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The Journal of Immunology, 1999, 163: 4253-4261.
Copyright © 1999 by The American Association of Immunologists

LFA-1 to LFA-1 Signals Involve {zeta}-Associated Protein-70 (ZAP-70) Tyrosine Kinase: Relevance for Invasion and Migration of a T Cell Hybridoma1

Ron D. M. Soede, Mariëtte H. E. Driessens2, Lisette Ruuls-Van Stalle, Paula E. M. Van Hulten, Arjen Brink and Ed Roos3

Division of Cell Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We previously showed that LFA-1-dependent in vitro invasion and in vivo migration of a T cell hybridoma was blocked in cells overexpressing a truncated dominant-negative {zeta}-associated protein (ZAP)-70. The truncated ZAP-70 also blocked LFA-1-dependent chemotaxis through ICAM-1-coated filters induced by 1 ng/ml stromal cell-derived factor-1, but not LFA-1-independent chemotaxis induced by 100 ng/ml stromal cell-derived factor-1. This suggested that LFA-1 engagement triggers a signal that amplifies a weak chemokine signal and that dominant-negative ZAP-70 blocks this LFA-1 signal. Here we show that cross-linking of part of the LFA-1 molecules with Abs causes activation of free LFA-1 molecules (not occupied by the Ab) on the same cell, which then bind to ICAM-2 on other cells. This causes cell aggregation that was also blocked by dominant-negative ZAP-70. Thus, an LFA-1 signal involving ZAP-70 activates other LFA-1 molecules, suggesting that the chemokine signal can be amplified by multiple cycles of LFA-1 activation. The chemokine and the LFA-1 signal were both blocked by a phospholipase C inhibitor and a calpain inhibitor, suggesting that one of the amplified signals is the phospholipase C-dependent activation of calpain. Finally, we show that both Src-homology 2 domains are required for inhibition of invasion, chemotaxis, and aggregation by the truncated ZAP-70, suggesting that ZAP-70 interacts with a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) sequence. Remarkably, this is not an ITAM in the TCR/CD3 complex because this is not expressed by this T cell hybridoma.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The integrin LFA-1 (CD11a/CD18) is involved in intercellular adhesion among leukocytes and between leukocytes and other cell types (1, 2, 3). It is essential for the initiation of an immune response as well as for many leukocyte effector functions. LFA-1 is one of the adhesion molecules involved in the influx of leukocytes into tissues (4, 5, 6, 7). This migration can be studied using T cell hybridomas as a model. Like the activated T cells or the T cell clones from which they were generated, T cell hybridomas are highly invasive and migrate into many different tissues upon i.v. injection. Because of the autonomous growth capacity derived from the lymphoma fusion partner, this can be easily detected because of the extensive proliferation of the cells in the invaded tissues (8, 9). LFA-1 is essential for this invasion, because LFA-1-deficient mutants do not disseminate at all (10).

Adhesion mediated by LFA-1 is tightly regulated (1). LFA-1 is usually not active, i.e., LFA-1-expressing leukocytes do not spontaneously adhere to LFA-1 ligands. Physiologically relevant signals that activate LFA-1 are triggered by the TCR/CD3 complex upon interaction with MHC molecules on APCs or on target cells of cytotoxic T cells (11). Furthermore, LFA-1 and other integrins are activated by cytokines such as IL-2 (12) and by chemokines and other inflammation mediators such as platelet-activating factor, relevant for leukocyte influx into inflamed tissues (13, 14, 15). This phenomenon has been termed "inside-out" signaling (16) and can be mimicked by binding of Abs against CD3 (17, 18) and against several other surface molecules, including CD2 (18), CD28 (19), and CD44 (20).

Upon ligand engagement, LFA-1 not only provides physical attachment but it also generates signals that, depending on the circumstances, may be required for proliferation (21) or prevention of apoptosis (22). Thus, binding to either the physiological ligand ICAM-1 or to Abs provides costimulatory signals for activation of T cells (21, 23, 24). This phenomenon is termed "outside-in" signaling (16). Some Abs directed against LFA-1 induce aggregation of cells. In part, these mAbs are directed against "activation epitopes" and may act by stabilizing the "active" conformation of the integrins (25, 26, 27, 28). However, aggregation can also be induced by mAbs against epitopes that are not activation sensitive (29, 30). This suggests that outside-in signals generated by Ab binding trigger inside-out signals that lead to integrin activation. These mAbs often block the interactions of their cognate integrins with ligand and it was thus thought that the aggregation was mediated by other integrins or nonintegrin adhesion molecules. Indeed, triggering of one integrin subtype by another does occur (31). However, Koopman et al. (29) showed that aggregation induced by an LFA-1 mAb occurred at low but not high mAb concentrations and was mediated by LFA-1 itself. This suggested that triggering of part of the LFA-1 molecules, at a subsaturating mAb concentration, led to activation of other LFA-1 molecules that were not occupied by the mAb. In this report, we demonstrate that this is in fact true for aggregation of mouse T cell hybridoma cells induced by the M17/4 and GAME-46 LFA-1-blocking mAbs.

Recently, we showed that the kinase activity of the {zeta}-associated protein-70 (ZAP-70)4 tyrosine kinase is essential for LFA-1-dependent in vitro invasion and in vivo dissemination of T cell hybridomas (32). Invasion and dissemination are also blocked by pertussis toxin (33), indicating that Gi protein-coupled receptors such as chemokine receptors are involved, suggesting that invasion is triggered by a chemokine. A potentially involved chemokine is stromal cell-derived factor (SDF)-1 (34), which is a potent chemoattractant for T cells (35) and also for these T cell hybridomas. SDF-1 is present in many noninflamed tissues and may therefore be involved in the migration of the T cell hybridoma cells into these tissues. We found that LFA-1 is required for migration through ICAM-1-coated filters induced by a low concentration (1 ng/ml) of SDF-1, whereas migration induced by a high SDF-1 concentration (100 ng/ml) was independent of LFA-1. ZAP-70 was essential for the LFA-1-dependent chemotaxis but not when LFA-1 was not required. Furthermore, we showed that ZAP-70 is involved in the tyrosine phosphorylation induced by LFA-1 engagement. Thus, the combination of low SDF-1 levels and an ICAM-1-coated filter appears to provide a suitable model for the in vivo migration of the T cell hybridoma, because both the in vivo and in vitro migration require the activity of the pertussis toxin-sensitive Gi proteins, LFA-1 and ZAP-70. The results suggested that the signal, initiated by the few SDF-1 receptors triggered at low SDF-1 levels, activates a limited number of LFA-1 molecules, which can then bind to ICAM-1. This then leads to ZAP-70 activation and amplification of the signal. We show here that ZAP-70 activity triggered by LFA-1 engagement leads to adhesion mediated by unengaged LFA-1 molecules. This combination of outside-in and inside-out signals may be responsible for the propagation and amplification of the chemokine signal.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

The Syk/ZAP-70-specific inhibitor piceatannol was obtained from Boehringer Mannheim (Mannheim, Germany); and the protein kinase C (PKC) inhibitor Ro 31-8220 and the calpain inhibitor calpeptin were from Calbiochem (La Jolla, CA). The phospholipase C (PLC) inhibitor U-73122 (1-{{6-{[(17ß-3-methoxestra-1,3,5(10)trien-17-yl]amino}hexyl}}-1H-pyrrole-2, 5-dione) and its inactive congener, U-73343 (1-{{6-{[17ß-3-methoxestra-1,3,5(10)trien-17-yl]amino}hexyl}}-2,5-pyrrolidine-dione) were from Biomol (Plymouth, MA). Both U-73122 and U-73343 were dissolved in chloroform and dried under a nitrogen stream. Just before use, the dried film was dissolved in DMSO to a concentration of 5 mM. Ro 31-8220 was dissolved in DMSO to 1 mM, aliquoted, and kept at -80°C. Calpeptin was dissolved in DMSO at 10 mg/ml and kept at -20°C. Piceatannol was dissolved in 30% DMSO at 2.4 mg/ml and kept at -20°C.

