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Blood, 1 December 2003, Vol. 102, No. 12, pp. 3890-3899. Prepublished online as a Blood First Edition Paper on August 7, 2003; DOI 10.1182/blood-2002-12-3807.
CHEMOKINES Chemokine stimulation of human peripheral blood T lymphocytes induces rapid dephosphorylation of ERM proteins, which facilitates loss of microvilli and polarizationFrom the National Cancer Institute, Experimental Immunology Branch, Bethesda, MD; SAIC-Frederick, Image Analysis Laboratory, Frederick, MD; National Institute of Dental and Craniofacial Research, Craniofacial Developmental Biology and Regeneration Branch, Bethesda, MD; University of Minnesota Medical School, Department of Genetics, Cell Biology, and Development, Minneapolis; and Institut COCHIN, Departement de Biologie Cellulaire, INSERM U567/CNRS UMR 8104, Paris, France.
Lymphocyte microvilli mediate initial rolling-adhesion along endothelium but are lost during transmigration from circulation to tissue. However, the mechanism for resorption of lymphocyte microvilli remains unexplored. We show that chemokine stimulation of human peripheral blood T (PBT) cells is sufficient to induce rapid resorption of microvilli. Microvilli in other cells are regulated by ezrin/radixin/moesin (ERM) proteins, which link the plasma membrane to the cortical F-actin cytoskeleton; maintenance of these linkages requires ERM activation, reflected by phosphorylation at a specific carboxy-terminal threonine residue. Carboxyphosphorylated-ERM (cpERM) proteins in resting PBT cells show a punctate peripheral distribution consistent with localization to microvilli. cpERM dephosphorylation begins within seconds of stimulation by chemokines (stromal derived factor 1  [SDF-1] or secondary lymphoid tissue cytokine), and ERM proteins lose their punctate distribution with kinetics paralleling the loss of microvilli. The cpERM proteins are preferentially associated with the cytoskeleton at rest and this association is lost with chemokine-induced dephosphorylation. Transfection studies show that a dominant-negative ERM construct destroys microvilli, whereas a construct mimicking cpERM facilitates formation of microvilli, retards chemokine-induced loss of microvilli, and markedly impairs chemokine-induced polarization. Thus, chemokine induces rapid dephosphorylation and inactivation of cpERM, which may in turn facilitate 2 aspects of cytoskeletal reorganization involved in lymphocyte recruitment: loss of microvilli and polarization.
The microvilli of circulating lymphocytes play an important role in the specialized adhesion cascade1-6 that mediates emigration from circulation to tissue during normal immunosurveillance and in response to inflammation. The initial contact and rolling of lymphocytes along endothelial cells uses adhesion receptors that are selectively concentrated at the tips of microvilli, such as L-selectin and very late activation antigen 4 (VLA-4).7-10 Rolling lymphocytes can be triggered by chemokines to undergo an abrupt transition to strong, integrin-mediated adhesion,1-6,11 ultimately leading to transmigration across the endothelium. In contrast to L-selectin, the integrin leukocyte function-associated antigen 1 (LFA-1; L 2), which often mediates strong adhesion, is confined to nonmicrovillar regions of the membrane.7,9 Such localization of receptors excludes them from initial leukocyte-endothelial interactions.10 Consequently, it has been suggested that microvillus retraction might be important during strong adhesion to allow the cell body of the lymphocyte to contact the endothelium.12 Lymphocyte adhesion to and migration across endothelial cell surfaces in vivo has in fact been shown to involve a loss of microvilli.13,14 