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Departments of
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Immunology and Medical Zoology and
Hygiene, School of Medicine, Kagoshima University, Kagoshima, Japan;
Kagoshima Red Cross Hospital, Center for Rheumatic Diseases, Kagoshima, Japan; and
§
Laboratory of Biochemistry, Aichi Cancer Center, Nagoya, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The adhesive function of very late Ag-4 (VLA-4) (a heterodimer of
4 and ß1 subunits), which is known as a
counter receptor for VCAM-1, has been shown to vary in the multiple
activation states of T cells (8). Chemokines and PMA rapidly induce a
high affinity conformation of VLA-4 on T cells in short cultures (9).
In a chronically activated state in vivo, the adhesion activity of
lymphocytes from RA SF to fibronectin and VCAM-1 are up-regulated via
VLA-4/5 with high affinity (7, 10). Studies according to the
differential adhesive activity of diverse leukemic cell lines revealed
that the epitope of ß1-subunit with high affinity
conformation of VLA integrins was identified by the 15/7 mAb and a
group of HUTS mAb (11, 12).
In this study, we demonstrate that sVCAM-1 exhibits chemotactic activity toward T cells with high affinity VLA-4 such as Jurkat cells and IL-2-dependent T (IL-2 T) cells, which express up-regulated 15/7 mAb epitope. Inhibition of the chemotactic activity of sVCAM-1 for T cells by the addition of anti-VCAM-1 and VLA-4 confirm that the chemotactic activity is mediated by the binding of sVCAM-1 to VLA-4 on the cell surface. Further studies using various kinase inhibitors and Western blot analyses suggest that the chemotactic activity of sVCAM-1 is mediated by Rho, Ca2+/calmodulin-dependent kinase II (CaM kinase II), and protein kinase C (PKC) signaling in T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recombinant sVCAM-1 that was truncated in the EcoRI site in the 5th Ig-like domain and has two binding sites for VLA-4 within its 1st and 4th Ig-like domains was prepared as previously described (7). The following reagents were purchased: H-7, genistein, cytochalasin B (Sigma, St. Louis, MO); C3 exoenzyme (Upstate Biotechnology, Lake Placid, NY); and KN62 (Seikagaku, Tokyo Japan). The following mAbs were purchased: anti-VLA-4 (HP2/1) and anti-VLA-5 (SAM1) (Immunotech, Marseille, France); anti-CD4 (NU-T H/I), anti-CD8 (NU-T S/C) (Nichirei, Tokyo, Japan); anti-CD11b (Mo1), anti-CD14 (Mo2), anti-CD16 (NK1), and anti-CD20 (B1) (Coulter Immunology, Hialeah, FL); anti-CD45RO (UCHL-1) (Nichirei); and anti-CD45RA (HI100) (PharMingen, San Diego, CA). OKT3 (CD3) hybridoma was obtained from the American Type Culture Collection (ATCC, Manassas, VA). 4B4, anti-integrin ß1/CD29 was a generous gift from Dr. S. F. Schlossman (Dana-Farber Cancer Institute, Boston, MA). Another anti-integrin ß1/CD29, 15/7 was kindly provided by Dr. Ted A Yednock (Athena Neurosciences, South San Francisco, CA) (11). Anti-VCAM-1/CD106 (2G7) was generously provided by Dr. D. Beall, American Otsuka Pharmaceuticals, Rockville, MD. Anti-ICAM-1 (CD54, YH-370) was generously provided by Dr. A. Yamada, Kurume University, Kurume, Japan.
Cell preparations and cultures
Mononuclear cells were isolated from normal volunteers and from the SFs of 12 patients with RA who met the American College of Rheumatology revised criteria for classification of RA (13) by Lymphoprep (Nycomed, Oslo, Norway) gradient centrifugation. After depletion of adherent cells by adhesion to plastic dishes, T cells were enriched by negative selection using anti-mouse IgG-coated magnetic beads (Dynal, Oslo, Norway) after incubation with an Ab mixture consisting of anti-CD14, anti-CD16, and anti-CD19 mAbs, as previously described (purity was typically 94% CD3 positive) (14). In some experiments, anti-CD4, anti CD-8, anti-CD45RA, or anti-CD45RO mAb was added to the previous mixture of mAbs to obtain a reciprocal CD8-, CD4-, CD45RO-, or CD45RA--enriched subpopulation of T cells, respectively. Reanalysis of sorted populations revealed a purity of >94%. IL-2-dependent T cells were obtained by initially stimulating mononuclear cells with PHA, followed by the addition of IL-2 (20 U/ml) twice a week in HEPES (25 mM)-buffered RPMI 1640 medium supplemented with 10% FCS, penicillin (100 U/ml), and streptomycin (100 µg/ml) (all from Life Technologies, Baltimore, MD). Jurkat and H9 T cell leukemic cell lines (Hayashibara Research Institute, Okayama, Japan) were also maintained in RPMI 1640 conditioned medium.