Cell lines and culture conditions

Murine TAM2D2 T cell hybridoma cells (8) and rat embryo fibroblasts were cultured as described previously (36).

Antibodies

Hybridomas producing rat anti-murine CD11a mAb M17/4, anti-ICAM-1 mAb YN1/1.7, and CD44 mAb KM201 were obtained from the American Type Culture Collection (Manassas, VA). Two new rat anti-mouse ß2 (CD18) hybridomas, GAME-46 (IgG1) and -245 (IgG2a), were generated previously by us using purified LFA-1 as immunogen (37). The GoH3 mAb against CD49f, the {alpha}6ß1 integrin {alpha} subunit, was kindly donated by A. Sonnenberg. Rat anti-mouse ICAM-2 mAb MIC2/4 (3C4) was purchased from PharMingen (San Diego, CA), and rat IgG was purchased from Nordic Immunology (Tilburg, The Netherlands). All mAbs were affinity purified. Fab fragments were generated using immobilized papain according to the manufacturer’s protocol (Pierce, Rockford, IL). F(ab')2 fragments were generated with pepsin and concentrated using a Centricon 100 (Millipore, Bedford, MA) to remove the Fc parts. The mAb 2F3.2 against human ZAP-70 (38) was kindly supplied by Dr. A. Weiss.

Aggregation assay

To induce aggregation, M17/4 mAb was added to TAM2D2 cells (2 x 106/ml in HBSS, pH 7.0, supplemented with 20 mM HEPES, 0.35 g/l NaHCO3, 1 mM CaCl2, and 1 mM MgCl2) to a final concentration of 0.2 µg/ml or as indicated. The total volume was 0.5 ml in 10-ml tubes. Alternatively, cells were incubated for 20 min on ice with different concentrations of M17/4 Fab fragments as indicated. Subsequently, rabbit anti-rat Ig Abs (RARa/7S, dilution: 1/500; Nordic Immunology) were added. The cells were incubated in a waterbath at 37°C for 2 h in an upright position and shaken at low speed. By mild agitation with a wide-bore pipette, the suspensions were then dispersed, and 100-µl samples were transferred to a flat-bottom dish and photographed using an inverted microscope. To test the effects of Abs or inhibitors, the cells were preincubated with the mAbs for 15 min or with inhibitors for 30 min at room temperature before addition of the M17/4 mAb.

Determination of number of free LFA-1 molecules by FACScan analysis

TAM2D2 cells were incubated with the M17/4 mAb or the M17/4 F(ab')2 or Fab fragments at different concentrations, ranging from 0.02 to 10 µg/ml. After 2 h of incubation, aggregation was scored as described above. A sample of the cells (2 x 105) was removed, vigorously pipetted to disperse the aggregates, washed with FACS buffer (PBS with 0.5% BSA and 0.02% azide), and subsequently incubated with FITC-labeled M17/4 (10 µg/ml) for 30 min at 4°C. The sample was washed twice and fluorescence was measured on a FACScan (Becton Dickinson, San Jose, CA) using the lysis II program. Cells that had not been exposed to Abs were used as a negative control. The median fluorescence of cells that had not been preincubated with unlabeled M17/4 mAb at all but only with the FITC-labeled mAb minus the median background fluorescence of control cells (F) was set at 100%. The median fluorescence of the samples minus the background fluorescence as a percentage of F is a measure of the percentage of LFA-1 molecules that was not occupied by the nonlabeled M17/4 mAb.

Generation and transduction of DNA constructs

Both the construction and the transduction of constructs have been described previously (32). Briefly, the cDNAs encoding the truncated human ZAP-701–276(1–276) (38) or the full-length wild-type (WT) ZAP-70 protein, cloned in the retroviral vector pMFG-IRES-geo (geo = fusion protein of lacZ and neoR), were transfected into the BOSC23 packaging cell line. TAM2D2 cells were infected with the retrovirus by coculture or incubation with BOSC23 supernatant, and clones with homogeneous high and stable lacZ expression were selected by subcloning and FACS sorting. The internal ribosome entry site (IRES) in the vector allows the translation of both the ZAP-70 and geo protein from one bicistronic mRNA (39). High lacZ activity correlated with high expression of ZAP-70, which was at least 10-fold higher than endogenous ZAP-70 in the clones used in this study, DN22 and DN38. As controls, we generated cells expressing similarly high levels of the WT full-length ZAP-70 protein. Truncated ZAP-70 constructs with a point mutation in either the N-terminal Src-homology domain 2 (SH2) (R37K) or the C-terminal SH2 domain (R190K) (40) were transduced similarly. The N-terminal SH2 domain mutant was generated by PCR, and the C-terminal SH2 mutant was kindly supplied by Dr. A. Weiss. The presence of the mutations was verified by sequencing.

Immunoblotting

SDS-PAGE-separated cell lysates were blotted to nitrocellulose, which was then blocked with 3% BSA and 0.4% Tween 20. The membranes were incubated for 1 h with the mAb 2F3.2 against human ZAP-70 at room temperature, followed by incubation with sheep anti-mouse HRP-coupled Ig (Amersham, Little Chalfont, U.K.). Stained proteins were visualized by chemiluminescence.

Adhesion to anti-LFA-1 mAb-coated substrates

Wells in 96-well plates were coated with 100 µl of Ab solution (5 µg/ml) overnight at 4°C, followed by blocking for 2 h at room temperature with 0.5% OVA (Sigma, St. Louis, MO). Cells were allowed to adhere for 10 min in 20 mM Tris buffer, pH 7.2 (containing 150 mM NaCl, 6 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 5 mM D-glucose). For rapid warming up of the plate, it was placed on a prewarmed metal block in an incubator at 37°C, 5% CO2. Nonadherent cells were washed off, and the number of adherent cells was determined by assaying hexosaminidase activity using known numbers of cells as standard essentially as described by Landegren et al. (41).