The elimination of microvilli is thus expected to increase the effectiveness of activated integrins in mediating lymphocyte adhesion and to facilitate the extended approximation of T-cell/endothelial cell membranes observed during transmigration. Microvilli are regulated by the widely expressed proteins of the ezrin-radixin-moesin (ERM) family,15-18 of which moesin is particularly important in formation of microvilli in varied cell types, including hematopoietic cells.19 ERM proteins concentrate within peripheral processes and regulate their shape by forming reversible links between the plasma membrane and the cortical cytoskeleton (for reviews, see Gautreau et al,15 Bretscher et al,17 and Tsukita and Yonemura20). These links are mediated by the interaction of the ERM C-terminus with F-actin and of its N-terminus (called the FERM domain) with lipid- and membrane-associated proteins.21-26 The majority of cellular ERM proteins exist in a functionally dormant state in which intramolecular association between FERM and the C-terminus masks their binding sites for cytoskeleton and membrane.15,17,20 ERMs become activated by molecular events such as phosphoinositide binding and phosphorylation that decrease inhibitory self-interactions and thereby unmask actin- and membrane-binding sites.21-26 In vitro, phosphorylation of a carboxy-terminal threonine residue conserved among ezrin, radixin, and moesin disrupts intramolecular interactions and exposes the C-terminal actin-binding site.21,23,26 In vivo, carboxy-terminal phosphorylation is thought to maintain the active conformation of ERM proteins that have been "opened" by other factors, particularly by interactions of the lipid phosphatidylinositol 4,5-bisphosphate (PIP2) with the N-terminus.25,27 Accordingly, changes in the levels of carboxy-threonine phosphorylated ERM (cpERM) proteins have been shown to directly correlate with the remodeling of microvilli in many cell types.25,28,29
Because chemokines trigger conversion from rolling adhesion to strong adhesion, we examined whether chemokine stimulation also contributes to remodeling of the microvilli. Using freshly isolated normal human peripheral blood T (PBT) cells, we found that stromal-derived factor 1
Cells and reagents
PBT cells were isolated by leukapheresis of blood from healthy human volunteers, centrifugation of cells on a lymphocyte separation medium (ICN Biochemical, Aurora, OH) gradient, and immunomagnetic negative selection as previously described.30 The resulting PBT cells (> 95% purity) were suspended in Hanks balanced salt solution without phenol red containing 10 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) and 0.2% bovine serum albumin (BSA). Cells were either used immediately or held in suspension by rotation at 4°C for up to 24 hours. Cells were placed in polypropylene microcentrifuge tubes at 1 to 3 x 107/mL and warmed to 37°C on a rocking platform for at least 1 hour before use. The following antibody reagents were used for Western blots and immunofluorescence: monoclonal antibody (mAb) 297s (generously provided by Sa Tsukita, Kyoto University, Kyoto, Japan) whose reactivity includes moesin pT558, ezrin pT564, and radixin pT56724; phosphoezrin/radixin/moesin (ERM) antibody (Cell Signaling Technology, Beverly, MA); moesin-specific mAb 38/87 (Lab Vision, Fremont, CA); goat polyclonal antiezrin (C-19) and antimoesin (C-15), which react/cross-react with ezrin, moesin, and radixin (Santa Cruz Biotechnology, Santa Cruz, CA); the ezrin-specific mAb clone 18 and the antimoesin mAb clone 38, which recognizes ezrin and moesin, respectively (Transduction Labs, San Jose, CA); antihemagglutinin (HA)–fluorescein isothiocyanate (FITC) "high affinity" (Roche, Indianapolis, IN); and rabbit antiphospho-p44/42 mitogen-activated protein (MAP) kinase (Cell Signaling Technology). Preadsorbed secondary antibodies conjugated with FITC, rhodamine, Cy5, and horseradish peroxidase were obtained from Jackson ImmunoResearch (West Grove, PA). Other reagents included poly-L-lysine (PLL), rhodamine-conjugated phalloidin (Sigma-Aldrich, St Louis, MO); calyculin A, staurosporine, and pertussis toxin (Calbiochem, San Diego, CA); and recombinant human SDF-1 Cell lysates and Western blots
