of tight B cell aggregates. This might indicate a need for separation of these responses in vivo.

We also found that microvilli are enriched in ICAM-1 and MHC class II molecules but not LFA-1 or CD40. Our studies further suggest that formation and maintenance of long microvilli require intact lipid rafts, even though the amount of GM1, a marker for lipid rafts, was not directly regulated by IL-4. However, IL-4 can modulate the composition of lipid rafts, such as directing MHC class II molecules to DRM.

Mutual adhesion and clustering of membrane receptors are key elements in co-activation of B and T lymphocytes. We previously found that LFA-1-mediated adhesion was differentially regulated depending on conditions of B cell activation (23). In the same study, we described that LPS and LPS plus IL-4 induced a similar increase in LFA-1 avidity, as monitored by B cell adhesion to ICAM-1-expressing fibroblasts. Our present study indicates that IL-4 plays a dual role in the functional regulation of LFA-1–ICAM-1 interactions. First, expression of ICAM-1 is higher on cells activated by LPS plus IL-4 than on LPS-activated cells (Fig. 5). Second, ICAM-1 is preferentially localized to the microvilli, with increased local density and more flexible contact surface. These results are in agreement with the study of Ganpule et al. indicating that a certain coating density and geometry of ICAM-1-containing surfaces are required for optimal LFA-1 activation (28).

Distinct recruitment of ICAM-3 (29) and CD43 (30) along with cytoskeletal components to uropods of polarized T cells is well recognized. However, in non-polarized cells, microvilli can also serve as molecular clustering sites. Preferential recruitment to microvilli was described for CCR5, CXCR4 and CD4 in T cells (3) and complement receptor 1 (CD21) (4) in B cells. Moreover, localization on microvilli seems to be important for insulin receptor (31,32) and CD4 signaling (33). In the present study, we found that ICAM-1 is co-expressed on microvilli together with MHC class II molecules and CD86. Interestingly, a similar enrichment in ICAM-1, CD86 and MHC class II is observed in exosomes, vesicles generated by B lymphocytes (34) and dendritic cells (35) that can present antigen (36). In addition, our findings also indicate that CD40 and LFA-1 are preferentially localized on smooth cell surfaces, implying that the overall geometry of receptor distribution can play a role in IL-4-induced direct cellular interactions.

Clustering of signaling receptors to cholesterol-ordered lipid rafts is an important step in cellular signaling. It has been shown that depletion of cholesterol perturbs LFA-1-dependent adhesion and inhibits antigen presentation by MHC class II molecules (21,37,38). We also found that disruption of lipid rafts by MCD abolished homotypic aggregation of B lymphocytes activated by either LPS or LPS plus IL-4. Furthermore, this effect of MCD was linked to a decreased proportion of long, IL-4-induced microvilli. As reported by Mobius et al. (39), filopodium-like processes of lymphoblastoid cells are enriched in cholesterol. This provides a notion that microvilli on B lymphocytes might be a morphological representation of lipid raft assemblies. In fact, we detected IL-4-induced localization of MHC class II molecules on both microvilli and in DRM fractions in the absence of cross-linkers. However, we failed to detect ICAM-1 and CD86 in DRM fractions, even though they were also localized on microvilli. In addition, LFA-1 and CD40 were also found only in detergent soluble fractions. Increased LFA-1-mediated lymphocyte aggregation, which is observed in our system, induced clustering of ICAM-1 in cell–cell contacts (data not shown). Nevertheless, we did not detect any mobilization of ICAM-1 to DRM without antibody mediated cross-linking. Such translocation was previously described in endothelial cells by Tilghman et al. (40) after cross-linking with antibodies to ICAM-1.

Our data indicate that the cellular level of GM1 might modulate lipid raft formation and signaling events. We found that GM1 levels are increased upon stimulation, but are not regulated by IL-4. Interestingly, a pronounced decrease in GM1 levels was described in T lymphocytes and peripheral blood mononuclear cells from WAS patients (27). Surprisingly, we did not observed any decrease in GM1 levels in activated WASP-deficient B lymphocytes. These results may explain the somewhat milder defects observed in activation of B lymphocytes from WASP-deficient mice.

Even though microvilli are observed in different hematopoietic cells, their functional role in B cell biology remains to be elucidated. Leukocytes from WAS patients are defective in microvilli formation (41). We also found that B lymphocytes from WASP-deficient mice have decreased amounts of microvilli after activation with anti-CD40 and IL-4 (10). Considering that microvilli are grouping sites of key molecules required for antigen presentation, it is possible that such defects contribute to the abnormal splenic architecture of WAS patients (42) as well as immunodeficiency (43). B lymphocytes activated in the presence of IL-4 were reported to be rather poor antigen-presenting cells (44). Our studies (45) indicated that cells activated by LPS plus IL-4 were not different from LPS-activated cells in antigen-presenting capacity. Nevertheless, the fact that IL-4 induced recruitment of MHC class II molecules to lipid rafts opens the possibility that IL-4 may regulate antigen uptake and processing. Regulation of membrane plasticity and microvilli by IL-4 and CD40 are most likely important in B cell interactions with surrounding cells, and may be important for the germinal center reaction. We have previously found that expression of microvilli correlates well with increased homotypic B cell adhesion (9). Further studies will explore whether these changes in the membrane regulate interactions with other immune cells as well.


    Acknowledgements
 
This work was supported by grants from the Swedish Research Council, the Swedish Heart Lung Foundation, the King Gustaf V 80th Birthday Fund, the Magnus Bergvall Foundation, the Åke Wiberg Foundation, Karolinska Institutet, the Council for Work Life Sciences, Asthma and Allergy Association’s Research Foundation, and the Vårdal Foundation for Health Care Sciences and Allergy Research.


    Abbreviations
 
ANCOVA—analysis of covariance

BSS—balanced salt solution

DRM—detergent-resistant membranes

LPS—lipopolysaccharide

MCD—ß-methyl-cyclodextrin

WAS—Wiskott–Aldrich syndrome

WASP—Wiskott–Aldrich syndrome protein


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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