Invasion and migration assays

Invasion assays were performed as described (36). Briefly, TAM2D2 cells or TAM2D2 transfectants were added to confluent rat embryo fibroblast monolayers in serum-free medium. After 1 h at 37°C and 5% CO2, the monolayers were extensively washed and then paraformaldehyde-fixed. The invaded cells were counted using phase-contrast microscopy, and the percentage of invaded cells was calculated. Migration was assayed in Transwells, as described (32). Briefly, Transwells with 8-µm pore filters, coated with 1 µg/ml recombinant monomeric truncated soluble mouse ICAM-1 (without transmembrane and cytoplasmic domains) were used. The lower chamber was filled with 250 µl RPMI 1640 containing 0.1% OVA and either 1 or 100 ng/ml SDF-1{alpha} (Pepro Tech, Rocky Hill, NJ). The Transwell was placed on top, and 150 µl medium with 105 cells was inserted into the upper chamber. The data presented are the percentages of added cells that have been collected from the lower chamber after 2 h at 37°C, 5% CO2.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
M17/4 mAb induces LFA-1/ICAM-2-mediated aggregation of TAM2D2 cells

TAM2D2 T cell hybridoma cells express the adhesion molecules LFA-1, {alpha}6ß1, CD44, and ICAM-2 but not ICAM-1 (9). LFA-1 is the only ß2 integrin present. We found that the M17/4 mAb against the {alpha} subunit of LFA-1 induced aggregation of TAM2D2 cells. Aggregation was noticeable within 30 min, but the aggregates reached their maximum size usually after 2 h of incubation with 0.2 µg/ml M17/4 mAb. To determine which adhesion molecules were involved, we preincubated the cells with inhibitory mAbs against ICAM-2, the {alpha}6 integrin subunit, and CD44, as well as rat IgG as a negative control. The anti-murine ICAM-2 mAb MIC2/4 prevented aggregation almost completely (Fig. 1Go), whereas rat IgG and the blocking mAbs GoH3 against {alpha}6 (Fig. 1Go) and KM201 against CD44 (data not shown) had no effect. This shows that aggregation is mediated by the binding of LFA-1 to ICAM-2 and is not caused by agglutination of the cells by the M17/4 mAb. Indeed, two anti-ß2 (CD18) mAbs generated by us, GAME-46 and -245 (37), blocked aggregation completely (Fig. 1Go and data not shown). By using flow cytometry with FITC-labeled M17/4 mAb, we established that the GAME-46 and -245 mAb did not inhibit binding of M17/4 to LFA-1 (data not shown), so that inhibition is not due to interference with the binding of M17/4 to LFA-1.



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  		FIGURE 1. M17/4-induced aggregation of TAM2D2 cells is mediated by LFA-1 to ICAM-2 binding. Aggregation was induced by addition of M17/4 mAb at 0.2 µg/ml. The picture is representative of aggregation 2 h after addition of M17/4 and shows that preincubation with saturating concentrations (10 µg/ml) of anti-ß2 mAb GAME-46 and anti-ICAM-2 mAb MIC2/4, but not by anti-{alpha}6 mAb GoH3, blocks M17/4-induced aggregation.

 
Aggregation is caused by cross-linking of part of the LFA-1 molecules

The induction of LFA-1/ICAM-2-dependent aggregation by the M17/4 Ab was surprising because M17/4 is a function-blocking mAb. We found, however, that aggregation only occurred at M17/4 concentrations lower than the saturating concentration of 10 µg/ml and most optimally at 0.2–0.5 µg/ml. To determine the number of free LFA-1 molecules at this subsaturating concentration, we used FACS analysis on cells treated with different concentrations of unlabeled M17/4, and subsequently, after 2 h, with 10 µg/ml FITC-labeled M17/4. The results are shown in Fig. 2GoA. We found that ~25% of LFA-1 molecules were occupied by M17/4 mAb at 0.2 µg/ml and ~50% at 0.5 µg/ml. Aggregation was reduced at 1 µg/ml, with ~80% of LFA-1 occupied. F(ab')2 fragments induced aggregation at similar concentrations (data not shown), whereas Fab fragments did not induce aggregation at similar occupation percentages (determined similarly, see Fig. 2GoB). We conclude that signals triggered by cross-linking of LFA-1 molecules by the M17/4 Ab cause the unoccupied LFA-1 molecules to bind to ICAM-2, leading to aggregation of the cells.



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  		FIGURE 2. Aggregation of TAM2D2 cells is induced at subsaturating concentrations of the anti-LFA-1 mAb M17/4 and requires cross-linking of LFA-1 molecules. The extent of aggregation of TAM2D2 cells was scored as: +, small; ++, medium size; +++, large; ++++, very large aggregates, after 2 h of incubation with the M17/4 mAb (A) or M17/4 Fab fragments (B) in a concentration range from 0.01 to 10 µg/ml. To induce aggregate formation, M17/4 Fab fragments were cross-linked with polyclonal anti-rat Ig Abs. For comparison, the effect of cross-linking the intact M17/4 mAb is also shown in A. Triangles, M17/4 mAb; stars, M17/4 mAb + anti-rat Ig; hexagons, M17/4 Fab alone; diamonds, M17/4 Fab + anti-rat Ig. Also shown are the percentages of occupied LFA-1 molecules (squares) after 2 h of incubation with the indicated concentrations of M17/4 Abs (A) or M17/4 Fab fragments (B). Data are averages from three independent experiments.

 
To demonstrate that aggregation was due to cross-linking, we preincubated cells with M17/4 Fab fragments, followed by incubation at 37°C with anti-rat Ig Abs. Aggregation comparable with that maximally induced by intact M17/4 required a slightly higher extent of occupation (~40%), but between ~50 and 80% occupation aggregation was more extensive, with larger aggregates (Fig. 2GoB). This may be due to more efficient cross-linking. Indeed, aggregation by intact M17/4 was also enhanced by anti-rat Ig Abs: it started at slightly lower M17/4 concentrations and the aggregates were very large at the optimal concentration (Fig. 2GoA). Incubation with anti-rat Ig Abs alone did not induce aggregation (data not shown).

Aggregation induced by LFA-1 engagement is blocked by dominant-negative ZAP-70

To study the signal pathways involved in the M17/4-induced LFA-1 activation, we tested several inhibitors. Piceatannol, an inhibitor of the Syk tyrosine kinase (42), blocked aggregation completely (Table IGo). We have shown previously that the Syk homologue ZAP-70, but not Syk, is expressed by TAM2D2 cells. Furthermore, we showed that piceatannol inhibits ZAP-70 activity with the same dose dependence as reported for Syk with a complete block at 50 µg/ml (32).


