Article |
Address correspondence to Dr. Tatsuo Kinashi, Bayer-chair, Dept. of Molecular Immunology and Allergy, Graduate School of Medicine, Kyoto University, Yoshida-konoe, Sakyo-ku, Kyoto 606-8501, Japan. Tel.: 81-75-771-8159. Fax: 81-75-771-8184. E-mail: tkinashi{at}mfour.med.kyoto-u.ac.jp
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Abstract |
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Key Words: chemokines; LFA-1; ICAM-1; lymphocyte; transmigration
* Abbreviations used in this paper: HUVEC, human umbilical vascular endothelial cells; ICAM-1, intercellular adhesion molecule 1; LFA-1, lymphocyte functionassociated antigen 1; LN, lymph node; PTX, pertussis toxin; SDF-1, stromal-derived factor 1; SLC, secondary lymphoid tissue chemokine; VCAM-1, vascular cell adhesion molecule 1; VLA-4, very late antigen 4.
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Introduction |
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Chemokines signal through heptahelical receptors that activate pertussis toxin (PTX)*-sensitive Gi-type heterotrimeric G proteins. Gi proteinlinked receptors trigger a diversified cascade of second messengers (Thelen, 2001). Although PI3K is important in neutrophil chemotaxis, lymphocytes did not show a significant dependence on this mediator (Hirsch et al., 2000; Li et al., 2000; Sasaki et al., 2000). Inhibition of PI3K activity has little effect on lymphocyte homing in vivo (Constantin et al., 2000) or on transendothelial migration in laminar flow chambers (Cinamon et al., 2001). These findings imply that chemokine receptors can induce lymphocyte chemotaxis through PI3K-independent pathways.
The small GTPase Rap1 is a potent inside-out signal that functions in a distinct manner from PI3K and PKC, and increases the adhesive activity of both lymphocyte functionassociated antigen 1 (LFA-1) and very late antigen 4 (VLA-4; Katagiri et al., 2000; Reedquist et al., 2000). Rap1 activation occurs in response to a variety of external stimuli (Bos et al., 2001), including T cell receptor engagement (Katagiri et al., 2002), CD31 stimulation (Reedquist et al., 2000), and CD98 ligation (Suga et al., 2001). In this paper, we investigate the possibility that Rap1 is involved in lymphocyte adhesion and transmigration by chemokines. We show that rapid Rap1 activation by chemokines is required for adhesion and transmigration of lymphocytes through endothelium. Furthermore, Rap1 has the unique functions that stimulate robust cell migration and induce lymphocyte polarization. Our work suggests that Rap1 plays a pivotal role in lymphocyte trafficking.
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Results |
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Furthermore, we examined the effect of Spa1 inhibition of Rap1 on the arrest of rolling lymphocytes in under-flow adhesion assays using a mouse endothelial cell line, BC1 (Tatsuta et al., 1992). Pretreatment of BC1 with SDF-1 drastically increased firm adhesion of rolling lymphocytes to the BC1 monolayer (Fig. 2 C), as seen with human umbilical vascular endothelial cells (HUVEC; Cinamon et al., 2001). Firm attachment was significantly inhibited by treatment with antibodies against either LFA-1 or ICAM-1. Spa1 expression abrogated firm adhesion of rolling T cells. Conversely, expression of a constitutively activated Rap1 (Rap1V12) in T cells increased adhesion to immobilized ICAM-1 (see following paragraph) and induced T cell arrest on endothelial cells in the absence of SDF-1 (Fig. 2 C). However, neither Spa1 nor Rap1V12 expression in T cells affected rolling or tethering adhesion on BC1 monolayers (unpublished data). These results suggest that Rap1 activation is both necessary and sufficient for chemokine-induced T cell arrest on endothelial layers via the LFA-1ICAM-1 interaction.
Activation of Rap1 induced robust cell migration on immobilized ICAM-1
Next, we examined the effect of Rap1V12 on T cell adhesion and migration. Rap1V12 expression in primary T cells increased adhesion to ICAM-1 (Fig. 3 A), as previously seen for T cell clones (Katagiri et al., 2002) or lymphocytes derived from Rap1V12-transgenic mice (Sebzda et al., 2002). Surprisingly, Rap1V12 also stimulated robust cell migration on ICAM-1, comparable to that seen after SLC stimulation (Fig. 3 B). Rap1V12-expressing cells moved on ICAM-1 as fast as 25 µm/min (Videos 1 and 2, available at http://www.jcb.org/cgi/content/full/jcb.200301133/DC1). PMA treatment increased adhesion levels, but did not stimulate migration (Fig. 3, A and B), in contrast to the stimulatory effect of Rap1 on both adhesion and migration.