Chemotaxis assay
Cell migration was measured using 48-well chemotaxis chambers (Neuro Probe, Cabin John, MD) and polyvinylpyrrolidone-free polycarbonate filters (Nucleopore, Pleasanton, CA) with a pore size of 5 µM for PBL and 8 µM for leukemic cells, as previously described, with slight modifications (15). To avoid adsorption of recombinant sVCAM-1 to filters in the migration assay, filters were coated on both surfaces with 1% BSA in Dulbecco’s PBS overnight at 4°C, then extensively washed with DPBS followed by distilled water. The filters were subsequently air dried. Triplicates of lower wells were filled with different concentrations of recombinant sVCAM-1 diluted in chemotaxis medium (serum-free RPMI 1640 supplemented with 1 mg/ml BSA) whereas the upper wells were aliquoted with 50 µl of 3 x 106 cells/ml in the chemotaxis medium. Checker board analysis was also performed by varying the concentration of sVCAM-1 in both the lower and upper wells. Where indicated, mAbs and inhibitors were either preincubated with cells or added to the wells at the start of the assay. The assembled chemotaxis chambers were placed in a 5% CO2 incubator at 37°C for 3 h, the top chambers were disassembled, and nonmigrating cells were removed from the upper side of the filters by drawing the filter up over the wiper blade. The filters were fixed and stained with Giemsa. The number of migrated cells was counted by light microscopy in nine high power fields in triplicate. The results are expressed as the number of migrated cells per high power field. To confirm that the migration activity of sVCAM-1 was not mediated by the absorbed ligand on the filter, we tested the migration assay employing a three step incubation as follows: 1st step, filters are blocked with 1% BSA overnight in the culture dish; 2nd step, the lower surface of each blocked filter was immersed for 3 h with 3.3 nM sVCAM-1 or medium alone in the culture dish; 3rd step, after being rinsed with PBS and air dried, each filter was placed between the assembled chemotaxis chamber. IL-2-dependent T cells in the upper wells were allowed to migrate across the filter to the lower wells filled with sVCAM-1 (3.3 nM) for 3 h.
sVCAM-1 ELISA
The recombinant sVCAM-1 and sVCAM-1 in the peripheral blood and SF were quantitated by sandwich ELISA using a proprietary kit (R&D Systems, Minneapolis, MN). Statistical analysis of the data was performed using the nonparametric Wilcoxon signed rank test.
Flow cytometric analysis
Flow cytometry was performed on an EPICS Elite (Coulter) after indirect immunofluorescence staining of the cells using the indicated mAbs. Control cells were treated with isotype-matched myeloma proteins (IgG1:MOPC21, IgG2a:UPC10), washed, then stained with FITC-conjugated goat F(ab')2 anti-mouse Ab (Tago, Burlingame, CA).