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  		Table I. Role of ZAP-70 in aggregation and effects of PLC and calpain inhibitors1

 
To prove that ZAP-70 is involved in aggregation, we used TAM2D2 cells expressing high levels of a truncated ZAP-70 protein (32), which inhibits TCR signaling in a dominant-negative fashion (38). We have shown that this truncated ZAP-70 also blocks the induction of ZAP-70 tyrosine kinase activity upon engagement of LFA-1 (32). We used two independent clones, DN22 and DN38, with similar LFA-1 levels as TAM2D2 cells (data not shown). Neither of the two aggregated upon incubation with 0.2 µg/ml M17/4 (see Table IGo and Fig. 4Go), except for the occasional formation of a few small cell clumps. Cross-linking of Fab fragments with anti-Ig Abs did not induce aggregation of these cells either (data not shown). In contrast, WT cells expressing similarly high levels of the full-length WT human ZAP-70 protein (32), as well as cells transduced with empty vector, aggregated as extensively as the parental TAM2D2 cells (Table IGo). Furthermore, we recently generated cells that express a truncated ZAP-70 with a point mutation in either the C-terminal SH2 domain (R190K) or the N-terminal SH2 domain (R37K) at similar levels as the nonmutated truncated ZAP-70 (Fig. 3Go). Two independently generated clones of each (C-SH2.7, C-SH2.16 and N-SH2.10, N-SH2.15) were examined. Aggregation of these cells was comparable to that of TAM2D2 cells (Table IGo and Fig. 4Go), showing that both SH2 domains are required for the dominant-negative effect and therefore both are essential for the participation of ZAP-70 in LFA-1 function. The ZAP-70-transduced cells, including WT cells that overexpress the intact ZAP-70, did not aggregate spontaneously.



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  		FIGURE 4. M17/4-induced aggregation of TAM2D2 cells is blocked by dominant-negative ZAP-70. Aggregation was induced by addition of M17/4 mAb at 0.2 µg/ml. The picture is representative of aggregation 2 h after addition of M17/4 and shows that overexpression of dominant-negative truncated ZAP-70 (DN22) blocks aggregation, whereas the N-terminal (N-SH2.10) and C-terminal (C-SH2.7) SH2 domain mutants of this truncated ZAP-70 have no effect.

 


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  		FIGURE 3. Expression levels of dominant-negative truncated ZAP-70 and SH2 mutants. Levels of overexpressed dominant-negative human ZAP-70 in clones DN22 and DN38, the C-terminal SH2 domain mutant of this truncated ZAP-70 in clones C-SH2.7 and C-SH2.16, and the N-terminal SH2 domain mutant in clones N-SH2.10 and N-SH2.15, detected by the mouse anti-human ZAP-70 mAb 2F3.2.

 
Aggregation is blocked by inhibitors of PLC and calpain

A potential downstream effector of ZAP-70 is PLC-{gamma}. To investigate its possible involvement, we used the PLC inhibitor U-73122. This inhibited aggregation completely, whereas the inactive structural analogue U-73343 had no effect (Table IGo). PLC generates two products: diacylglycerol, which activates PKC, and inositol-trisphosphate which releases Ca2+. The PKC inhibitor Ro 31-8220 did not affect aggregation (Table IGo), and PKC is therefore probably not involved. One of the possible effectors activated by Ca2+ is the calcium-dependent protease calpain, which was recently proposed to be involved in LFA-1 activation (43). In agreement with this, the calpain inhibitor calpeptin inhibited aggregation completely (Table IGo).

Adhesion to immobilized LFA-1 mAb: roles of ZAP-70, PLC, and calpain

To further study the effects of signals induced by the binding of the M17/4 anti-LFA-1 mAb, we allowed cells to adhere to an M17/4-coated substrate. This adhesion is mediated by LFA-1, because preincubation of cells with M17/4 Abs prevented adhesion completely, whereas control Abs did not (data not shown). TAM2D2 cells readily adhered to these surfaces (Fig. 5Go). In contrast, the DN22 and DN38 clones, overexpressing dominant-negative ZAP-70, did not adhere at all, i.e., did not adhere strongly enough to resist the shear forces of the washing steps. Furthermore, piceatannol completely blocked adhesion of TAM2D2 cells (Fig. 5GoA). In contrast, truncated ZAP-70 with a mutation in either of the two SH2 domains had no effect at all (Fig. 5GoB). In addition, adhesion was completely blocked by U-73122 but not U-73343 and calpeptin but not Ro 31-8220 (Fig. 5GoC).



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  		FIGURE 5. Adhesion of TAM2D2 cells to immobilized M17/4 mAb requires ZAP-70 and is blocked by PLC and calpain inhibitors. A, Adhesion is blocked by treatment of TAM2D2 cells with piceatannol and by expression of dominant-negative truncated ZAP-70 (DN22 and DN38). B, Overexpression of N-terminal and C-terminal SH2 domain mutants of truncated ZAP-70 does not block adhesion to M17/4 (clones N-SH2.10 and N-SH2.15 and C-SH2.7 and C-SH2.16, respectively). C, Inhibitors of PLC and calpain, but not PKC, block adhesion to M17/4. TAM2D2 cells were pretreated with inhibitors as described in Materials and Methods. Data are averages + SEM of three experiments performed in triplicate.

 
T cell hybridoma cells do not adhere to an immobilized nonblocking LFA-1 Ab

In addition to M17/4, which reacts with the {alpha}L subunit, the blocking GAME-46 Ab against the ß2 subunit induced aggregation at subsaturating concentrations (data not shown). In contrast, the nonblocking M18/2 Ab against the ß2 subunit did not. Indeed, TAM2D2 T cell hybridoma cells adhered extensively to and spread on the immobilized blocking Abs M17/4 and GAME-46, but not the nonblocking Ab M18/2 (Figs. 6Go and 7). This was not due to a difference in affinity, which was similar for all three Abs. As determined by FACS analysis (similarly as in Fig. 2Go), half-maximal binding occurred at ~0.5 µg/ml (M17/4, 0.48 µg/ml; GAME-46, 0.56 µg/ml; and M18/2, 0.54 µg/ml).



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  		FIGURE 6. TAM2D2 cells adhere to and spread on immobilized LFA-1 blocking Abs but not to a nonblocking Ab. TAM2D2 cells adhered to the blocking mAbs M17/4 (A) and GAME-46 (B) against the {alpha}L and ß2 subunit of LFA-1, respectively, but did not adhere to the nonblocking mAb M18/2 (C) against the ß2 subunit. Shown are representative micrographs.

 
Effects of ZAP-70, PLC, and calpain inhibitors on invasion and migration

Invasiveness of cells was tested in rat embryo fibroblast cultures (36). TAM2D2 cells invaded the monolayers within 1 h. As shown previously (32), invasion was completely inhibited by piceatannol at 50 µg/ml, and both DN22 and DN38 cells expressing truncated dominant-negative ZAP-70 had lost invasive capacity (Fig. 8GoA). Here we show that the cells expressing the truncated ZAP-70 with a mutation in either of the two SH2 domains invaded normally (Fig. 8GoA). Furthermore, we show that invasion is blocked by U-73122 but not U-73343 and by calpeptin but not by Ro 31-8220 (Fig. 8GoB).



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  		FIGURE 8. LFA-1-dependent invasion of TAM2D2 cells requires ZAP-70 and is blocked by PLC and calpain inhibitors. A, Expression of dominant-negative truncated ZAP-70 blocks invasion of TAM2D2 cells into fibroblast monolayers (clones DN22 and DN38), whereas N-terminal and C-terminal SH2 domain mutants of this truncated ZAP-70 have no effect (clones N-SH2.10 and N-SH2.15 and C-SH2.7 and C-SH2.16, respectively). B Invasion of TAM2D2 cells is blocked by inhibitors of PLC (U73122) and calpain (calpeptin) but not by a PKC inhibitor (Ro 31-8220). Shown are the percentages of cells that have invaded fibroblast monolayers after 1 h. Data are averages + SEM of three experiments performed in duplicate.