PBT whole cell lysates were generated by brief centrifugation of cell suspensions, aspiration of supernatants, and direct lysis of pellets in 2 x reducing sample buffer. Samples were vortexed to disrupt cell pellets, boiled for 3 minutes, and homogenized by sonication. Equal sample volumes were resolved by 8% or 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by Western blot using enhanced chemiluminescence (ECL; Amersham, Piscataway, NJ). PBT cells were fractionated into soluble and insoluble/cytoskeletal pools using a modification of a previously described extraction procedure using the cationic detergent dodecyl-trimethyl ammonium chloride (DOTMAC; ICN Biochemical), which preserves actin association of cpERM proteins.22 Following treatment with SDF-1 Immunofluorescence and microscopy
Immunofluorescent staining of lymphocytes with mAb 297s was performed using the trichloroacetic acid (TCA; ICN) fixation method described previously.31 PBT cell suspensions at 37°C were aliquoted at 5 x 106 cells/well into prewarmed 24-well tissue-culture plates containing 12-mm round, no. 1 glass coverslips coated with 10 µg/mL PLL. Cells were allowed to settle for 1 minute at 37°C and were then stimulated by adding SDF-1
Flow cytometry
PBT cells were stimulated in suspension with 100 ng/mL SDF-1 Scanning electron microscopy
PBT cells were analyzed either by field emission scanning electron microscopy (SEM; Figure 1) or conventional SEM (Figures 7, 8). For field emission SEM, PBT cells were stimulated with SDF-1
Transfection with moesin expression vectors
Expression vectors (pEF-BOS-HA) containing cDNAs encoding N-terminally truncated wild-type and mutant mouse moesins (moesin
SDF-1 induces rapid resorption of PBT cell microvilli
Resting PBT cells exhibit heterogeneity of shape and surface features, but the majority of cells are spherical and have abundant microvilli of varying lengths and densities (Figure 1A). Within 1 to 2 minutes of SDF-1
Because the breakdown of microvilli in several cell types correlates with the inactivation of ERM cross-linking activity,25,28,29 we examined whether the loss of PBT cell microvilli in response to SDF-1
Many intracellular events triggered by SDF-1
To examine the kinetics of ERM dephosphorylation in more detail, we adapted cpERM staining methods to allow analysis by flow cytometry. Single-cell analysis shows that the dephosphorylation of cpERM is evident in a significant proportion of cells as early as 5 seconds after SDF-1
Calyculin A inhibits SDF-1 Investigations in other cell types have identified kinase and phosphatase inhibitors that perturb ERM phosphorylation.21,42 Our findings confirm that such agents are also effective and informative in PBT cells (Figure 4). Brief treatment with the kinase inhibitor staurosporine reduces cpERM to undetectable levels within 30 seconds (Figure 4A). Conversely, treatment with the serine/threonine phosphatase inhibitor calyculin A increases carboxy-threonine phosphorylation of moesin and ezrin; at the concentrations used in these studies, calyculin A effects are slower than staurosporine effects, becoming evident within 1 to 5 minutes. Thus, basal cpERM phosphorylation in lymphocytes reflects a dynamic kinase/phosphatase balance.
