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,§
*
First Department of Internal Medicine, University of Occupational and Environmental Health, Japan, School of Medicine, Kitakyushu, Japan;
Department of Biochemistry, Kobe University School of Medicine, Kobe, Japan;
Department of Biochemistry, University of Occupational and Environmental Health, Japan, School of Medicine, Kitakyushu, Japan;
§
School of Allied Health Science, Faculty of Medicine Osaka University, Osaka, Japan;
¶
Department of Tumor Immunology, University Hospital Nijmegen, Nijmegen, The Netherlands; and
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Department of Physiology, Kobe University School of Medicine, Kobe, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and MIP-1ß was
inhibited by pertussis toxin, wortmannin, and cytochalasin B,
suggesting that both G protein-sensitive phosphatidylinositol (PI)
3-kinase activation and cytoskeletal assemblies are involved. The
chemokine-induced T cell adhesion could be mimicked by expression of
small G proteins, fully activated H-RasV12, or
H-RasV12Y40C mutant, which selectively binds to PI
3-kinase, in T cells, inducing activated form of LFA-1 and
LFA-1-dependent adhesion to ICAM-1. H-Ras expression also induced
F-actin polymerization which colocalized with profilin in T cells.
Adult T cell leukemia (ATL) cells spontaneously adhered to ICAM-1,
which depended on endogenous MIP-1 and MIP-1ß through activation
of G protein-sensitive PI 3-kinase. H-Ras signal pathway, leading to PI
3-kinase activation, also induced active configuration of LFA-1 and
LFA-1-mediated adhesion of ATL cells, whereas expression of a
dominant-negative H-Ras mutant failed to do. Profilin-dependent
spontaneous polymerization of F-actin in ATL cells was reduced by PI
3-kinase inhibitors. In this paper we propose that H-Ras-mediated
activation of PI 3-kinase can be involved in induction of
LFA-1-dependent adhesion of T cells, which is relevant to
chemokine-mediated signaling, and that profilin may form an important
link between chemokine- and/or H-Ras-mediated signals and F-actin
polymerization, which results in triggering of LFA-1 on T cells or
leukemic T cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and MIP-1ß trigger integrins and
induce adhesion of circulating T cells and leukemic T cells to
endothelial cell integrin ligands (5, 6, 7, 8, 9, 10). Several recent
studies have emphasized the potential importance of chemokines in
inflammatory responses; various chemokines including MIP-1ß produced
in large amounts at the site of inflammation activate integrins on
leukocytes and result in leukocyte migration and accumulation in
inflamed tissues. Recent findings indicate that integrin-triggering can be induced by multiple signaling pathways which involve different integrin regulators including G proteins, tyrosine kinases, protein kinase C, cAMP pathway, and phosphoinositide 3 (PI 3)-kinases, which results in actin polymerization and association of integrins with cytoskeletal proteins (5, 11, 12, 13, 14, 15, 16, 17, 18). Furthermore, chemokine receptors belong to the "serpentine" receptor family with seven transmembrane domains and are a G protein-coupled proteins involved in integrin triggering. G protein-coupled receptors are known to activate PI 3-kinases and integrin adhesiveness through ligation of the receptor with FMLP and certain chemokines such as RANTES and monocyte chemotactic protein-1. Among several small G proteins, recent findings suggest that Ras plays an important role in signal transduction radiating multiple pathways. For instance, ectopic expression of an active form of R-Ras was found to enhance cell adhesion to extracellular matrix via activation of several integrins (19). In contrast, expression of an active form of H-Ras in Chinese hamster ovary (CHO) cells stably expressing an active chimeric integrin suppresses the function of the chimeric integrin (20). However, the relevance of Ras-mediated signaling to chemokine-induced activation of adhesive function of integrins on T cells is still unknown.