 
As we have shown previously (32), a simplified model for this invasion is migration induced by low SDF-1 concentrations through ICAM-1-coated filters, which is blocked by both pertussis toxin and by LFA-1 Abs, similar to invasion. SDF-1 is a major candidate for being involved, because it is constitutively expressed in many tissues, unlike most CC-chemokines, which are mainly found in inflamed tissues. In fact, chemokines such as monocyte chemoattractant protein-3 and RANTES do not trigger migration by the T cell hybridoma used in this study (data not shown), most likely because it does not express the receptors as indicated by RT-PCR (data not shown).

Similarly as described before (32), 1 ng/ml SDF-1 induced substantial LFA-1-dependent migration through ICAM-1-coated filters. This was completely blocked by 50 µg/ml piceatannol, and dominant-negative ZAP-70-expressing DN22 and DN38 cells did not migrate at all (Fig. 9GoA). The truncated ZAP-70 SH2 domain mutants did not affect migration (Fig. 9GoB). Furthermore, migration was blocked by U-73122 but not U-73343 and by calpeptin but not by Ro 31-8220 (Fig. 9GoC). In contrast, migration induced by 100 ng/ml SDF-1, which was completely blocked by pertussis toxin, did not depend on LFA-1 because it was not inhibited by LFA-1 mAb and occurred to the same extent through ICAM-1-coated and uncoated filters (32). This migration was not at all affected by piceatannol or by the truncated ZAP-70 (Fig. 9GoA). However, U-73122 did inhibit this LFA-1-independent migration induced by 100 ng/ml SDF-1 (Fig. 9GoC), suggesting the involvement of PLC-ß, triggered by SDF-1 receptor-coupled G-proteins. Furthermore, the calpain inhibitor calpeptin completely blocked this migration (Fig. 9GoC), suggesting that calpain is also required for LFA-1-independent migration.



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  		FIGURE 9. Both LFA-1-dependent and -independent migration of TAM2D2 cells are blocked by PLC and calpain inhibitors, whereas ZAP-70 is specifically involved in LFA-1-dependent migration. A, LFA-1-dependent (1 ng/ml SDF-1{alpha}) but not LFA-1-independent (100 ng/ml SDF-1{alpha}) migration is blocked by overexpression of dominant-negative truncated ZAP-70 (clones DN22 and DN38). B, N-terminal and C-terminal SH2 domain mutants of truncated ZAP-70 do not block LFA-1-dependent migration (clones N-SH2.10 and N-SH2.15 and C-SH2.7 and C-SH2.16, respectively). C, Inhibitors of PLC (U73122) and calpain (calpeptin), but not PKC (Ro 31-8220), block both LFA-1-dependent (1 ng/ml SDF-1{alpha}) and -independent (100 ng/ml SDF-1{alpha}) migration. Data are averages + SEM of two experiments performed in duplicate.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this paper we show that cross-linking of LFA-1 integrins by a blocking Ab promotes adhesion mediated by other LFA-1 molecules. The effector molecules activated by LFA-1 cross-linking are the tyrosine kinase ZAP-70 and probably PLC-{gamma} (44) and the calcium-dependent protease calpain, as suggested by the effects of inhibitors. ZAP-70 is also required for LFA-1-dependent migration through ICAM-1-coated filters and for LFA-1-dependent invasion into fibroblast monolayers, and these processes are also blocked by PLC and calpain inhibitors. This indicates that similar signals are triggered by cross-linking of LFA-1 with blocking Abs as by binding of LFA-1 to its ligand ICAM-1, in line with previous reports on LFA-1 as well as other integrins (45, 46, 47). Thus, the outside-in signals (48) induced by interaction of LFA-1 with ICAM-1 (or ICAM-2) lead to inside-out signals that promote binding of other LFA-1 molecules to additional ligand molecules. This binding may lead to a new cycle of outside-in and inside-out signals. This repetitive process, illustrated in Fig. 10Go, seems perfectly suitable for the propagation of signals triggered by the sequential contacts of cell protrusions with ligand molecules when a cell moves on a surface. However, because the T cell hybridoma cells do not bind spontaneously to ICAM-1, a different signal is required to initiate the process, which is provided by low concentrations of a chemokine such as SDF-1. Migration induced by higher chemokine concentrations does not require the LFA-1 signal and is not blocked by dominant-negative ZAP-70. Therefore, this does not seem a relevant model for in vivo migration, because both the in vitro invasion into fibroblast monolayers and the in vivo invasion of many different tissues by the T cell hybridoma cells requires LFA-1 and is completely blocked by the dominant-negative ZAP-70.



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  		FIGURE 10. Model of signaling during invasion of T cell hybridoma cells into fibroblast monolayers (and into multiple tissues in vivo). A chemokine such as SDF-1 (bound to a surface proteoglycan) binds to its receptor (CXCR4 for SDF-1) on the T cell hybridoma cell. This triggers signal 1, among others involving PLC-ß and calpain. A sufficiently strong signal induces migration independent of LFA-1. A weak signal causes local activation of LFA-1 molecules that then bind to ICAM-1 or -2, and this triggers signal 2, among others involving ZAP-70, PLC-{gamma}, and calpain. This signal activates additional LFA-1 molecules and this cycle may be repeated. Thus, the signal that is initiated by a limited amount of chemokine is amplified and propagated upon LFA-1 engagement.

 
It should be noted that the cells interact with monomeric ICAM-1 on substrates and filters. It has recently been shown by Stupack et al. (49) that lymphocytes require activation to bind to monomeric integrin ligands but not to polymeric forms. This suggests that the chemokine signal is required for clustering of LFA-1. The signal induced by the Ab-induced cross-linking of LFA-1 is therefore likely to mimic the signal triggered by LFA-1 clusters interacting with ICAM-1.

Signals amplified via LFA-1 and ZAP-70 may include PLC and calpain

LFA-1-independent chemotaxis induced by high SDF-1 levels was blocked by U-73122, which inhibits PLC (3), as well as by calpeptin, which inhibits calpain. This suggests that PLC-ß activated by the chemokine receptor is involved and that calpain activated by calcium elicited by the PLC product inositol-trisphosphate is also required. However, because these drugs are unlikely to be specific for only these targets, the involvement of PLC and calpain will have to be confirmed by more direct approaches. Ab-induced aggregation is also blocked by U-73122 and calpeptin, suggesting that LFA-1 engagement triggers in part similar signals that are thus amplified. A likely possibility again is that PLC is involved, but rather than the PLC-ß activated by the chemokine receptor, it would be PLC-{gamma}, which is activated by ZAP-70 (50), and by Ab engagement of LFA-1 (44). Thus, chemokines and integrins may collaborate in providing sufficient PLC activity by activation of the two distinct phospholipases.