Western blot analysis
IL-2-dependent T cells were lysed in immunoprecipitation buffer (PBS (pH7.4), 1% Nonidet P-40 (NP40), 0.01% SDS, 0.5% deoxycholate, 10 µg/ml aprotinin, and 10 µg/ml leupeptin, 1 mM PMSF, 10 mM sodium fluoride, and 1 mM sodium orthovanadate). Lysates were cleared by centrifugation to remove NP40-insoluble protein and saved for tyrosine phosphorylation analysis. The insoluble pellet was solubilized with 1% SDS lysis buffer for vimentin phosphorylation analysis (16). Protein concentrations were determined with the Bio-Rad (Hercules, CA) protein assay, and equivalent amounts of protein were denatured by boiling in sample buffer and then separated by SDS-PAGE (10%) under nonreducing conditions. Proteins were transferred onto nitrocellulose membranes (Hybond-ECL, Amersham, Buckinghamshire, U.K.) and probed with either anti-phosphotyrosine mAb for the NP40-soluble lysates (4G10; Upstate Biotechnology) or with anti-vimentin (1B8) or anti-phosphoserine mAb (MO82, which specifically reacts with Ser82 in phosphorylated vimentin by calmodulin-dependent kinase II) for the NP40-insoluble protein (17). Peroxidase-conjugated goat anti-mouse Ab (Tago) was then applied, and the detection was conducted using the enhanced chemiluminescence (ECL) detection system (Amersham). The relative intensities of protein bands in the Western blots were determined using an LKB laser densitometer (Uppsala, Sweden).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The effect of sVCAM-1 on lymphocyte chemotaxis was examined in
three cases. sVCAM-1 induced significant levels of migration of
IL-2-dependent T cells, but not in unstimulated PBL-T cells (Fig. 1
). The chemotactic effect of sVCAM-1 on
IL-2-dependent T cells was apparently dose dependent, and the maximal
effect was obtained at a concentration of 3.3 nM. A leukemic T cell
cell line (Jurkat cells) showed strong migration toward sVCAM-1,
whereas another T cell line (H9 cells) showed little migration (Fig. 1). As reported earlier (7), sVCAM-1 does not bind to PBL-T cells, but
it does bind to IL-2-dependent T cells via VLA-4 molecules in the
presence of divalent cations (Ca2+ and/or Mn2+)
from the same donor; moreover, Jurkat cells adhere strongly to
immobilized sVCAM-1 whereas H9 adheres to a much lesser
extent. Therefore, the differential effect of sVCAM-1-induced
chemotaxis on these T cells may be correlated with the variable
affinity states of VLA-4 expressed on the T cells. We also confirmed,
by checkerboard analysis and by varying the concentrations of
chemoattractant in the upper and lower chemotaxis chambers, that, while
sVCAM-1 was chemotactic for T cells, it was not chemokinetic for T
cells (Table I). These studies also
showed that lymphocyte chemotaxis was completely abrogated when sVCAM-1
was at an equal concentration in both lower and upper chambers.
Finally, we performed studies to show that the amount of sVCAM-1
absorbed to its filter in our chamber assay was negligible. In these
studies we prepared BSA-blocked filters preincubated with either
sVCAM-1 (3.3 nM) or with medium alone for 3 h at 37°C, and then
washed the filters with PBS and air dried the filters. We then measured
the chemotaxis of IL-2-dependent T cells toward the lower wells filled
with medium alone. We found equal levels of migration using filters
preincubated with either sVCAM-1 or medium alone (23 ± 4 vs
20 ± 3) (Table II).
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In the next study, surface adhesive molecules on PBL-T and
IL-2-dependent T cells from normal donors, and T cells from SF in
patients with RA (RA SF), were studied by flow cytometry.
Representative flow cytometric profiles from normal donor A.K. and from
RA patient S.Y. are shown in Figure 2A. In normal donors, the
expression of VLA-4 (HP2/1) is up-regulated in IL-2-dependent T cells
as compared with PBL-T cells in donors studied. Although total
ß1 subunit expression detected by 4B4 was not
significantly increased in IL-2-dependent T cells in comparison with
PBL-T cells, the high affinity epitope of the ß1 subunit
detected by 15/7 is up-regulated in IL-2-dependent T cells as compared
with that in PBL-T, which is consistent with the previous finding of
sVCAM-1 binding to IL-2 T cells (7). ICAM-1 expression was not
significantly different between PBL-T and IL-2 T cells in normal
donors. CD4+ T cells are dominant among PBL-T cells
(CD4+ T cells, 42–57%; CD8+ T cells,
24–39%), whereas the CD4+/CD8+ T cell ratio
among IL-2 T cells varied (CD4+ T cells, 22–73%;
CD8+ T cells, 25–75%) in the three cases studied. With
regard to T cells from RA SF, VLA-4 (HP2/1) and, most notably, 15/7
ß1-subunit epitope and ICAM-1 expression were
up-regulated when compared with levels found in normal PBL-T cells. In
leukemic T cells, both Jurkat and H9 T cells in comparison with PBL-T
cells showed greater expression of VLA-4 and ß1-subunit
(4B4). While 15/7 expression was up-regulated in Jurkat cells, it was
not up-regulated in H9 T cells (Fig. 2B). It was estimated
that, when the mean fluorescence intensity (MFI) was over 2.2 in 15/7
expression, the cells possessed high affinity VLA-4 in parallel assays
of cell attachment (data not shown).