It is thought that PI 3-kinase is controlled by G protein-coupled chemoattractant receptors and is involved in cytoskeletal rearrangement associated with localized polymerization of actin filaments and highly cross-linked membrane-associated fibers (14). The mechanism underlying activation of integrin is thought to involve conformational changes of the ectodomain of integrins and/or clustering of integrins on the cell membrane, which may induce adhesion, resulting from cytoskeletal actin-polymerization associated with cytoplasmic domain of integrins (5, 6, 8, 9, 10, 11, 14, 16, 21, 22, 23). Recent reports also indicate that profilin, a 12- to 15-kDa cytoplasmic protein, promotes actin polymerization by converting ADP-actin to ATP-actin, thus stimulating polymerization (24, 25, 26). Because profilin is physically associated with phosphatidylinositol 4,5-bisphosphate (PIP2) and it is proposed to function as a "linker" between cytoplasmic signaling and actin assembly (27, 28, 29, 30). However, the pathways downstream of PI 3-kinase are unknown at present; also unknown are their roles in these cytoskeletal changes and subsequent activation of integrins on T cells.
The present study was performed to investigate the relevance of cytoplasmic signaling and cytoskeletal assembly to chemokine-induced adhesive function of integrins, with special emphasis on signaling through H-Ras and profilin-mediated actin polymerization in T cells and leukemic T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Peripheral blood from 10 normal healthy volunteers and 5 patients with ATL, and an established human T cell leukemia virus (HTLV)-I-infected T cell line MT-2 (from K. Sagawa, Kurume University Medical School, Kurume, Japan) were used. ATL was diagnosed according to the clinical features, hematological findings, serum Abs against HTLV-1, and monoclonal integration of HTLV-1 proviral genome (31, 32). Highly purified T cells and ATL cells were prepared by exhaustive negative selection (3, 33) from PBMC of normal donors and ATL patients using magnetic beads (Dynal, Oslo, Norway) and multiple Ab mixture consisting of CD19 mAb FMC63, CD16 mAb 3G8, CD11b mAb NIH11b-1, and CD14 mAb 63D3.
Synovial tissues obtained from patients with active rheumatoid arthritis (RA), who were treated by joint replacement surgery, were dissected under sterile conditions in PBS and immediately prepared for culture of synovial endothelial cells as described (10). Briefly, the tissue sample was minced into small pieces and digested with collagenase (Sigma Aldrich Japan, Tokyo, Japan) in serum-free DMEM (Nissui, Tokyo, Japan). After filtering through a nylon mesh and washing, purification of endothelial cells was achieved by a magnetic cell separation technique using endothelial lectin Ulex europaenus agglutinin type I (UEA-I; Seikagaku, Tokyo, Japan) which was covalently coupled to the tosyl-activated megnetizable polystyrene beads (Dynal) as previously described (10, 34). The cells were incubated with the UEA-I-coated magnetic beads in DMEM, supplemented with 10% FCS (Bio-Pro, Karlsruhe, Germany) at 37°C for 30 min. The synovial endothelial cells were obtained by magnetization. HUVEC were purified as previously described (3, 35). After the pre-incubation on plastic dishes for 24 h, the synovial endothelial cells and HUVEC were applied to 48-well culture plates (Costar, Cambridge, MA) coated with 2% gelatin and were cultured to confluence in DMEM containing 100 U/ml penicillin G, 100 U/ml streptomycin (Sigma Aldrich), 20% heat-inactivated FCS, 20 µg/ml endothelial mitogen (Biomedical Technologies, Stoughton, MA), and 10 U/ml heparin in a humidified 5% carbon dioxide atmosphere.
mAbs and reagents
The following mAbs were used as purified Ig in preparation of T
cells and ATL cells, staining and analysis of cell surface molecules,
and blocking of cellular adhesion; anti-activated form of LFA-1 mAb
NKI-L16 (36), CD19 mAb FMC63 (H. Zola, Bedford Park,
Australia), CD11b mAb NIH11b-1, CD49d (VLA-4) mAb NIH49d-1, CD54
(ICAM-1) mAb 84H10 (S. Shaw, Bethesda, MD), CD49d mAb HP2/1 (F.
Sanchez-Madrid, Madrid, Spain) (37, 38), CD16 mAb 3G8 (D.