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Discussion |
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Integrin adhesiveness is regulated by ligand binding affinity and/or lateral mobility/clustering (avidity; van Kooyk and Figdor, 2000). Previous studies indicated that chemokines (SDF-1, SLC, and EBII-ligand chemokine) regulate LFA-1 adhesive activity by modulating avidity and affinity of LFA-1, and demonstrated the existence of a PI3K-independent pathway responsible for the attachment of lymphocytes to high density ICAM-1 molecules or high endothelial venules (Constantin et al., 2000). Rap1V12 up-regulates soluble ICAM-1Fc binding and a conformational epitope associated with the high affinity state of LFA-1 (Katagiri et al., 2000; Reedquist et al., 2000) and clustering (Sebzda et al., 2002). These findings support the notion that Rap1 plays an essential role in chemokine-induced lymphocyte adhesion. Although both Rap1 and PI3K are thought to function in chemokine-stimulated integrin activation, we did not find a significant effect of PI3K inhibitors on lymphocyte attachment and transmigration through endothelial layers in our experimental system. The relative contribution of these signaling pathways may vary according to the expression levels of Rap1 and PI3K
in leukocytes and the density of ICAM-1 on endothelial cells.
Our work demonstrates that Rap1 is the major inside-out signal for LFA-1 and VLA-4 by chemokines, thus playing a critical role in lymphocyte attachment to immobilized ICAM-1, VCAM-1, and endothelial cells under flow. Rap1 activation by chemokines occurs in seconds, which is followed by the integrin-triggering effect of Rap1 in a minute. Thus, the integrin-triggering effect of Rap1 occurs within a time window of chemokine-induced conversion of rolling lymphocytes to firm arrest. It has recently been shown that endothelial chemokines also trigger earlier VLA-4mediated capture by subsecond modulation of integrin avidity (Grabovsky et al., 2000). We showed the requisite involvement of Rap1 in lymphocyte arrest under the suboptimal shear stress (0.1 dyne/cm2). Our experimental system failed to support efficient lymphocyte rolling or tethering at a higher shear stress (15 dyne/cm2). This precludes us from examining whether Rap1 is also involved in extremely rapid integrin modulation. Therefore, it is still an open question of whether Rap1 could function as a subsecond integrin modulator in lymphocyte arrest. It is also possible that Rap1 activation by apical endothelial chemokines could convert transient lymphocyte attachment to shear-resistant adhesion necessary for the later progression to lymphocyte transendothelial migration.
The Rho family of small GTPases regulates cytoskeletal rearrangements underlying morphological transformations such as lamellipodia, filopodia, and focal adhesion (Hall, 1998). These signal transducers likely contribute to leukocyte adhesion and migration through such rearrangements, but little is known about their contributions to rapid integrin activation after chemokine stimulation. Rho was previously reported to be involved in IL-8induced adhesion of leukocytes via VLA-4 and Mac1 (Laudanna et al., 1996). However, it remains to be determined whether Rho modulates either integrin affinity or clustering. Rac1 was reported to regulate integrin-mediated spreading in T cells, resulting in the enhancement of cell adhesion without affecting integrin affinity (D'Souza-Schorey et al., 1998). In BAF/LFA-1 cells, Rac1V12, Cdc42V12, and RhoV14 expression stimulates membrane ruffling, dendrite extension, or cell rounding, respectively (unpublished data). However, these GTPases stimulated little or only mild adhesion to ICAM-1, which was previously shown to be dependent on PI3K activity (Katagiri et al., 2000). Recent work by McLeod and colleagues showed both Rap1 and Rap2 activation after SDF-1 stimulation of B cell lines and the expression of RapGAPII inhibited chemotaxis toward SDF-1 in Transwell assays using bare membranes (McLeod et al., 2002). It was not clear from this paper whether Rap1 and Rap2 are critically involved in integrin-mediated adhesion and migration induced by chemokines. Although this work could identify a stimulatory effect of Rap2 on chemotaxis, our work could not identify an effect of constitutively active Rap2 on LFA-1 activation and migration on immobilized ICAM-1. Rap2 likely acts in a manner unrelated to integrin-mediated adhesion and migration. Thus, our work indicates the unique ability of Rap1 to rapidly trigger LFA-1 activation on chemokine stimulation.