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The specificity of IL-2 T and Jurkat cell migration by sVCAM-1 was
confirmed by various mAbs, including mAbs that blocked cell adhesion
between VCAM-1/VLA-4. As shown in Figure 3, anti-CD3 stimulation during the
chemotaxis assay period did not enhance sVCAM-1-mediated IL-2 T cell
migration. As expected, pretreatment of sVCAM-1 with anti-VCAM-1
mAb (2G7) (74% decrease) or pretreatment of IL-2 T cells with
anti-VLA-4 mAb (HP2/1) (82% decrease) abrogated IL-2-dependent T
cell migration toward sVCAM-1. An mAb for ß1-subunit
(4B4) also showed strong inhibition (85%) of IL-2 T cell migration,
suggesting that 4ß7 might have little, if
any, role in its migration. Unexpectedly, anti-ICAM-1 mAb
considerably inhibited IL-2-dependent T cell migration, suggesting that
the signal triggered through ICAM-1 might perturb VLA-4-mediated
signaling in IL-2-dependent T cells. Regarding the effect of mAbs on
Jurkat T cells, similar inhibition of Jurkat T cell migration was
obtained using anti-VCAM-1, anti-VLA-4, and
anti-ß1-subunit (4B4). However, anti-ICAM-1 did
not inhibit Jurkat T cell migration toward sVCAM-1. Collectively,
sVCAM-1-induced T cell migration was mediated by its binding to VLA-4
on the cell surface and signaling through VLA-4-triggered cytoplasmic
mobility.
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It was reported that T cells in RA SF express increased 15/7
ß1 epitope (18). Therefore, it was presumed that
chronically activated T cells from RA SF with high affinity VLA-4 may
migrate toward sVCAM-1. T cells in RA SF spontaneously migrated without
sVCAM-1 (Fig. 4). The numbers
of migrated T cells from RA SF donors (48 ± 13 cells/field) were
significantly higher than the numbers of migrated T cells from RA PBL
(22 ± 5 cells/field) and normal PBL (20 ± 3 cells/field)
without the presence of chemoattractant. Moreover, RA SF T cells showed
significantly increased migration in response to sVCAM-1 (95 ± 21
cells/field), compared with medium without sVCAM-1 (48 ± 13
cells/field). The presence or absence of sVCAM-1 had no discernable
effect on the migration of T cells from both RA PBL (with sVCAM-1,
25 ± 7; without sVCAM-1, 22 ± 5 cells/field) and normal
PBL (with sVCAM-1, 21 ± 3; without sVCAM-1, 20 ±
3 cells/field). Furthermore, a subset study using negative selection by
magnetic beads showed that both CD4+- and
CD8+-enriched subsets were chemotactic for sVCAM-1 (Fig. 5). Synovial T cells are known to be
enriched with CD45RO+ memory T cells as compared with PB T
cells (19, 20). Furthermore, this study showed that a CD45RO-enriched
memory T cell subset from RA SF exhibited the most notable migratory
response to sVCAM-1, while the CD45RA enriched T cell subset showed
less migration than unseparated T cells from RA SF.
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In the next series of studies, we examined the signal transduction
pathways involved in sVCAM-1-induced chemotaxis using various
inhibitors. Since the cell permeability of C3 exoenzyme is quite low,
cells were preincubated with C3 exoenzyme at the concentration of 25
µg/ml overnight as previously reported (21). Other inhibitors were
added at the indicated concentrations 30 min before the chemotaxis
assay. sVCAM-1-induced chemotaxis in both IL-2-dependent and Jurkat T
cells were significantly inhibited by C3 exoenzyme (Rho inhibitor), H7
(protein kinase C inhibitor), and KN62 (CaM kinase II inhibitor) (Fig. 6). These results suggest that the
GTP-binding protein Rho, protein kinase C, and CaMII kinase activation
pathways were involved in sVCAM-1-induced chemotaxis. Although the
inhibitory effect of genistein (45% inhibition in IL-2-dependent T
cells; 21% inhibition in Jurkat cells) was not as great an effect as
that observed with other inhibitors, such as C3 exoenzyme, H7, and
KN62, the tyrosine kinase pathway may as well be involved in
sVCAM-1-induced signaling. As predicted, cytochalasin B, which inhibits
actin polymerization and cell motility, completely abolished
IL-2-dependent T and Jurkat T cell migration. In addition, the complete
abrogation of T cell chemotaxis in the presence of EDTA confirmed that
the binding of sVCAM-1 to VLA-4 was divalent cation dependent.