Induction of LFA-1-mediated adhesion: possible mechanisms

Adhesion triggered by inside-out signals has been ascribed either to changes in the conformation of integrins that increase the affinity for ligand or to clustering of integrins that leads to increased avidity (1, 16, 43, 48). However, the same inhibitors that block LFA-1-dependent adhesion during invasion and migration (dominant-negative ZAP-70, piceatannol, U-73122, and calpeptin) also inhibit adhesion of T cell hybridoma cells to immobilized Abs. In the latter case a change in affinity or avidity for the ligand, which is an Ab, cannot explain the enhanced adhesion, because the Ab binds to nonactivated LFA-1 with an affinity that is not increased when the active conformation is induced by Mn2+ (data not shown). In this respect, "activation" of LFA-1 by Ab is comparable with activation by PMA, which does not involve a change in integrin conformation either (51, 52). An alternative explanation, as suggested above, is that clustering plus binding of a single ICAM-1 is sufficient to trigger the required signals. The role of calpain in LFA-1 function has been described (43), and it was proposed that calpain releases a cytoskeletal constraint and thus facilitates the redistribution of LFA-1 molecules to the contact area. Indeed, it was recently shown that activation of leukocytes leads to proteolysis of talin that links the ß2-chain to the cytoskeleton and this is blocked by calpeptin (53). It is therefore conceivable that the outside-in signal leads to increased lateral mobility and consequently to accumulation of LFA-1 molecules in the area of contact with the Ab-coated substrate. It should be noted, however, that calpeptin affects the migration induced by high SDF-1 concentrations as well, and that this is independent of LFA-1 (see Fig. 9GoC). If this is also due to calpain inhibition, calpain should have other relevant effects than only the release of LFA-1 constraints. A potential alternative explanation is that the elicited signals cause the cells to spread on the substrate (see Fig. 6Go). This would increase the contact area and thereby the number of LFA-1-ligand interactions. This possible explanation is supported by a recent report on NIH 3T3 cells overexpressing the endogenous calpain inhibitor calpastatin in which the capacity to spread was greatly reduced (54). Other explanations are not excluded. For instance, it is conceivable that the outside-in signals induce the interaction of LFA-1 molecules with cytoskeletal or signaling complexes.

T cell hybridoma cells do not adhere to immobilized nonblocking LFA-1 Ab

The cells adhered to the blocking Abs M17/4 and GAME-46 directed against the murine LFA-1 {alpha}L and ß2 subunits, respectively. In contrast, the cells did not bind to the nonblocking mAb M18/2, which has similar affinity for the ß2-chain as GAME-46. Furthermore, both blocking Abs induced aggregation, whereas M18/2 did not. This is in line with the report by Miyamoto et al. (47) that cross-linking of the integrin {alpha}5ß1 by blocking Abs leads to formation of large signaling complexes, similar to binding to the {alpha}5ß1 ligand fibronectin, whereas cross-linking by nonblocking Abs does not. Blocking Abs usually bind at or near the ligand-binding site, although they may also affect the binding site conformation allosterically. For the Abs used in this study, the binding sites have not been mapped. However, assuming that these are located near the binding site, it may affect integrin conformation in a manner comparable with ligand binding. This may be required, in addition to cross-linking, to mimic the effect of ligand engagement. The unexpected complete lack of adhesion to immobilized M18/2 may thus be explained by the inability of this nonblocking Ab to trigger the appropriate outside-in signals.

ZAP-70 activation depends on SH2 domains but not on the TCR or CD3

The T cell hybridoma cells used in this study express ZAP-70 but not the homologous Syk tyrosine kinase (32). Our conclusion that ZAP-70 is involved in LFA-1-dependent migration, invasion, and metastasis, as well as Ab-induced aggregation and adhesion to immobilized LFA-1 Abs, is based on two considerations. First, these processes are blocked by the inhibitor piceatannol with the exact same dose dependence as inhibition of ZAP-70 activity as we showed previously (32) and, second, these processes are blocked in cells overexpressing a truncated dominant-negative ZAP-70. This truncated protein consists mainly of the two SH2 domains. Triggering of ZAP-70 by the ß2 integrin LFA-1 is in line with the previously reported activation of Syk upon engagement of ß1 and ß3 integrins (55, 56, 57, 58). However, overexpression of a truncated Syk containing the SH2 domains did not affect the integrin-induced activation of Syk, whereas it blocked Fc-receptor-induced Syk activation (59). This led to the conclusion that the SH2 domains are not required for the signals triggered by the ß1 and ß3 integrins. In contrast, we show here that inhibition of LFA-1-mediated processes by the truncated ZAP-70 depends on both SH2 domains, because the inhibitory activity was blocked by a mutation in either domain that impairs the binding to tyrosine-phosphorylated proteins (40). Individually, the two SH2 domains bind very poorly to their tyrosine-phosphorylated target sequences and with distinct preferences, and the affinity of the tandem SH2 domains for doubly phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) sequences is ~1000-fold higher (60, 61). Hence, it is most likely that a tyrosine-phosphorylated protein, containing two phosphotyrosines in an ITAM sequence comparable with those in the TCR-{zeta} and the CD3 chains (59), is involved in LFA-1-induced ZAP-70 activity. This protein is distinct from the TCR and CD3 chains, however, because these are not expressed by the T cell hybridoma used in this study. This protein remains to be identified.

The ITAMs in the TCR/CD3 complex are phosphorylated by Src family kinases (62). However, these kinases do not appear to be involved in invasion and migration of the T cell hybridoma because Src inhibitors such as herbimycin (32) and overexpression of a kinase-dead Lck mutant (R. Soede et al., unpublished observations) had no effect. This indicates that the ITAM involved is phosphorylated by a non-Src-like kinase. It is noteworthy that ZAP-70 becomes associated with the focal adhesion kinase (FAK) (3) upon integrin signaling in NK cells (63), and that in a complex containing paxillin, FAK, and ZAP-70, the latter two become highly phosphorylated upon chemokine signaling in T cells (64). Because the FAK homologue Pyk2 also binds paxillin (65), it is likely to form part of such complexes. How ZAP-70 binds to the complex is unknown but the presence of an ITAM-containing protein seems a likely option. If so, FAK and Pyk2 are likely candidates for the ITAM-phosphorylating kinase.

Conclusion

In conclusion, we have provided evidence that signals induced by engagement of the integrin LFA-1 promote adhesion mediated by unengaged LFA-1 molecules. The effector molecules triggered by these signals are ZAP-70 and probably PLC-{gamma} and calpain. This LFA-1 signaling cascade may propagate and amplify the migration-initiating signals induced by low chemokine levels. Gi proteins, LFA-1, and ZAP-70 are required for T cell hybridoma invasiveness in vitro as well as for dissemination of these cells in vivo, as we have shown previously. These three components are also essential for LFA-1-dependent migration initiated by low chemokine concentrations, which apparently involves amplification of the chemokine signal. LFA-1 to LFA-1 signaling may therefore be important for T cell migration in vivo.