Siegel, Bethesda, MD), anti-MIP-1 polyclonal Ab,
anti-MIP-1ß Ab (U. Siebenlist, Bethesda, MD) (6),
CD14 mAb 63D3, CD11a (LFA-1) mAb TS1/22, MHC class I mAb W6/32, and
control mAb Thy1.2 (American Type Culture Collection, Manassas, VA).
ICAM-1 was purified by affinity column chromatography from the
Reed-Sternberg cell line L428 as previously described (3, 8).
Multiple inhibitors for intracytoplasmic signaling were applied to each assay systems, and all reagents were used at an indicated concentration, which did not induce cytotoxic effects on T cells and ATL cells assessed by trypan blue staining; wortmannin (Wako Pure Chemical, Osaka, Japan) and LY294002 (Cosmo-Bio, Tokyo, Japan), PI 3-kinase inhibitors; pertussis toxin, uncoupler of certain G proteins from their complex; H88 and H89, A-kinase inhibitors; H7 and staurosporine (Seikagaku, Tokyo, Japan), C-kinase inhibitors; herbimycin A (Sigma, St. Louis, MO) and genistein (Carbiochem, San Diego, CA), tyrosine kinase inhibitors; and cytochalasin B and cytochalasin D (Sigma), cytoskeleton-disrupting reagents.
Sense and antisense oligonucleotide sequences of MIP-1 and MIP-1ß
were 5'-CACCTGCTCAGAATCA-3', 5'-TGATTCTGAGCAGGTG-3',
5'-ATGAAGCTCTGCGTG-3', and 5'-CACGCAGAGCTTCAT-3', respectively. We
synthesized 15-base deoxyribonucleotides on an automated solid-phase
synthesizer (Sawady Technology, Tokyo, Japan). The oligomers were
purified by affinity-gel chromatography embedded ether-toyopearl
(Tosoh, Tokyo, Japan) carrying hydrophobic affinity and gel filtration
effect (DNA stec-1000: ASTEC, Fukuoka, Japan), precipitated with
ethanol, lyophilized to dryness, and dissolved in the culture medium.
The obtained oligonucleotides, a human wild-type H-Ras expression
plasmid pEF-BOS-HA-Ras, a human active form of H-Ras expression plasmid
pEF-BOS-HA-RasV12, a human dominant-negative form
of H-Ras expression plasmid
pEF-BOS-HA-RasV12S17N, and a human PI 3-kinase
binding/activating form of H-Ras expression plasmid
pEF-BOS-HA-RasV12Y40C were introduced into T
cells and ATL cells using a cationic liposome-mediated transfection
method (39, 40, 41). Oligonucleotides and plasmids dissolved
in 100 µl of serum-free medium, OPTI-MEM (Life Technologies,
Gaithersburg, MD), were mixed with 5 µl of lipofectin reagent
(LipofectAMINEplus, Life Technologies) in the same volume of OPTI-MEM
and incubated for 10 min at room temperature. The oligonucleotide and
liposome complex was added to T cells and ATL cells plated in a 6-well
culture dish (3 x 105 cells/well),
incubated for 6 h in OPTI-MEM, and then replaced with a 10% FCS
containing RPMI 1640 (Nissui) for 24 h. The concentration of
oligonucleotides in the conditioned medium was 2.2 µM and the
expression of each H-Ras was confirmed by staining with
anti-HA Ab.
Adhesion assay
Adhesion of T cells or ATL cells to RA-synovial endothelial
cells, HUVEC, or purified ICAM-1 glycoproteins was performed as
previously described (3, 9). Endothelial cells were
applied to 48-well culture plates (Costar, Cambridge, MA) coated with
2% gelatin and were cultured to confluence in DMEM (Nissui) containing
100 U/ml penicillin G, 100 U/ml streptomycin, 20% heat-inactivated
FCS, 20 µg/ml endothelial mitogen (Biomedical Technologies) and 10
U/ml heparin. After washing with PBS, HUVEC were stimulated with 20
ng/ml IL-1ß (Otsuka, Tokyo, Japan) for 4 h at 37°C. The
endothelial cells were also immobilized with MIP-1 or MIP-1ß at
37°C for 2 h, and subsequently free chemokines or reagents were
washed with PBS twice. Purified T cells were labeled with
51Cr (DuPont-NEN, Wilmington, DE) in RPMI 1640
(Nissui) with 10% FCS at 37°C for 2 h. Purified ICAM-1 (50
ng/well) was applied to the 48-well plates in Ca/Mg-free PBS at 4°C
overnight. Binding sites on the plastic were subsequently blocked with
Ca/Mg-free PBS/3% human serum albumin (Green-Cross, Osaka, Japan) for
2 h at 37°C to reduce nonspecific attachment. The plates were
washed three times with PBS before the addition of T cells.