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Several recent studies have established that the clustering of
ß1-integrins at focal adhesion sites results in increased
tyrosine phosphorylation of pp125FAK (22, 23). However, substrates
immunoprecipitated with anti-FAK showed that sVCAM-1 did not induce
phosphorylation of pp125FAK in IL-2-dependent T cells (data not shown).
It was recently shown that phosphorylated peptide-specific Abs enabled
several kinase-specific activations, such as cdc2, MAPK, and CaM kinase
II (17, 24). Using MO82 mAb, which detects phosphorylated
Ser82 by activated CaM kinase II in vimentin, CaM kinase II
activity was assessed in sVCAM-1-induced T cell chemotaxis. Western
blot analysis with an anti-vimentin mAb showed that vimentin (58
kDa) is constitutively synthesized in IL-2-dependent T cells (Fig. 7A). In regard to the
Ser82 phosphorylation site in vimentin, a time-dependent
increase in phosphorylation was observed (from 5 to 30 min) in
the presence of sVCAM-1 (Fig. 7B). Thus, the
CaM kinase II activation pathway is involved in sVCAM-1 signaling which
induces T cell-chemotaxis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, RANTES, and MIP1
and -ß) or as a result of exposure to numerous costimulatory and
adhesion molecules such as CD28, CD44, and various integrins (18, 25, 26, 27). In fact, the above-mentioned chemokines are known to
up-regulate the ß1 integrin affinity rapidly, as seen in
PMA stimulation through PKC (6, 9). Thus, it is possible that sVCAM-1
acts as a chemotactic factor for partially activated T cells exposed to
these substances as they pass through the synovium.
T cells from RA synovium were found to be enriched in CD4+
memory T cells that exhibited an enhanced intrinsic capacity for
transendothelial migration as assayed in a HUVEC monolayer over
collagen gel (20). In another report, RA T cells from SF and synovial
tissue were found to be markedly enriched in CD45RAdim,
CD45RO+, CD45RBdim, and CD27-
mature memory cells, had low proliferative ability, but exhibited
potent helper activity for B cell Ig production (28). We found that RA
SF T cells were spontaneously (intrinsically) chemokinetic without
chemoattractant and showed a significantly higher chemotactic response
to sVCAM-1. The observed high chemokinesis in the absence of
chemoattractant seems partly due to the binding of sVCAM-1 to the cell
surface of RA SF T cells (7). We further demonstrate that
CD45RO-enriched SF T cells are most notably chemotactic toward sVCAM-1.
These results indicate that the migration of SF T cells mediated by VLA
integrin may be involved in a memory T cell state that is
characteristic for T cells responsive to CC chemokine, RANTES, or
MIP1 and -ß (29, 30). Roth et al. found that RANTES and
MIP1 and -ß induced significant transendothelial chemotaxis of
CD4+, CD8+, and CD45RO+ T cell
subsets. Very recently, it was reported that CXCR3- and CCR5-expressing
T cells were mostly CD45RO memory type Th1 cells and generally
expressed high levels of ß1 integrins. Furthermore, most
T cells in SF express CXCR3 and represent a high enrichment of
CXCR3+ T cells in blood. sVCAM-1 effect on T cells with
high affinity VLA-4 showed migration activity comparable to that of
chemokines that bind to CXCR3 (IP-10, Mig) and CCR5 (RANTES, MIP1,
and MIP1ß) (31). These findings support the idea that the high
migration activity of T cells in SF is mediated by sVCAM-1 in concert
with the chemokines that bind to CXCR3 and CCR5.
A number of soluble forms of adhesion molecules have been identified in SF, e.g., soluble intercellular adhesion molecule-1 (sICAM-1), sE-selectin, and sL-selectin, in addition to sVCAM-1, indicating inflammatory interactions between endothelial cells, leukocytes, and other synovial cells in the diseased joint (32, 33, 34). These soluble adhesion molecules may modulate the recruitment and retention of inflammatory cells into SF as well as tissue. In fact, there is a gradient of sVCAM-1 concentration between plasma (9.8 ± 2.8 nM) and SF (17.3 ± 4.4 nM) in patients with RA (n = 11, p < 0.005) consistent with an earlier report (35). These concentrations of sVCAM-1 are within the range of eliciting chemotactic activity in T cells. In this regard, it was also shown that the chemotactic activity of rheumatoid SF for endothelial cells, and its angiogenic activity, were blocked by Abs to either sVCAM-1 and sE-selectin (5).
