Published online before print November 21, 2003
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Department of Molecular Biomedicine, Centro de Investigación y Estudios Avanzados, I.P.N., México
1 Correspondence: Departamento de Biomedicina Molecular, CINVESTAV-IPN, Av. IPN #2508, Col. Zacatenco, Apartado Postal 14-740, CP 07360, México, D.F., México. E-mail: lesantos{at}mail.cinvestav.mx
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Key Words: cytoskeleton • hyaluronic acid • microtubule-organizing center • motility
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CD44 is a polymorphic group of class I transmembrane glycoproteins (80–200 kDa) [4 5 6 ], and one of its ligands is hyaluronic acid (HA), a component of extracellular matrix (ECM) [7 ]. CD44 participates in many cellular processes, which include regulation of growth, survival, differentiation and motility of normal cells, and tumoral cell migration and invasion [8 9 10 11 12 13 ]. CD44 null mice have mild abnormalities in myeloid-progenitor migration and bone marrow colonization [14 ] and in the homing of lymphocytes to lymph nodes or the thymus [15 ]. In agreement with a possible role in cellular migration, CD44 is located in the leading edges and lamellipodia of several cell types in vitro. Moreover, CD44 interacts with the actin cytoskeleton through the association with proteins such as ankyrin [16 ] and the ezrin, radixin, moesin family [17 18 19 ], although the significance of these interactions is not entirely understood.
Cross-linking CD44 with monoclonal antibodies (mAb), which recognize different epitopes, including HA domain, induced spreading (characterized by the formation of dendrites and lamellipodia) of preactivated B lymphocytes [20 21 22 23 24 ]. Preactivation with any of the following stimuli, such as lipopolysaccharide (LPS), anti-µ antibodies, anti-CD40 antibodies, or anti-CD38 antibodies plus interleukin-4 (IL-4), prepares the cells for spreading on culture plates precoated with anti-CD44 antibodies [20 21 22 23 24 ]. The spreading of a T cell line by cross-linking CD44 has also been reported [25 ]. These phenomena are dependent on an increase of intracellular calcium [21 ], activation of protein kinase C [21 ], signal transducer and activator of transcription 6 (STAT-6) [23 ], Cdc42, Rac1, Wiskott-Aldrich syndrome protein [24 , 25 ], and actin polymerization [21 , 25 ]. In addition, it has been observed that during T cell spreading, CD44 is colocalized with F-actin in the lamellipodia protusions [25 ].
Conversely, it is known that in response to chemoattractant gradients, motile cells (such as neutrophils and T cells) adopt a polarized morphology, with distinctive leading and trailing edges oriented with respect to the gradient. Actin preferentially accumulates at the leading edge [26 27 28 29 30 ], whereas the microtubules are in the trailing edge (uropod) [31 32 33 ]. Additionally, microtubules and intermediate filaments reorganization has been observed during migration of T lymphocytes [1 ]. However, knowledge of how the components of the cytoskeleton participate in the morphological changes of B lymphocyte upon spreading is very scarce.
The present study was mainly focused on the participation of actin, microtubules, and vimentin in spreading of activated B lymphocytes induced by anti-CD44 antibodies. The results obtained in this work strongly suggest that the generation of dendrites is dependent on the coordinated polymerization of actin, tubulin, and vimentin.
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Medium
RPMI 1640 (Gibco, Grand Island, NY) was supplemented with 1% nonessential amino acids (Gibco), 5 x 10-5 M 2-mercaptoethanol, 1 mM sodium pyruvate, and 2 mM glutamine (all from Sigma Chemical Co., St. Louis, MO), and 10% (v/v) fetal calf serum (FCS; Gibco).
B lymphocyte isolation and activation
Mononuclear cells were isolated from spleen by Ficoll-density gradient separation, and B cells were enriched, eliminating T cell by panning using plastic Petri dishes coated with anti-Thy-1 mAb ascites (NIM-R1) [34
]. For activation (priming), 2 x 106 purified B cells were incubated in 1 ml medium containing LPS from Escherichia coli serotype O55:B5 at 50 µg/ml (Sigma Chemical Co.) plus 10 U/ml murine IL-4 (Genzyme, Cambridge, MA). Cells were incubated for 18–24 h at 37°C in a 24-well plate.