Leukocyte emigration from the bloodstream across the microvessel wall into tissues is an essential step in the inflammatory response and lymphocyte homing. This process is based on robust leukocyte cell motility. In addition to an effect on integrin triggering, our paper demonstrates a promigratory effect of Rap1 on lymphocytes. Rap1V12 expression stimulated lymphocyte motility on ICAM-1 and VCAM-1 at similar levels observed for chemokine stimulation. Cell migration requires the coordination of front adhesion and rear de-adhesion. Detachment of the cell rear of Rap1V12- or SDF-1stimulated migratory cells was impaired by mutation of the tyrosine-based endocytosis motif of the ß2 integrin subunit (Tohyama et al., 2003). This mutation did not affect LFA-1 adhesive activity, suggesting that detachment is regulated by endocytosis of LFA-1 rather than a switch between on and off states of integrin activity during migration. Therefore, firm attachment does not necessarily hamper cell migration. Our paper demonstrates that the activation of Rap1 induces both integrin-mediated cell adhesion as well as migration.
Chemokines critical for T cell homing such as SLC are distributed on the apical side of high endothelial venule (Stein et al., 2000; Warnock et al., 2000). Thus, it is unlikely that a chemokine gradient across the endothelium stimulates transmigration of T cells in vivo. Retention of chemokines on the apical side is instrumental both to prevent dilution of chemokines by the flowing blood and to restrict the site of lymphocyte emigration. Our work indicated that both soluble chemokines and Rap1V12 expression were able to trigger initial arrest and promote subsequent diapedesis. Thus, a chemokine gradient is not necessarily required for transendothelial migration, which is in agreement with other works (Cinamon et al., 2001; Cuvelier and Patel, 2001).
Our paper suggests that haptokinesis, rather than chemotaxis, is important for transmigration. We showed in this paper that Rap1V12 expression in lymphocytes induced robust cell motility and transmigration without exogenous chemokines. PTX treatment did not inhibit transmigration of Rap1V12-expressing lymphocytes, excluding the possibility that Rap1 enhances transmigration by promoting endogenous PTX-sensitive Gi machineries. The transmigration stimulated by soluble chemokines and Rap1V12 still required the application of shear stress, consistent with results observed for lymphocyte transmigration through chemokine-immobilized HUVEC layers (Cinamon et al., 2001). This result suggests that chemokine activation of Rap1 and shear activation of transmigration are two separable and probably sequential processes. Shear stress dependency of transmigration implies that shear-induced mechanotransduction in lymphocytes and/or endothelial cells is required in addition to Rap1 signaling. Alternatively, shear stress may induce cell deformation, helping to redirect free membrane protrusions at the leading edge to junctions in the endothelium.
Given the presence of chemokines on the apical side of the endothelium, it is feasible that attached lymphocytes are stimulated by chemokines to migrate over endothelial cells under the control of the Rap1 promigratory signal, as seen in our work. When migrating lymphocytes reach intercellular junctions, they begin diapedesis between tightly apposed endothelial cells. Although the detailed process of diapedesis is still unclear, several adhesion molecules of the immunoglobulin superfamily, including CD31, CD99 (Schenkel et al., 2002), and JAM-1 (Martin-Padura et al., 1998; Ostermann et al., 2002) that are concentrated at endothelial junctions, have been reported to play a critical role in leukocyte transmigration. Antibody cross-linking of CD31 and CD99 on leukocytes also activates LFA-1 and VLA-4, acting through Rap1 signaling in the case of CD31 (Hahn et al., 1997; Reedquist et al., 2000). Thus, it is conceivable that chemokines and junctional adhesion molecules sequentially activate Rap1, stimulating haptotactic migration through endothelial cell layers.