51Cr-labeled T cells or ATL cells were also
pretreated with or without multiple inhibitors for intracytoplasmic
signaling. A total of 2 x 105 T cells were
added to the culture plates coated with the endothelial cells with or
without relevant adhesion-blocking mAb in the presence or absence of
MIP-1 and MIP-1ß. All mAbs were used at a saturating concentration
of 10 µg/ml, which was shown in previous studies to maximally inhibit
the relevant adhesive interaction (3). After a settling
phase of 30 min at 4°C, which also allowed mAb binding, plates were
rapidly warmed to 37°C for 30 min. Then the plates were gently washed
twice with RPMI 1640 at room temperature to remove nonadherent T cells
completely. The contents of each well containing adherent T cells were
lysed with 250 µl of 1% Triton X-100 (Sigma Aldrich), and gamma
emissions of well contents were determined. Data are expressed as mean
percentage of binding of indicated cells from a representative
experiment.
Flow microfluorometry
Staining and flow cytometric analyses of freshly obtained T cells were conducted by standard procedures as previously described using a FACScan (Becton Dickinson, Mountain View, CA) (3, 33). Briefly, cells (2 x 105) were incubated with negative control mAb Thy1.2, LFA-1 (CD11a) mAb TS1/22, and anti-activated form of LFA-1 mAb NKI-L16 in FACS media consisting of HBSS (Nissui), 0.5% human serum albumin, and 0.2% NaN3 (Sigma) for 30 min at 4°C. After washing the cells with FACS medium three times, the cells were further incubated with FITC-conjugated goat anti-mouse IgG Ab for 30 min at 4°C. The staining of cells with the mAbs were detected using a FACScan. Amplification of the mAb binding was provided by a three-decade logarithmic amplifier. Quantification of cell surface Ags on one cell was calculated using standard beads, QIFKIT (Dako Japan, Kyoto, Japan).
Confocal microscopic analysis of cytoskeleton and cytoplasmic proteins
For microscopic analysis, T cells, ATL cells, and cell lines were settled for 30 min at 4°C on fibronectin-coated slides. After the cells were incubated for 1 min at 37°C, they were fixed with 3% formaldehyde. F-actin was stained with rhodamine-phalloidin (1 U/slide, Molecular Probes, Eugene, OR), and profilin was stained with anti-profilin Ab and second FITC-conjugated anti-rabbit Ig. F-actin polymerization and localization of cytoplasmic profilin were analyzed with a confocal laser microscope system LSM 410UV (LD ACHROPLAN x20 objective lens, Carl Zeiss, Oberkochen, Germany) as previously described (5, 9).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and MIP-1ß-induced T cell adhesion to
RA-endothelial cells
Leukocyte integrins cannot mediate adhesion unless activated, and
therefore regulation of integrin-dependent adhesion is critical to the
migration of virtually all hematopoietic cells (2, 3). We
and others have reported that chemokines such as MIP-1ß and MIP-1
trigger T cell integrin functions (5, 6, 42). Resting T
cells cannot adhere to RA-endothelial cells. When MIP-1 or MIP-1ß
was immobilized on RA-endothelial cells and subsequently soluble
chemokines left in culture supernatant was washed out, T cells adhered
to RA-endothelial cells in a concentration-dependent manner within
30-min incubation. The immobilized MIP-1 and MIP-1ß-induced
integrin-dependent adhesion of T cells to RA-endothelial cells was
clearly reduced by pretreatment of T cells with 1 µg/ml pertussis
toxin, which uncouples certain G proteins from their complex. The
chemokine-induced T cell adhesion was also decreased by the treatment
with 100 µM wortmannin, a PI 3-kinase inhibitor. However, T cell
adhesion was not affected by 10 µM genistein, a tyrosine kinase
inhibitor, 30 µM H7, a protein kinase C inhibitor, or 10 µM H89, an
A-kinase inhibitor. Furthermore, pretreatment of T cells with
cytochalasin B, a cytoskeleton-disrupting agent, reduced
chemokine-induced adhesion of T cells to the endothelial cells (Fig. 1
). These results suggest that the
endothelial immobilized MIP-1 and MIP-1ß-induced
integrin-dependent adhesion of T cells to RA-endothelial cells might
depend on cytoskeletal rearrangement induced through G
protein-sensitive PI 3-kinase activation stimulated by these
chemokines.