Assay of dendrite formation ("spreading")
Glass slides or polystyrene-96 plates were coated with 20 µg/ml in phosphate-buffered saline (PBS) of NIM-R8 [rat immunoglobulin G (IgG)2a anti-mouse CD44; ref. 20
] or rat IgG (isotype control) for 1 h at 37°C. The plates were "blocked" with PBS–10% FCS (v/v) for 1 h at 37°C and then washed extensively with medium before use. Activated B cells were transferred without washing to the precoated glass slides or plates and were incubated for 1–2 h at 37°C. Afterwards, they were observed with an inverted microscope at 10x or 40x magnification. For each experiment, 200 cells in each well were counted. The percentage was obtained comparing dendrite-forming cells (DFC) obtained in control wells (100% DFC) with DFC obtained for each condition.
Cells presenting thin, long membrane extensions (usually 2x or 3x the size of their spherical body) were quantified as DFC. In contrast, cells with spherical or pleomorfic morphology but without extension as described above were not considered DFC.
Inhibition of dendrite formation
For these experiments, the cells were divided in three groups: For the first group, the following drugs were added after priming and 30 min before the dendrite formation assay: colchicine and nocodazole (microtubule polymerization inhibitors) and cytochalasin E (actin polymerization inhibitor; all from Sigma Chemical Co.). Dimethyl sulfoxide (DMSO) at 0.05% (Sigma Chemical Co.; representing the highest concentration of the solvent used during any experiment) was used as a toxicity control. Cells with and without treatment were transferred to anti-CD44 antibodies (NIM-R8) and were incubated for 1 h more to induce dendrite formation. Finally, the percentage of DFC was calculated. For each experiment, 200 cells in each well were counted. The percentage was obtained comparing DFC obtained in control wells (DMSO and without treatment; 100%) with DFC obtained for each condition, where no differences were found between DMSO-treated or cells without treatment. In parallel wells, cell viability was also evaluated in each experiment by trypan blue exclusion.
For the second group, cells with dendritic morphology (activated B lymphocytes that were incubated for 1 h with insolubilized anti-CD44 mAb, as described above) were treated with cytochalasin E or DMSO to revert the morphology. After incubation (0–60 min), the cells were fixed and stained for F-actin and ß-tubulin and were then observed under confocal microscopy.
For the third group, cytochalasin E or DMSO was added at the beginning of the spreading (10–60 min), and all of the cultures were further incubated to complete 2 h.
Finally, the cells were fixed and stained for F-actin and ß-tubulin and were then observed under confocal microscopy.
Actin and microtubule localization
Cells in spreading and control cells were washed with PBS, fixed with 3.7% paraformaldehyde in PBS for 10 min at room temperature, permeabilized with 0.05% Triton X-100 (Sigma Chemical Co.) in PBS at room temperature for 10 min, and then extensively washed with PBS. For staining, 40 µl mouse anti-ß-tubulin antibody (Sigma Chemical Co.), diluted 1:40, or a similar amount of mouse IgG (isotype control) was added to each sample and incubated for 30 min at room temperature. After incubation, cells were washed three times with PBS and incubated for 20 min in the dark with 40 µl fluorescein isothiocyanate (FITC)-anti-mouse IgG (Cappel, West Chester, PA), diluted 1:100. Cells were then counterstained with 40 µl rhodamine-phalloidin (Molecular Probes, Eugene, OR), diluted 1:100, and were incubated in the dark for 15 min at room temperature. Finally, cells were washed with PBS, dried, mounted in Vecta-Shield (Vector Laboratories, Burlingame, CA), and observed by epifluorescence or confocal microscopy.
Intermediate filament localization
Cells in spreading and control cells were washed with PBS, fixed for 5 min at room temperature in 2% paraformaldehyde in PBS, permeabilized with 0.05% Nonidet-P40 (Sigma Chemical Co.) in PBS at room temperature for 5 min, and then extensively washed with PBS. Cells were then incubated with 40 µl goat anti-mouse vimentin (ICN Pharmaceuticals, Costa Mesa, CA) antibody, diluted 1:20, or goat IgG (isotype control) for 2 h at room temperature. After incubation, cells were washed three times with PBS and stained with FITC-anti-goat IgG (Cappel), diluted 1:300, for 30 min in the dark. Finally, cells were washed again with PBS, dried and mounted in Vecta-Shield (Vector Laboratories), and observed by confocal microscopy.