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  		FIGURE 7. Quantification of the adhesion to immobilized blocking mAbs M17/4 and GAME-46 against the {alpha}L and ß2 subunit of LFA-1 and the nonblocking mAb M18/2 against the ß2 subunit. Data are averages + SEM of three experiments performed in triplicate.

 

   Acknowledgments
 
We thank Dr. A. Weiss for the mouse anti-human ZAP-70 2F3.2 mAb, the pBJ1-ZAP-70(1–276), pBJ1-ZAP-70 WT vectors, and the C-terminal SH2 domain mutant of truncated ZAP-70.


   Footnotes
 
1 This work was supported by Grant NKI 95-969 from the Dutch Cancer Society (to R.D.M.S.) and Grant NKI 91-04 from the Dutch Cancer Society (to M.H.E.D.). Back

2 Current address: Medical Research Council Laboratory for Molecular Biology, University College London, London, U.K. Back

3 Address correspondence and reprint requests to Dr. E. Roos, Division of Cell Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands. E-mail address: Back

4 Abbreviations used in this paper: ZAP, {zeta}-associated protein; FAK, focal adhesion kinase; PKC, protein kinase C; PLC, phospholipase C; SDF, stromal cell-derived factor; WT, wild type; SH2, Src-homology domain 2; ITAM, immunoreceptor tyrosine-based activation motif. Back