We extended the analysis of phosphatase inhibitor by investigating whether calyculin A is also able to inhibit SDF-1
SDF-1
Recent studies22,25,42 suggest that the activation of ERM proteins is primarily driven by N-terminal interactions with polyphosphoinositides such as PIP2. Carboxy-threonine phosphorylation may not be necessary for ERM activation, but instead may serve to stabilize ERM proteins in their active conformation. Carboxy-threonine phosphorylation is, however, indicative of active ERM proteins, and dephosphorylation correlates with loss of ERM cross-linking function and the breakdown of ERM-stabilized surface features such as microvilli.28,29 We therefore examined the cytoskeletal association of lymphocyte ERM before and after stimulation with SDF-1
The spatial distribution of ERM was examined in PBT cells before and after chemokine stimulation using confocal microscopy. In resting PBT cells, cpERM proteins exhibit a fine punctate cortical distribution (Figure 6). Such punctate cortical staining is characteristic of proteins found in microvilli. In contrast, staining with conventional anti-ERM antibodies in resting cells shows a combination of diffuse cortical staining, punctate cortical staining, and cytoplasmic localization. There is negligible signal in the nucleus, which occupies the majority of intracellular space in resting PBT cells. The cpERM staining is rapidly and dramatically decreased following SDF-1 Kinase and phosphatase inhibitors influence microvilli in a manner predicted by their effects on ERM phosphorylation
The foregoing findings are consistent with a model in which SDF-1 Transfection studies demonstrate the importance of ERM for PBT microvilli PBT cells were transfected with a construct containing the N-terminal domain of ezrin (FERM) but lacking the C-terminal actin-binding domain. This FERM construct has been used as a dominant-negative in various model systems, and it most likely blocks ERM functions by occupying binding sites on transmembrane proteins without coupling them to actin16 and efficiently competing with endogenous active ERM. The absence of the C-terminus eliminates auto-inhibition, and therefore bypasses the need for C-terminal phosphorylation to form stable membrane interactions. Expression of the FERM construct markedly changes both the surface features and the F-actin distribution of PBT cells (Figure 8). The punctate F-actin staining characteristic of cells with fine peripheral processes such as microvilli and filopodia is consistently observed in untransfected PBT cells as well as in PBT cells transfected with control constructs (Figure 8E), but is absent in FERM-transfected cells (Figure 8B), suggesting that expression of FERM domain disrupts peripheral processes in PBT cells. SEM analysis of FERM-transfected PBT cells reveals a unique cellular phenotype—intact spherical cells devoid of peripheral processes (Figure 8C). This phenotype is observed in 15% to 30% of cells "nucleoporated" in the presence of FERM DNA but has not been observed in cells "nucleoporated" with control constructs (Figure 8F). These results are consistent with previous studies in which suppression of ERM expression in thymoma cells through the introduction of antisense probes resulted in the disruption of microvilli.19 Thus, suppression of peripheral processes is the distinctive outcome of transfection with FERM, consistent with the involvement of ERM proteins in lymphocyte peripheral processes including microvilli. Role of cpERM phosphorylation in formation and dephosphorylation in chemokine-induced loss of PBT microvilli To directly address the importance of ERM carboxy-terminal threonine phosphorylation to PBT microvilli, cells were transfected with point-substitution mutant constructs of moesin in which residue T558 was replaced by either aspartic acid (T558D) to mimic phosphorylated moesin or alanine (T558A) to mimic dephosphorylated moesin. By fluorescence microscopy, a distinctive phenotype is seen in cells transfected with the T558D construct; of those T558D-transfected cells, 20% to 30% (depending on the experiment) have long peripheral processes rich in actin and moesin-T558D (Figure 9D-E). SEM images confirm the presence of long, coarse microvilli on such cells (Figure 9F). Such processes were not observed in moesin-T558A–transfected PBT cells (Figure 9A-C). The ability of T558D but not T558A to augment microvilli indicates the critical role of phosphorylation at T558 in their establishment/maintenance in resting PBT cells.