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Figure 1. Coordinated reorganization of actin and microtubules during dendrite formation in CD44-dependent B lymphocyte spreading. Splenic B lymphocytes stimulated overnight with LPS plus IL-4 were transferred to glass slides precoated with monoclonal anti-CD44 antibodies (NIM-R8) or rat IgG (as isotype control), and they were incubated for 1 h at 37°C to induce dendritic processes. Control cells (A) or DFC (B-D) were fixed with paraformaldehyde, permeabilized with Triton X-100, indirectly stained with anti-ß-tubulin antibodies (B) or mouse IgG as isotype control (C), and observed by confocal microscopy (100x original magnification; D is a Nomarski photograph). (E) DFC were fixed at different time points, as stated in each panel, and stained with rhodamine-phalloidin (left panels) and indirectly stained with anti-ß-tubulin antibodies (right panels). Cells were observed by fluorescence microscopy at x100 original magnification. Arrows show the F-actin accumulated in the lamellipodias. (F) Inhibitory drugs (nocodazole and colchicine) were independently added after priming and 30 min before the dendrite assay. Cells were transferred to NIM-R8 and were further incubated for 1 h. Finally, percentage of DFC was evaluated, and cells presenting thin, long membrane extensions (usually 2x or 3x the size of their spherical body) were quantified as DFC. In contrast, cells with spherical or pleomorfic morphology but without extension were considered negative. These experiments were done by triplicate, and the results shown in the graphs represent three independent assays. For each experiment, 200 cells in each well were counted. The percentage was obtained comparing DFC obtained in control wells (DMSO and without treatment; 100%) with DFC obtained for each condition, where no differences between DMSO-treated or cells without treatment were found. Cell viability, evaluated by trypan blue exclusion, was always >95% at the end of each experiment.
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As a result of these findings, the influence of the organization and integrity of microtubules on actin cytoskeleton, and vice versa during B cell spreading, were analyzed. Therefore, the cells were pretreated with colchicine (tubulin polymerization inhibitor) or cytochalasin E (actin polymerization inhibitor) 30 min before spreading assay, as described previously. Then, the cells were stained with rhodamine-phalloidin and anti-ß-tubulin antibodies, and finally, they were analyzed by confocal microscopy. Figure 2A (top panels) shows that control cells treated with vehicle, F-actin, and microtubules were distributed, as it was observed in previous experiments. When B lymphocytes were pretreated with colchicine, the cells were able to polymerize actin and began the spreading, but the formation of dendrites was not possible in the absence of intact microtubules (middle panels). Finally, when the cells were pretreated with cytochalasin E, they were unable to begin the spreading (Fig. 2A , bottom panels); however, they presented multiple filopodia with a clear staining for ß-tubulin. In addition, the cells accumulated F-actin around the nucleus, and the MTOC was displaced from the center. In control cells and cells treated with colchicine, F-actin was observed as a diffuse but strong staining in lamellipodias, suggesting a fast and active actin polymerization-depolymerization. These results suggest an important role of actin cytoskeleton in the definition of the B lymphocyte morphology, as it forms different cellular structures that may have diverse physiological roles in the cell.
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Figure 2. Actin cytoskeleton determinates the stability of the dendrites. (A) B lymphocytes stimulated overnight with IL-4 plus LPS were treated with the vehicle (DMSO; top panels) or the inhibitory drugs colchicine (middle panels) or cytochalasin E (bottom panels), 30 min before the dendrite assay. Cells were then transferred to glass slides precoated with anti-CD44 antibodies and incubated for 1 h at 37°C. DFC were fixed and stained with rhodamine-phalloidin (red) and indirectly stained with anti-ß-tubulin antibodies (green). (B) DFC were exposed (10–60 min) to the inhibitory drug cytochalasin E or the vehicle. After treatment, the cells were fixed and stained with rhodamine-phalloidin (left panels) and indirectly stained with anti-ß-tubulin antibodies (middle panels). Finally, the cells were observed by confocal microscopy at 100x original magnification.
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Intermediate filaments were reorganized in dendrite-forming B lymphocytes
Intermediate filaments are expressed in cell type-specific patterns. Besides, these molecules harbor binding sites for high molecular weight proteins interconnecting intermediate filaments with microtubules and microfilaments [1
]. T lymphocytes express filaments of vimentin that are reconfigured during the polarization of the cell [35
, 36
]. Thus, it was necessary to verify if intermediate filaments were reorganized during B cell spreading. Therefore, DFC or control cells were fixed, indirectly stained with anti-vimentin antibodies, and analyzed by confocal microscopy. It was observed that intermediate filaments of vimentin were distributed as a coarsely woven cage in round cells (Fig. 3A
). In contrast, filaments were redistributed to the dendrite (Fig. 3B) , with a distribution similar to the microtubules in DFC, as previously shown. These results suggest some interaction or coordination between microtubule bundles and intermediate filaments of vimentin during B cell spreading.