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Chemokine receptors belong to the serpentine family of seven
transmembrane G protein-coupled receptors (43). Among
several small G proteins, Ras is known to play a central role in signal
transduction radiating multiple pathways. To determine whether Ras is
involved in the induction of T cell adhesion to endothelium, we
analyzed the ability of H-Ras and its mutants to induce adhesion when
expressed in resting T cells. Resting T cells did not adhere to
purified ICAM-1. However, T cells transfected with the expression
vector encoding fully activated H-RasV12 mutant
strongly adhered to ICAM-1. In contrast, adhesion of T cells expressing
the wild type H-Ras was comparable to that of resting T cells. It is
noteworthy that T cells expressing the
H-RasV12Y40C mutant, which selectively binds to
PI 3-kinase, also strongly bound to ICAM-1. We also showed that
adhesion of T cells expressing H-RasV12 or
H-RasV12Y40C to ICAM-1 was integrin-mediated
because it could be inhibited by the addition of anti-LFA-1 mAb
(Fig. 2). These results imply that H-Ras
signals, especially those followed by PI 3-kinase activation, play an
important role in the induction of LFA-1-mediated adhesion of T
cells.
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LFA-1 requires an active configuration to bind to its ligand, a
process that can be induced by a variety of stimuli and can be reported
by NKI-L16 mAb which reacts with a Ca2+-dependent
activation epitope located on the ectodomain of -chain of LFA-1
(44). Resting T cells did not express the activated form
of LFA-1 as recognized by NKI-L16 mAb using flow cytometry. However,
the expression of the activated form of LFA-1 was clearly observed on
most of T cells expressing H-RasV12 or
H-RasV12Y40C. T cells expressing the wild-type
H-Ras did not express the NKI-L16 epitope, although they expressed
significant amounts of LFA-1 as recognized by a conventional CD11a mAb
TS1/22, which was comparable to that expressed by resting T cells as
well as T cells expressing H-RasV12 (Fig. 3). These results suggest that
LFA-1-mediated adhesion of T cells induced by
H-RasV12 and H-RasV12Y40C
depends on an active conformation of LFA-1 on these T cells.
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Actin polymerization is a dynamic process and LFA-1 function
is associated with polymerized F-actin (22, 45). Resting T
cells seeded on fibronectin did not spread and their F-actin content
remained distributed as observed by confocal microscopy. In contrast, T
cells expressing the H-RasV12 or
H-RasV12Y40C showed increased expression of
F-actin in the cell cortex, and marked polymerization of F-actin was
observed (Fig. 4A). Profilin,
a 12- to 15-kDa cytoplasmic protein, is known to promote actin
polymerization by converting ADP-actin to ATP-actin
(24, 25, 26). Although resting T cells showed constant and
slight distribution of profilin, T cells expressing the
H-RasV12 or H-RasV12Y40C
represented increased expression of profilin in their cortex (Fig. 4B). Furthermore, it is noteworthy that polymerized F-actin
clearly colocalized with profilin in T cells expressing
H-RasV12 or H-RasV12Y40C by
double staining of them (Fig. 4C). These results suggest
that profilin-dependent F-actin polymerization may be induced by
signaling mediated by H-Ras-mediated activation in T cells.