The microvilli of moesin-T558D–transfected PBT cells were also analyzed by SEM following SDF-1 stimulation. Cells fully polarized after 1 to 2 minutes of chemokine stimulation do not have the coarse microvilli characteristic of unstimulated moesin-T558D–transfected cells (data not shown). Thus, moesin-T558D does not prevent polarization or microvillus breakdown in response to chemokine stimulation. However, evidence for delayed loss of microvilli is observed in round moesin-T558D–transfected cells 30 to 60 seconds after chemokine stimulation. At those times, PBT cells are often not distinctly polarized, but the initiation of a lamellipodium is evident as the formation of one or more large ruffles on the cell surface. During this early phase of polarization, the moesin-T558A–transfected cells have largely lost their normal microvilli (Figure 10). In contrast, among moesin-T558D transfectants, many cells responding to SDF-1
Analysis of the samples by fluorescence microscopy demonstrates a substantial inhibition of polarization by T558D transfection. In samples transfected with moesin-T558D, a 2- to 4-fold decrease in the fraction of cells fully polarized by 60 seconds of SDF-1
Lymphocyte adhesion to endothelium is a multistep process in which chemokines play the critical role of inducing strong adhesion by activating integrins. Although disappearance of microvilli is a predictable element in the adhesion cascade,13,14 the molecular basis is not known. Using freshly purified human PBT cells, we demonstrate that stimulation with a physiologically relevant chemokine alone is sufficient to induce the rapid loss of lymphocyte microvilli. This is a novel function of chemokines that is distinct from their role as integrin activators; we propose that these 2 chemokine functions cooperate in the adhesion cascade. Furthermore, our studies identify a key molecular process contributing to this loss of microvilli, the dephosphorylation of ERM proteins. Moreover, our studies indicate that chemokine-induced dephosphorylation of moesin plays another important role in the cascade, facilitation of lymphocyte polarization. It has recently been shown that stimulation of resting neutrophils with 2 chemotactic and chemokinetic cytokines induces ERM dephosphorylation.43 Although the central focus of that report was on the role of Rho kinase in regulating ERM phosphorylation (and uropod retraction), Yoshinaga-Ohara et al's finding that formyl-methionine-leucine-phenylalanine (FMLP) or granulocyte-macrophage colony-stimulating factor (GM-CSF) rapidly induces ERM dephosphorylation in neutrophils parallels our findings that chemokines induce rapid dephosphorylation of lymphocyte ERM protein. The combination of our findings and those of Yoshinaga-Ohara et al suggests that ERM dephosphorylation is a molecular pathway that is commonly used during hematopoietic cell polarization in response to diverse chemotactic/chemokinetic agents. Discussion hereafter will focus on 3 issues: (1) the evidence that dephosphorylation of ERM per se is a contributor to the loss of microvilli and polarization in response to chemokine, rather than simply being associated with it; (2) the molecular mechanisms regulating ERM dephosphorylation; and (3) the potential functional relevance of the loss of microvilli. Dephosphorylation of cpERM per se is a contributor to loss of microvilli Our findings indicate that ERM proteins are involved in formation of lymphocyte microvilli and specifically that dephosphorylation of cpERM proteins is causally related to their loss in PBT cells. The general importance of ERM to lymphocyte microvilli is clearly demonstrated by transfection studies with the dominant-negative FERM construct of ezrin, which results in ablation of lymphocyte surface processes (Figure 8). Because of the strong sequence conservation between the FERM domains of ezrin and moesin (94% amino acid similarity) and the overlap in the molecules to which they bind, the FERM domain from ezrin is believed to preempt binding sites normally used by both moesin and ezrin proteins, thereby preventing ERM proteins from establishing normal linkage between cytoskeleton and membrane proteins including L-selectin, CD43, and CD50. Such membrane anchorage for the cortical cytoskeleton is understood to be critical for organization of peripheral processes. Our finding is consistent with results of transfection of FERM in kidney epithelial cells16 and with an extensive literature documenting the importance of ERM in formation of microvilli in other cell types,15,17,20 including the loss of microvilli in mouse thymoma cells following exposure to ERM-antisense oligonucleotides.19 