The acquisition of front-rear polarity is critical for cell migration. Chemokines induce lymphocyte polarization, associated with the development of both a leading edge and uropod. The polarized characteristics induced by chemokines and Rap1V12 are indistinguishable in terms of both morphology and cell surface receptor distribution, and appear to be inherent to lymphocytes because it occurs without the spatial cues such as adhesion or chemokine gradients. Many lines of evidence indicate that the Rho family of GTPases is involved in leukocyte chemotaxis through cytoskeletal remodeling and cell polarity (Sanchez-Madrid and del Pozo, 1999; Worthylake and Burridge, 2001). Constitutively activated Rac1, Cdc42, or Rho failed to stimulate cell polarization and migration in our system. This result is consistent with previous reports demonstrating their inhibitory effects on both CSF-1stimulated macrophage chemotactic migration (Allen et al., 1998) and lymphocyte polarization (del Pozo et al., 1999). As yet, no constitutively active Ras/Rho family GTPases, with the exception of Rap1, have been reported to stimulated lymphocyte polarization and migration, suggesting a unique function for Rap1 in lymphocyte polarization and migration. The relationship of Rap1 with cell polarity determination and migration was previously indicated from studies in yeast (Chant and Stowers, 1995) and flies (Asha et al., 1999). In budding yeast, Bud1 (closest homologue to Rap1) determines the bud site by recruiting polarity-determining factors, such as Cdc42 and Cdc24 (a guanine exchange factor for Cdc42; Gulli and Peter, 2001). However, Rap1 does not directly activate Cdc42 or Rac in lymphoid cells (unpublished data). There is little evidence indicating a pathway triggered by chemokines inducing the hierarchical activation of GTPases. Chemokines may activate Rap1 and Rho family GTPases in parallel. It is also conceivable that Rap1 may control spatial regulation of Rho family GTPases and regulatory proteins, as implied from studies in budding yeast.
Our paper demonstrates the critical contributions of Rap1 to integrin activation, enhancement of integrin-mediated migration, and cell polarization, resulting in chemokine-triggered lymphocyte transmigration. The unexpected relationship between integrin triggering and cell polarization and migration suggests that Rap1 governs not only integrins, but also lymphocyte migration machinery. The mechanism by which Rap1 accomplishes this task awaits further study. In particular, the identification of Rap1 effector molecules is crucial to clarify whether Rap1 controls these biological effects through single or multiple biochemical events, which will provide important information facilitating a better overall understanding of Rap1 function.
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Materials and methods |
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Pull-down assays
LN cells from Balb/c mice suspended at 5 x 107/ml in RPMI 1640 were stimulated with 100 nM mouse SLC or SDF-1 (R&D Systems) at 37°C for the indicated times, and were stopped by resuspending the cells in 1% Triton X-100 containing lysis buffer (Katagiri et al., 2002). Active GTP-bound Rap1 was measured using a GSTRalGDSRBD fusion protein as described previously (Franke et al., 1997).
Spa1 or Rap1V12 expression in lymphocytes via adenovirus
Spa1 and Rap1V12 were expressed in lymphocytes using adenoviruses produced according to the manufacturer's instruction (AdEasyTM adenoviral vector system; Stratagene). LN cells from transgenic mice carrying the adenovirus receptor expressed in T cells (Wan et al., 2000) were infected with recombinant adenoviruses, cultured in RPMI 1640 containing 10% FCS and 50 µM 2-ME with 20 µg/ml 2C11 for 2 h, and then with 5 U IL-2 for 36 h. Infection efficiencies were estimated by lymphocyte GFP expression levels with FACSCaliburTM (Becton Dickinson). Greater than 80% of the lymphocytes constantly expressed GFP.
Plasmids and transfection
BAF/LFA-1 cells were transfected by electroporation with pcDNA3 (Invitrogen) containing FLAG-Spa1, or T7-Rap1AV12 (Katagiri et al., 2000), Cdc42V12 (a gift from Dr. S. Hattori, University of Tokyo, Tokyo, Japan), and Rap2AV12 (a gift from Drs. H. Kitamura and M. Noda, Kyoto University, Kyoto, Japan). H-Ras, Rac, and Rho were described previously (Katagiri et al., 2000).
Adhesion to immobilized ICAM-1 and VCAM-1
SLC-induced adhesion to ICAM-1 and VCAM-1 was measured at 37°C in a parallel plate flow chamber (FCS2 system; Bioptechs). 0.1 µg/ml recombinant mouse ICAM-1 (Katagiri et al., 2002) and 0.05 µg/ml VCAM-1 human IgG1-Fc was coated on polystyrene disks that were then blocked with 1% BSA. For production of recombinant mouse VCAM-1, the first three immunoglobulin domains (aa 1309) of mouse VCAM-1 was obtained from lung mRNA by RT-PCR, and a fusion protein of this fragment and human IgG1-Fc was generated essentially as described previously (Katagiri et al., 2002). The flow chamber was mounted on the stage of an inverted confocal laser microscope (model LSM510, Carl Zeiss MicroImaging, Inc.). Shear stress was generated with an automated syringe pump (Harvard Apparatus). 2 x 106 cells suspended in 500 µl Lefkovitz L15 medium (GIBCO BRL) containing 0.5% BSA were loaded with or without 100 nM SLC (R&D systems) and were incubated for the indicated time before applying shear stress at 2 dyne/cm2. Phase-contrast and GFP fluorescence images were recorded and processed to count cells using Image-Pro® Plus software (Media Cybernetics).