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- and
MIP-1ß-mediated adhesion of ATL cells to ICAM-1
We reported that chemokines produced by ATL cells regulated
to trigger LFA-1 and to induce LFA-1-mediated adhesion in an autocrine
manner (8) and thereby ATL cells appear to be a good model
to investigate the signaling pathways in chemokine-mediated adhesion of
T cells. ATL cells spontaneously bound to purified ICAM-1 and mAb
blocking studies, in which ATL cell-adhesion to ICAM-1 was inhibited by
anti-LFA-1 mAb, indicated that the adhesion was mediated by LFA-1.
The increased adhesion could be inhibited by pretreatment of ATL cells
with a mixture of anti-MIP-1 and MIP-1ß Abs and was also
reduced by transfection of antisense oligonucleotides, but not by sense
oligonucleotides, for either MIP-1 or MIP-1ß. These findings
indicate that ATL cell adhesion is mediated by endogenous MIP-1 and
MIP-1ß. Furthermore, pretreatment of cells with pertussis toxin,
wortmannin, or LY294002 reduced LFA-1-dependent adhesion of ATL cells
to purified ICAM-1. However, ATL cell adhesion was not affected by 10
µM genistein, 30 µM H7, or 10 µM H89. Finally, the
cytoskeleton-disrupting agents, cytochalasin B and cytochalasin D, also
reduced the adhesion (Fig. 5). Together
these data suggest that although the increased LFA-1-mediated adhesion
of ATL cells to ICAM-1 is mediated by a number of signaling pathways,
it mainly depends on activation of G protein-sensitive PI 3-kinase
which is stimulated by the endogenous MIP-1 and MIP-1ß.
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Next, we assessed the role of H-Ras in endogenous
chemokine-mediated adhesion of ATL cells that had been precultured in
the serum-free medium for 24 h to observe the effects of
expression of H-Ras and its mutants. The ATL cells slightly expressed
the activated form of LFA-1 -chain as recognized by NKI-L16 mAb.
However, the binding of NKI-L16 mAb was further increased on ATL cells
expressing the H-RasV12 or
H-RasV12Y40C, whereas it was inhibited on ATL
cells expressing the dominant-negative form of
H-RasV12S17N. In contrast, expression of LFA-1
-chain as recognized by CD11a mAb TS1/22 was similar on ATL cells
and ATL cells expressing the H-RasV12,
H-RasV12Y40C or
H-RasV12S17N (Fig. 6A). ATL cells that were
precultured in the serum-free medium did so weakly, but still adhered
to purified ICAM-1 proteins. However, the adhesion of ATL cells,
transfected with the H-RasV12 or
H-RasV12Y40C mutants, was further increased,
whereas the adhesion of ATL cells expressing the
H-RasV12S17N were markedly reduced to the basal
level. The induced adhesion of ATL cells, expressing the
H-RasV12 or H-RasV12Y40C,
to ICAM-1 was completely inhibited by the addition of anti-LFA-1
mAb (Fig. 6B). These results demonstrate that the H-Ras
signal pathway followed by PI 3-kinase activation, plays a role in the
induction of active configuration of LFA-1, resulting in enhanced
LFA-1-mediated adhesion of ATL cells stimulated by endogenous
chemokines.
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Freshly obtained ATL cells showed increased expression of F-actin
in the cell cortex and polymerization of F-actin, which was clearly
colocalized with profilin. However, when ATL cells were pretreated with
wortmannin, F-actin-polymerization and colocalization with profilin was
markedly reduced. Furthermore, the cytoskeleton-disrupting agent
cytochalasin B markedly reduced F-actin polymerization, whereas
distribution of profilin remained constant as observed in untreated ATL
cells (Fig. 7). These results suggest
that the profilin-dependent polymerization of F-actin in ATL cells may
be induced by signaling through G protein-dependent activation of PI
3-kinase.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. 3) Expression of
H-RasV12 also induced F-actin polymerization
which co-localized with profilin in T cells, as chemokines did. 4) ATL
cells that spontaneously adhered to ICAM-1 induced by endogenous
chemokines depended on activation of G protein-sensitive PI 3-kinase.