Moreover, it is the presence of carboxy-terminal phosphorylation of ERM that is critical to peripheral processes in lymphocytes. Multiple lines of evidence indicate a causal relationship between ERM phosphorylation and peripheral processes. First, direct demonstration is provided by the transfection studies with mutant ERM constructs that either mimic the phosphorylated form (eg, moesin-T558D) or the unphosphorylated form (eg, moesin-T558A). We find that moesin-T558D promotes long peripheral processes, but moesin-T558A does not (Figure 9); thus phosphorylated and nonphosphorylated forms differ markedly in their contribution to peripheral processes. These findings in lymphocytes are concordant with findings in epithelial cells and fibroblasts.15,35,44 Second, studies with pharmacologic agents provide supporting evidence. Staurosporine rapidly induces dephosphorylation of ERM proteins and associated loss of microvilli in lymphocytes (Figure 7); this confirms and extends analogous findings in L-cell fibroblasts.42 Conversely, calyculin A inhibits the dephosphorylation of cpERM (Figure 4) and prevents loss of peripheral processes in response to chemokine (Figure 7). Third, there is good correspondence between the rapid kinetics of ERM dephosphorylation and the disappearance of microvilli. Fourth, we demonstrate that chemokine stimulation induces both ERM dephosphorylation (Figure 2) and loss of ERM cytoskeletal association (Figure 5), which is central to their participation in microvilli.21,23,26 Fifth, overexpression of a moesin construct that mimics cpERM (but not a construct that mimics nonphospho ERM) retards loss of microvilli (Figure 10). In short, direct and indirect evidence converge to establish a causal relationship between ERM dephosphorylation and loss of microvilli in lymphocytes. Although the presence of cpERM favors microvilli, current models suggest that cpERM alone is not sufficient for retention of microvilli.42 This is also evident in our results that microvillus loss, although delayed, does still occur in moesin-T558D–transfected PBT cells. A requirement for elements other than cpERM for the maintenance of microvilli is plausible, given the combinatorial requirements for complex molecular processes. One element likely to cooperate with cpERM in control of lymphocyte microvilli is phosphoinositide turnover; phosphoinositides interacting with the N-terminal FERM domain of ERM proteins are thought to collaborate with C-terminal phosphorylation in ERM activation.42 Moreover, chemokine responses in T lymphocytes involves acute regulation of phosphoinositides.45 Thus, in chemokine-stimulated PBT cells, phosphoinositide hydrolysis may be a key regulator of ERM inactivation and the loss of microvilli, but the concurrent dephosphorylation of ERM proteins may serve to increase the efficiency and rapidity of this important morphologic transition. Molecular mechanisms involved in regulation of cpERM phosphorylation Our studies demonstrate that the steady-state level of phosphorylated ERM in resting lymphocytes is subject to a dynamic kinase/phosphatase balance (Figure 4), as has been shown previously in fibroblasts and platelets.21,42 Analysis of the effects of kinase and phosphatase inhibitors suggests that, among the cell types studied, there are fundamental similarities in the regulators of ERM phosphorylation. Calyculin A, an inhibitor of type I serine/threonine phosphatases, augments cpERM phosphorylation in both platelets and epithelial cells. Staurosporine, an inhibitor of certain serine/threonine kinase of the AGC subfamily, tips the balance in the opposite direction, resulting in decreased cpERM phosphorylation in those cell types. Cellular responses to certain physiologic stimuli induce rapid changes in cpERM phosphorylation reminiscent of the effects of staurosporine or calyculin A. Rapid phosphorylation of cpERM is induced in platelets by treatment with thrombin21 and in serum-starved fibroblasts by treatment with epidermal growth factor (EGF).44 Rapid dephosphorylation has been observed in T lymphocytes in response to stimulation via the antigen-specific </ | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Abstract
Introduction
N) were generously provided by Dr Kozo Kaibuchi (Nagoya University Graduate School of Medicine, Showa, Nagoya, Aichi, Japan). The substitution of alanine or aspartic acid for T558 by site-directed mutagenesis, producing moesin 
), or with moesin constructs that mimic the unphosphorylated form (moesin-T558A-HA; 
), or the phosphorylated form (moesin-T558D-HA, 
). Cells were fixed before stimulation or at varying intervals after stimulation with SDF-1