It was recently reported that the interaction between LFA-1 and ICAM-1
decreases T cell adhesion mediated by 4ß1
and 5ß1, suggesting that the integrin
cross-talk decreases 4ß1 integrin-mediated
binding of T cells to fibronectin and VCAM-1 (36). This cross-talk
between ß1 and ß2 integrins may be the
reason that anti-ICAM-1 ligation has an inhibitory effect on the
chemotaxis of IL-2-dependent T cells induced by sVCAM-1. The same
inhibition does not apply to the Jurkat T cell line, because this line
is known to have a defect in LFA-1 signaling (37).
Signal transduction in sVCAM-1-induced T cell chemotaxis should be considered as two components, 1) outside-in signaling triggered by VLA-4 generated through the binding of sVCAM-1 in the fluid phase, which induced only VLA receptor occupancy, not receptor clustering, and exhibits incomplete signaling when compared with the signaling typically generated in cell adhesion; and 2) a prerequisite signal for cell migration due to cytoplasmic mobilization induced by a G-protein-coupled receptor such as a chemokine receptor. With regards to outside-in signaling, most studies premised immobilized ligands such as fibronectin and anti-VLA mAbs (38, 39, 40). Adhesion-induced integrin engagement induced tyrosine phosphorylation of pp125FAK, followed by the subsequent phosphorylation and assembly of various signaling and cytoskeletal proteins that form a focal adhesion structure (22, 23, 41, 42). In contrast, we found that the signal triggered by soluble VCAM-1 in the fluid phase did not effectively induce tyrosine phosphorylation of pp125FAK. In addition, T cells migrated toward sVCAM-1 even in the presence of genistein. Thus, tyrosine phosphorylation of pp125FAK might not be a prerequisite for T cell migration.
With regard to a prerequisite signal for cell migration, our finding
that sVCAM-1-induced migration is related to signaling by Rho may well
be interpreted by the observation of Laundanna et al. (43). They found
that IL-8-stimulated guanine nucleotide exchange on RhoA induces
4ß1 adhesion to VCAM-1 in the Jurkat cells
transfected with IL-8R and that adhesion was blocked by C3 exoenzyme,
which blocked Rho activation. Their transfection experiment coupling
chemoattractant receptor-triggered signaling to the adhesion-inducing
pathway indicated that Rho participates in both cell migration and
adhesion signaling pathways. This notion is consistent with our finding
that T cell chemotaxis in response to sVCAM-1, an adhesion molecule
adhered to by T cells in the fluid phase, is inhibited by C3 exoenzyme.
The roles of Rho in adhesion triggering and chemotaxis are also
explained by the finding that Rho-microinjected Swiss 3T3 fibroblasts
induce phosphorylation of pp125FAK, p130, and paxillin, which are known
to localize to focal adhesion and formations of stress fiber. Also,
Rho-induced phosphorylation of pp125FAK, p130, and paxillin is observed
in the absence of stress fiber formation and is, therefore, independent
of Rho-induced actin polymerization (42, 44).
In regard to migration involved in protein kinase C activation, haptotaxis of the Ag-specific human T cell line and PBL-T cells triggered by immobilized anti-VLA-4 was inhibited by calphostin, an inhibitor of PKC, while the effect of tyrosine kinase inhibitor on the haptotaxis depended on the cell type (40).
CaM kinase II was first demonstrated to be required for the migration of vascular smooth muscle cells (45). We found that blocking the activation of CaM kinase II by KN62 inhibited T cell chemotaxis in response to sVCAM-1 by more than 80%, as well as inhibition of protein kinase C activation by H7. Using mAb specific for phosphorylated Ser82 in the vimentin by CaM kinase II, we showed that IL-2 T chemotaxis in response to sVCAM-1 generated CaM kinase II activation. Integrin-mediated elevation of intracellular Ca2+ levels in Jurkat T cells was recently described (46), and activation of CaM kinase II by calmodulin may follow after intracellular Ca2+ signaling. Moreover, it was reported that Jurkat T cells transfected with a constitutively active mutant of CaM kinase II showed a transcriptional block of IL-2 reminiscent of T cell anergy (47). In this respect, the rheumatoid synovium has been proposed as a model of T cell anergy (48), and we previously reported that the T cells in RA SF were bound by sVCAM-1 and anergic in T cell proliferative responses (7).