5) Activation of the H-Ras signal pathway, leading to activation of PI
3-kinase, induced an activation of LFA-1 and LFA-1-mediated adhesion
of ATL cells, whereas expression of a dominant negative H-Ras mutant
failed to do. 6) Profilin-dependent polymerization of F-actin
spontaneously occurred in ATL cells was inhibited by PI 3-kinase
inhibitors. Based on these findings, we document that H-Ras signal
pathway, leading to PI 3-kinase activation, mimics LFA-1-dependent
adhesion of T cells induced by chemokines and that profilin might form
a link that couples chemokine- or H-Ras-mediated signals and the
F-actin polymerization, resulting in active configuration of LFA-1.
It is well known that the adhesive capacity of integrins is tightly
regulated. In this regard, we have previously reported that the
chemokines MIP-1 and MIP-1ß induce integrin-mediated adhesion of T
cell subsets to endothelial integrin-ligands (5, 6, 7, 8, 9, 10).
Several recent studies have supported the finding that multiple
chemoattractants, including chemokines, activate integrins on
lymphocytes and leukocytes. Furthermore, because most receptors for
chemoattractants belong to a "serpentine" family with seven
transmembrane domains, and are a G protein-coupled protein, cytoplasmic
large G proteins, and/or small G proteins have been postulated to be
involved in chemokine-induced triggering of integrins (11, 12, 14, 15, 17, 43, 46). Among several small G proteins, Ras is
known to play a central role in signal transduction, from which
multiple pathways radiate. For instance, activated H-Ras induces
expression of LFA-1 by modulating the transcription of LFA-1 -chain
on B cells (47). Similarly, expression of active form of
R-Ras, which is related to H-Ras, was found to enhance cell adhesion to
extracellular matrix via activation of several integrins
(19). In this study, we demonstrated that
chemokine-mediated adhesion of T cells to ICAM-1 was mimicked by
ectopic expression of activated mutants of H-Ras in T cells which
induced dramatic F-actin polymerization, expression of an activated
epitope of LFA-1, and the LFA-1-dependent adhesion to ICAM-1. ATL
cells, which are characterized by a malignant expansion of peripheral
mature CD4+ T cells infected with HTLV-I, is a
unique model to investigate because they spontaneously express
activation epitope of LFA-1 and highly adhere to ICAM-1 stimulated by
endogenous chemokines. The expression of active H-Ras mutants in ATL
cells further enhanced integrin-mediated adhesion, whereas a
dominant-negative H-Ras mutant reduced the adhesion. These findings
suggest the relevance of H-Ras-mediated signaling to chemokine-induced
activation of adhesive function of LFA-1 on T cells and leukemic T
cells.
In contrast, it was also reported that expression of an active form of
H-Ras, and its effector kinase, Raf-1, in CHO cells stably expressing
an active chimeric integrin suppressed the function of the chimeric
integrin 6A, ß1, and
ß3. Suppression of integrin function correlated
with activation of the Ras/Raf/mitogen-activated protein (extracellular
signal-related kinase, ERK) kinase pathway (20). One
plausible explanation for such discrepant and complex nature of H-Ras
functions is that active form of H-Ras may exhibit distinct functions
in regulating different types of integrins, although cell type-specific
functions of H-Ras can also be considered. We indeed observed that the
expression of H-Ras efficiently induced LFA-1
(ß2)-mediated T cell adhesion but that H-Ras
expression did not augment ß1-dependent T cell
adhesion or expression of activated form of ß1
(data not shown). Alternatively, second signals induced by H-Ras may be
differently involved in "on and off switch" for integrin
triggering. Ras is known to be a "hub" that radiates multiple
signaling pathway including Raf-1 and PI 3-kinase (48). We
observed that H-RasV12Y40C mutant, which binds to
PI 3-kinase in T cells (41), induced the activated form of
LFA-1 and LFA-1-dependent adhesion to ICAM-1. Recently accumulating
evidence demonstrates that PI 3-kinase appears to play a central role
in integrin-triggering as well as cytoskeletal changes (12, 49, 50, 51). We here observed that spontaneous activation of LFA-1
as well as F-actin polymerization, which are stimulated by endogenous
chemokines, were inhibited by PI 3-kinase inhibitors. These results
suggest that H-Ras-sensitive PI 3-kinase activation is involved in
"on switch" for LFA-1 on T cells, whereas the H-Ras-ERK kinases may
function as an "off switch" for integrins.