Under-flow adhesion and transmigration assays with endothelial cells
Mouse endothelial cells, BC1 and MBEC4 (Tatsuta et al., 1992), were cultured on fibronectin-coated disks for 2 d. The BC1 and MBEC4 monolayers were pretreated with TNF for the last 6 and 24 h, respectively. The cultured disk was set in the flow chamber as described above. For lymphocyte attachment under flow, the BC1 monolayer was preincubated with SDF-1 for 10 min and then washed. Lymphocytes were resuspended at 2 x 106/ml in Lefkovitz L15 medium containing 5% FCS, and then loaded into the chamber under shear stress at 0.1 dyne/cm2 for 10 min. For transmigration, an MBEC4 endothelial cell line was used because MBEC4 efficiently supports transmigration, when compared with BC1 and other mouse endothelial cells. Because lymphocyte rolling on the MBEC4 monolayer treated with TNF
for 24 h was insufficient for lymphocyte accumulation, lymphocytes were loaded into the flow chamber with or without SLC, and incubated on the endothelial monolayer for 1, 5, and 10 min. Then, shear stress was applied at 2 dyne/cm2 for 20 min. Phase-contrast and GFP fluorescence images were recorded every 10 s. The GFP-positive cells that remained firmly adherent were scored for attachment. The GFP-positive cells that became phase-dark from phase-light during migration over the endothelial monolayer were scored for transmigration.
Cell migration assays
Random cell migration was recorded at 37°C with a culture dish system for live-cell microscopy (T culture dish system; Bioptechs). 0.1 µg/ml mouse and human ICAM-1Fc, and 0.05 mg/ml mouse VCAM-1Fc were coated on thermoglass-based dishes (Bioptechs). Cells were loaded in the ICAM-1Fc or VCAM-1Fccoated dish and mounted on an inverted confocal laser microscope (model LSM510, Carl Zeiss MicroImaging, Inc.). Phase-contrast and GFP fluorescence images were taken every 15 s for 20 min. GFP-positive cells were traced and calculated for velocity using ImagePro® Plus software (Media Cybernetics).
Immunofluorescent staining
BAF/LFA-1 or adenovirus-infected T cells were fixed with 3.3% PFA for 15 min at RT. Fixed cells were mounted on poly-L-lysinecoated slides. For double staining, fixed cells were first stained with 1 µg/ml SDF-1Fc, followed by FITC-conjugated goat antihuman IgG (1:100 dilution). Then, CD44 was detected by rat antimouse CD44 mAb (1:50 dilution; BD Biosciences), followed by Alexa® 546 conjugate goat antirat IgG (1:400 dilution; Molecular Probes, Inc.). Cells were incubated with each antibody for 1 h, and unbound antibodies were removed by washing with PBS/0.1% BSA five times. Stained cells were observed with an inverted confocal laser microscope (model LSM510, Carl Zeiss MicroImaging, Inc.).
Online supplemental material
Time-lapse images of control and Rap1V12 adenovirus-infected lymphocytes on ICAM-1 or VCAM-1 were collected every 15 s, and videos were created at 6 frames/s using QuickTime Pro (Apple Computer, Inc.). Video 1 shows random migration of GFP-positive lymphocytes (green) infected with control adenovirus on ICAM-1. Video 2 shows random migration of GFP-positive lymphocytes (green) infected with Rap1V12 adenovirus on ICAM-1. Video 3 shows random migration of GFP-positive lymphocytes (green) infected with control adenovirus on VCAM-1. Video 4 shows random migration of GFP-positive lymphocytes (green) infected with Rap1V12 adenovirus on VCAM-1. Online supplemental material available at http://www.jcb.org/cgi/content/full/jcb.200301133/DC1.
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Acknowledgments |
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This work was supported in part by a grant-in-aid from the Ministry of Education, Science, Sport, and Culture of Japan.
Submitted: 30 January 2003
Revised: 7 March 2003
Accepted: 7 March 2003
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