Finally, two distinct features, highly chemotactic and anergic responsiveness of T cells in RA SF, have been characterized. sVCAM-1 generated in the lining layer of rheumatoid synovium might participate in the acquisition of these functional features of T cells in RA SF.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Atsushi Kitani at the current address: Bldg 10, 11N238, Mucosal Immunity Section, Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892.
3 Abbreviations used in this paper: sVCAM-1, soluble form of VCAM-1; VLA-4, very late antigen-4; CaM kinase II, Ca2+/calmodulin-dependent kinase II; PKC, protein kinase C; IL-2 T, IL-2-dependent T cells; RA, rheumatoid arthritis; SF, synovial fluid; MIP, macrophage inflammatory protein.
Received for publication September 25, 1997. Accepted for publication June 22, 1998.
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References |
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4ß1 integrin-dependent cell adhesion is regulated by a low affinity receptor pool that is conformationally responsive to ligand. J. Biol. Chem. 270:28740.
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 B. P. Harvey, S. S. Banga, and H. L. Ozer Regulation of the Multifunctional Ca2+/Calmodulin-dependent Protein Kinase II by the PP2C Phosphatase PPM1F in Fibroblasts J. Biol. Chem., June 4, 2004; 279(23): 24889 - 24898. [Abstract] [Full Text] [PDF]
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 G. C. Koo, K. Shah, G. J. F. Ding, J. Xiao, R. Wnek, G. Doherty, X. C. Tong, R. B. Pepinsky, K.-C. Lin, W. K. Hagmann, et al. A Small Molecule Very Late Antigen-4 Antagonist Can Inhibit Ovalbumin-induced Lung Inflammation Am. J. Respir. Crit. Care Med., May 15, 2003; 167(10): 1400 - 1409. [Abstract] [Full Text] [PDF]
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A. Poggi, M. R. Zocchi, R. Carosio, E. Ferrero, D. F. Angelini, S. Galgani, M. D. Caramia, G. Bernardi, G. Borsellino, and L. Battistini Transendothelial Migratory Pathways of V{delta}1+TCR{gamma}{delta}+ and V{delta}2+TCR{gamma}{delta}+ T Lymphocytes from Healthy Donors and Multiple Sclerosis Patients: Involvement of Phosphatidylinositol 3 Kinase and Calcium Calmodulin-Dependent Kinase II J. Immunol., June 15, 2002; 168(12): 6071 - 6077. [Abstract] [Full Text] [PDF]
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 Z. Li, M. J. Calzada, J. M. Sipes, J. A. Cashel, H. C. Krutzsch, D. S. Annis, D. F. Mosher, and D. D. Roberts Interactions of thrombospondins with {alpha}4{beta}1 integrin and CD47 differentially modulate T cell behavior J. Cell Biol., April 29, 2002; 157(3): 509 - 519. [Abstract] [Full Text] [PDF]
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 J. R. Chan, S. J. Hyduk, and M. I. Cybulsky Chemoattractants Induce a Rapid and Transient Upregulation of Monocyte {alpha}4 Integrin Affinity for Vascular Cell Adhesion Molecule 1 Which Mediates Arrest: An Early Step in the Process of Emigration J. Exp. Med., May 14, 2001; 193(10): 1149 - 1158. [Abstract] [Full Text] [PDF]
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D. M. Rose, P. M. Cardarelli, R. R. Cobb, and M. H. Ginsberg Soluble VCAM-1 binding to alpha 4 integrins is cell-type specific and activation dependent and is disrupted during apoptosis in T cells Blood, January 15, 2000; 95(2): 602 - 609. [Abstract] [Full Text] [PDF]
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Z. Li, M. J. Calzada, J. M. Sipes, J. A. Cashel, H. C. Krutzsch, D. S. Annis, D. F. Mosher, and D. D. Roberts Interactions of thrombospondins with {alpha}4{beta}1 integrin and CD47 differentially modulate T cell behavior J. Cell Biol., April 29, 2002; 157(3): 509 - 519. [Abstract] [Full Text] [PDF]
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