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Figure 3. Intermediate filaments are redistributed during CD44-induced spreading of activated B lymphocytes. Splenic B lymphocytes stimulated overnight with IL-4 plus LPS were transferred to glass slides or tissue-culture plates precoated with anti-CD44 mAb (NIM-R8) or rat IgG and were incubated for 1 h at 37°C to induce spreading. Control B cells (A) or dendrite-forming B cells (B and C) were fixed, permeabilized, indirectly stained with rabbit anti-vimentin antibodies (A and B) or rabbit IgG (C, isotype control), and observed by confocal microscopy at 100x original magnification.
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Dendrite formation and B cell spreading are also observed by cross-linking major histocompatibility complex class II (MHC-II), CD45R, CD23, lymphocyte function-associated antigen-1 (LFA-1), very late antigen-4 (VLA4), and intercellular adhesion molecule-1 (ICAM-1) [21 , 22 ]. However, cross-linking molecules such as CD22, CD38, CD40, or B cell receptor are unable to induce spreading of activated B lymphocytes [20 ].
In this study, evidences are being provided that dendrite formation and CD44-induced B cell spreading were also dependent on the coordinated rearrangements of actin, microtubules, and intermediate filaments. It was found that B cell spreading began with F-actin accumulation in a zone of the membrane, forming a lamellipodia that later was elongated by reorganization of microtubules and intermediate filaments in this growing zone. Thus, the dendrite was characterized by accumulation of microtubule bundles. It is interesting that the MTOC was observed in the growing base of the dendrites.
Migratory stimuli such as the chemokines stromal cell-derived factor-1
(SDF-1), regulated on activation, normal T expressed and secreted (RANTES), macrophage-inflammatory protein-1 (MIP-1), MIP-1ß, or monocyte chemoattractant protein-1 (MCP-1) [38
39
40
] or the recognition of immobilized, recombinant ICAM-1 [32
, 33
] induce morphological modifications of T and B lymphocytes. During chemotaxis, the amount of cellular F-actin is increased, as well as the accumulation of F-actin at the leading edge of the migratory cell [41
]. It has also been observed that microtubules are organized in the trailing edge (uropod) [31
32
33
].
Conversely, when microtubules were disrupted, the percentage of DFC was deeply affected, suggesting that reorganization and active assembly of tubulin are necessary for dendrite formation. B cells treated with tubulin polymerization inhibitors were still able to begin spreading, as actin polymerization was not affected, showing accumulation in lamellipodias as seen in control cells. These results suggest that the enforced locomotion of B lymphocytes may be produced by actin polymerization only. Similar observations have been registered from fibroblasts lacking microtubules [42 ]. However, even when actin polymerization is used to push the membrane, structures such as dendrites are never formed in the absence of tubulin polymerization. In the uropod formation in migrating T cells, microtubules are apparently not necessary [31 ]. It is possible that the B cell spreading morphology corresponds to a migratory phenotype, representing migratory cells during extravasation or in the process of invasion (metastasis) for CD44-expressing tumoral cells.
When B cells were treated with cytochalasin E, they were unable to begin spreading and to form dendrites. Moreover, these cells showed long filopodia based in microtubules and accumulated F-actin around the nuclei. These results suggest that actin cytoskeleton integrity is extremely important to maintain the stability of the dendrites.
Conversely, intermediate filaments are important molecules, as they have binding sites for microtubules and microfilaments interconnecting them [1 ]. They also confer rigidity to circulating lymphocytes and control their deformability [35 , 36 ]. In this work, we showed that vimentin, an intermediate filament, was reconfigured during spreading and dendrite formation of activated B lymphocytes. The distribution of vimentin was similar to the microtubule bundles, and it was accumulated primarily close to the MTOC. These results suggest a coordinated reorganization of vimentin, which may be one of the intermediate filaments connecting microtubule bundles with other elements of the cytoskeleton.
In summary, this paper provides evidence of the concerted participation of actin, microtubules, and intermediate filaments, which act as a dynamic network favoring the morphological modifications of B lymphocytes.
The biological relevance of the modifications in the B lymphocyte cytoskeleton is not clear yet, but the plasticity in the morphology of the cells has been observed in vivo in intact lymph nodes by two-photons microscopy [43 44 45 46 ]. This plasticity may favor the interaction of B lymphocytes with other cells and/or with the ECM, regulating the activation and/or migration of activated B lymphocytes.
Received August 27, 2003; revised October 14, 2003; accepted October 15, 2003.
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J. Immunol. 161,6487-6495-induced lymphocyte polarization and chemotaxis J. Immunol. 163,4001-4012 triggers a chemotactic response and induces cell polarization in human B lymphocytes Eur. J. Immunol. 28,2197-2207[CrossRef][Medline]This article has been cited by other articles:
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