Actin polymerization is a dynamic process critical for cell adhesion.
Furthermore, LFA-1 function is associated with polymerized F-actin
(45, 52). The expression of H-RasV12
or H-RasV12Y40C in T cells showed an apparent
increase of F-actin in the cell cortex and marked polymerization and
rearrangement of F-actin as well as highly augmented expression of the
activated form of LFA-1 as recognized by NKI-L16 mAb which reacts with
a Ca2+-dependent activation epitope located on
the ectodomain of -chain of LFA-1 (53). The epitope of
LFA-1 is thought to be induced by a conformational change of LFA-1 due
to multimerization of the LFA-1 molecules. It has been also suggested
that multimerization of integrins, mediated by F-actin polymerization,
results in the induction of an active conformation of integrins
(12, 54). However, the pathways downstream of PI 3-kinase
are still unknown at present as well as their role in these
cytoskeletal changes and subsequent activation of integrins.
Profilin is a widely and highly expressed cytoplasmic 14 kDa protein that binds actin monomers. Profilin, a representative actin binding protein, modulates the steady-state monomer-polymer cycle of actin in the presence of ATP, thereby determining the F-actin/G-actin ratio and the turnover rate of actin filaments (24, 25, 26, 55). Furthermore, profilin is known to be physically associated with PIP2 and PI 3-kinase products and it is known to function as a "linker" between cytoplasmic signaling and actin assembly (27, 28, 29, 30). PI 3-kinase products exhibit much higher affinity for the profilin-actin complex than the primary products, PIP and PIP2, and activated PI 3-kinase initiates massive actin polymerization through profilin. It is also reported that integrin-mediated adhesion of endothelial cells to fibronectin was increased by profilin overexpression, which resulted in increased recruitment of fibronectin receptors to the plasma membrane where focal contacts are being formed and focal adhesion proteins are located (56). By using immunofluorescence with confocal laser-scanning microscopy, we found profilin in areas of polymerized F-actin induced by the expression of H-RasV12 or H-RasV12Y40C in T cells. We also observed that colocalization of profilin with spontaneously polymerized F-actin in ATL cells was reduced by pretreatment of the cells with PI 3-kinase inhibitors or by expression of dominant-negative H-RasV12S17N (data not shown, in part). These results suggest that G protein-sensitive PI 3-kinase activation plays a role in cortical actin assembly composed by profilin, resulting in the triggering of LFA-1-mediated adhesion.
The potential importance of chemokines in inflammatory responses is well accepted. Various chemokines including MIP-1ß produced in large amounts at the site of inflammation activate integrins on leukocytes and result in their accumulation in the tissues. Based on these results, we propose that H-Ras-sensitive PI 3-kinase activation and subsequent profilin-mediated actin polymerization, may be involved in chemokine-induced LFA-1-dependent adhesion of T cells or leukemic T cells. The concept would warrant further studies in terms of cytoplasmic signaling not only in inflammatory processes but also in leukemic cell infiltration.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Yoshiya Tanaka, First Department of Internal Medicine, University of Occupational and Environmental Health, Japan, School of Medicine, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555 Japan. E-mail address:
3 Abbreviations used in this paper: VLA-4, very late Ag-4; MIP, macrophage inflammatory protein; PI 3-kinase, phosphoinositide 3 kinase; PIP2, phosphatidylinositol-4,5-bisphosphate; ATL, adult T cell leukemia; HTLV, human T cell leukemia virus; RA, rheumatoid arthritis.
Received for publication February 22, 1999. Accepted for publication September 15, 1999.
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