Received for publication March 1, 1999. Accepted for publication August 6, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Springer, T. A.. 1990. Adhesion receptors of the immune system. Nature 346:425.[Medline]
  2. Arnaout, M. A.. 1990. Leukocyte adhesion molecules deficiency: its structural basis, pathophysiology and implications for modulating the inflammatory response. Immunol. Rev. 114:145.[Medline]
  3. Dustin, M. L., T. A. Springer. 1991. Role of lymphocyte adhesion receptors in transient interactions and cell locomotion. Annu. Rev. Immunol. 9:27.[Medline]
  4. Heagy, W., C. Walterbangh, E. Martz. 1984. Potent ability of anti-LFA-1 monoclonal antibody to prolong allograft survival. Transplantation 37:520.[Medline]
  5. Harning, R., J. Pelletier, K. Lubbe, F. Takei, V. J. Merluzzi. 1991. Reduction in the severity of graft-versus-host disease and increased survival in allogenic mice by treatment with monoclonal antibodies to cell adhesion antigens LFA-1{alpha} and MALA-2. Transplantation 52:842.[Medline]
  6. Isobe, M., H. Yagita, K. Okumura, A. Ihara. 1992. Specific acceptance of cardiac allograft after treatment with antibodies to ICAM-1 and LFA-1. Science 255:1125.[Abstract/Free Full Text]
  7. Hasegawa, Y., K. Yokono, T. Taki, K. Amano, Y. Tominaga, R. Yoneda, N. Yagi, S. Maeda, H. Yagita, K. Omura, M. Kasuga. 1994. Prevention of autoimmune insulin-dependent diabetes in non-obese diabetic mice by anti-LFA-1 and anti-ICAM-1 mAb. Int. Immunol. 6:831.[Abstract/Free Full Text]
  8. Roos, E., G. La Rivière, J. G. Collard, M. J. Stukart, P. De Baetselier. 1985. Invasiveness of T-cell hybridomas in vitro and their metastatic potential in vivo. Cancer Res. 45:6238.[Medline]
  9. La Rivière, G., J. W. T. M. Klein Gebbinck, C. A. Schipper, W. J. Mooi, E. Roos. 1993. In vitro invasiveness of CTL clones and in vivo dissemination of CTL hybridomas. J. Leukocyte Biol. 53:381.[Abstract]
  10. Roossien, F. F., D. De Rijk, A. Bikker, E. Roos. 1989. Involvement of LFA-1 in lymphoma invasion and metastasis demonstrated with LFA-1-deficient mutants. J. Cell Biol. 108:1979.[Abstract/Free Full Text]
  11. Spertini, F., T. Chatila, R. S. Geha. 1992. Engagement of MHC class I molecules induces cell adhesion via both LFA-1-dependent and LFA-1-independent pathways. J. Immunol. 148:2045.[Abstract]
  12. Nielsen, M., A. Svejgaard, S. Skov, P. Dobson, K. Bemdtzen, C. Geisler, N. Ø dum. 1996. IL-2 induces ß2-integrin adhesion via a wortmannin/LY294002-sensitive, rapamycin-resistant pathway: phosphorylation of a 125-kilodalton protein correlates with induction of adhesion, but not mitogenesis. J. Immunol. 157:5350.[Abstract]
  13. Lorant, D. E., K. D. Patel, T. M. McIntyre, R. P. McEver, S. M. Prescott, G. A. Zimmerman. 1991. Coexpression of GMP-140 and PAF by endothelium stimulated by histamine or thrombin: a juxtacrine system for adhesion and activation of neutrophils. J. Cell Biol. 115:223.[Abstract/Free Full Text]
  14. Butcher, E. C.. 1991. Leukocyte-endothelial cell recognition: three (or more) steps to specificity and diversity. Cell 67:1033.[Medline]
  15. Shimizu, Y., S. W. Hunt. 1996. Regulating integrin-mediated adhesion: one more function for PI3-kinase?. Immunol. Today 17:565.[Medline]
  16. Schwartz, M. A., M. D. Schaller, M. H. Ginsberg. 1995. Integrins: emerging paradigms of signal transduction. Annu. Rev. Cell Dev. Biol. 11:549.[Medline]
  17. Dustin, M. L., T. A. Springer. 1989. T-cell receptor cross-linking transiently stimulates adhesiveness through LFA-1. Nature 341:619.[Medline]
  18. Van Kooyk, Y., P. Van de Wiel-van Kemenade, P. Weder, T. W. Kuijpers, C. G. Figdor. 1989. Enhancement of LFA-1-mediated cell adhesion by triggering through CD2 or CD3 on T lymphocytes. Nature 342:811.[Medline]
  19. Zell, T., S. W. Hunt, J. L. Mobley, L. D. Finkelstein, Y. Shimizu. 1996. CD28-mediated up-regulation of ß1-integrin adhesion involves phosphatidylinositol-3-kinase. J. Immunol. 156:883.[Abstract]
  20. Koopman, G., Y. Van Kooyk, M. De Graaff, C. J. Meyer, C. G. Figdor, S. T. Pals. 1990. Triggering of the CD44 antigen on T lymphocytes promotes T cell adhesion through the LFA-1 pathway. J. Immunol. 145:3589.[Abstract]
  21. Van Seventer, G. A., Y. Shimizu, K. J. Horgan, S. Shaw. 1990. The LFA-1 ligand ICAM-1 provides an important costimulatory signal for T cell receptor-mediated activation of resting T cells. J. Immunol. 144:4579.[Abstract]
  22. Koopman, G., R. M. J. Keehnen, E. Lindhout, W. Newman, Y. Shimizu, G. A. Van Seventer, C. De Groot, S. T. Pals. 1994. Adhesion through the LFA-1 (CD11a/CD18)-ICAM-1 (CD54) and the VLA-4 (CD49d)-VCAM-1 (CD106) pathways prevents apoptosis of germinal center B cells. J. Immunol. 152:3760.[Abstract]
  23. Van Noesel, C., F. Miedema, M. Brouwer, M. A. De Rie, L. A. Aarden, R. A. Van Lier. 1988. Regulatory properties of LFA-1 {alpha} and ß chains in human T-lymphocyte activation. Nature 333:850.[Medline]
  24. Damle, N. K., K. Klussman, A. Aruffo. 1992. Intercellular adhesion molecule-2, a second counter-receptor for CD11a/CD18 (leukocyte function-associated antigen-1), provides a costimulatory signal for T-cell receptor-initiated activation of human T cells. J. Immunol. 148:665.[Abstract]
  25. Keizer, G. D., W. Visser, M. Vliem, C. G. Figdor. 1988. A monoclonal antibody (NKI-L16) directed against a unique epitope on the {alpha}-chain of human leukocyte function-associated antigen 1 induces homotypic cell-cell interactions. J. Immunol. 140:1393.[Abstract]
  26. Robinson, M. K., D. Andrew, H. Rosen, D. Brown, S. Ortlepp, P. Stephens, E. C. Butcher. 1992. Antibody against the Leu-CAM ß-chain (CD18) promotes both LFA-1- and CR3-dependent adhesion events. J. Immunol. 148:1080.[Abstract]
  27. Andrew, D., A. Shock, E. Ball, S. Ortlepp, J. Bell, M. Robinson. 1993. KIM185, a monoclonal antibody to CD18 which induces a change in the conformation of CD18 and promotes both LFA-1- and CR3-dependent adhesion. Eur. J. Immunol. 23:2217.[Medline]
  28. Petruzzelli, L., L. Maduzia, T. A. Springer. 1995. Activation of the lymphocyte function-associated molecule-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) mimicked by an antibody directed against CD18. J. Immunol. 155:854.[Abstract]
  29. Koopman, G., M. De Graaff, A. C. Huysmans, C. J. Meijer, S. T. Pals. 1992. Induction of homotypic T cell adhesion by triggering of leukocyte function-associated antigen-1{alpha} (CD11a): differential effects on resting and activated T cells. Eur. J. Immunol. 22:1851.[Medline]
  30. Wuthrich, R. P.. 1992. Monoclonal antibodies targeting murine LFA-1 induce LFA-1/ICAM-1-independent homotypic lymphocyte aggregation. Cell. Immunol. 144:22.[Medline]
  31. Pacifici, R., J. Roman, R. Kimble, R. Civitelli, C. M. Brownfield, C. Bizzarri. 1994. Ligand binding to monocyte {alpha}5ß1 integrin activates the {alpha}2ß1 receptor via the {alpha}5 subunit cytoplasmic domain and protein kinase C. J. Immunol. 153:2222.[Abstract]
  32. Soede, R. D. M., Y. M. Wijnands, I. Van Kouteren-Cobzaru, E. Roos.. 1998. ZAP-70 tyrosine kinase is required for LFA-1-dependent T-cell migration and invasion. J. Cell Biol. 142:1371.[Abstract/Free Full Text]
  33. Driessens, M. H. E., E. A. M. Van Rijthoven, G. La Rivière, E. Roos. 1996. Expression of pertussis toxin adenosine diphosphate-ribosyltransferase in a T-cell hybridoma reduces metastatic capacity. Blood 88:3116.[Abstract/Free Full Text]
  34. Tashiro, K., T. Hideaki, R. Heilker, M. Shirozu, T. Nakano, T. Honjo. 1993. Signal sequence trap: a cloning strategy for secreted proteins and type I membrane proteins. Science 261:600.[Abstract/Free Full Text]
  35. Bleul, C. C., R. C. Fuhlbrigge, J. M. Casasnovas, A. Aiuti, T. A. Springer. 1996. A highly efficacious lymphocyte chemoattractant, stromal cell-derived factor 1 (SDF-1). J. Exp. Med. 184:1101.[Abstract/Free Full Text]
  36. La Rivière, G., C. A. Schipper, J. G. Collard, E. Roos. 1988. Invasiveness in hepatocyte and fibroblast monolayers and metastatic potential of T-cell hybridomas in mice. Cancer Res. 48:3405.[Abstract/Free Full Text]
  37. Driessens, M. H. E., P. E. M. Van Hulten, G. La Rivière, A. Zuurbier, E. Roos. 1996. Inhibition and stimulation of LFA-1 and Mac-1 functions by antibodies against murine CD18. Evidence that the LFA-1 binding sites for ICAM-1 and ICAM-2 and -3 are distinct. J. Leukocyte Biol. 60:758.[Abstract]
  38. Qian, D., M. N. Mollenauer, A. Weiss. 1996. Dominant-negative {zeta}-associated protein 70 inhibits T cell antigen receptor signaling. J. Exp. Med. 183:611.[Abstract/Free Full Text]
  39. Staal, F. J. T., A. Q. Bakker, M. Verkuijlen, E. Van Oort, H. Spits. 1996. Use of bicistronic retroviral vectors encoding the LacZ gene with a gene of interest: a method to select producer cells and follow transduced target cells. Cancer Gene Ther. 3:345.[Medline]
  40. Iwashima, M., B. A. Irving, N. S. C. Van Oers, A. C. Chan, A. Weiss. 1994. Sequential interactions of the TCR with two distinct cytoplasmic tyrosine kinases. Science 263:1136.[Abstract/Free Full Text]
  41. Landegren, U.. 1984. Measurement of cell numbers by means of the endogenous enzyme hexosaminidase: applications to detection of lymphokines and cell surface antigens. J. Immunol. Methods 67:379.[Medline]
  42. Oliver, J. M., D. L. Burg, B. S. Wilson, J. L. McLaughlin, R. L. Geahlen. 1994. Inhibition of mast cell Fc{epsilon}R1-mediated signaling and effector function by the Syk-selective inhibitor, piceatannol. J. Biol. Chem. 269:29697.[Abstract/Free Full Text]
  43. Stewart, M. P., A. McDowall, N. Hogg. 1998. LFA-1-mediated adhesion is regulated by cytoskeletal restraint and by a Ca2+-dependent protease, calpain. J. Cell Biol. 140:699.[Abstract/Free Full Text]
  44. Kanner, S.B., L. S. Grosmaire, J. A. Ledbetter, N. K. Damle. 1993. ß2-integrin LFA-1 signaling through phospolipase C-{gamma}1 activation. Proc. Natl. Acad. Sci. USA 90:7099.[Abstract/Free Full Text]
  45. Carrera, A. C., M. Rincón, F. Sánchez-Madrid, M. López-Botet, M.O. De Landazuri.. 1988. Triggering of co-mitogenic signals in T cell proliferation by anti-LFA-1 (CD18, CD11a), LFA-3, and CD7 monoclonal antibodies. J. Immunol. 141:1919.[Abstract]
  46. Pardi, R., J. R. Bender, C. Dettori, E. Gianazza, E. G. Engleman. 1989. Heterogeneous distribution and transmembrane signaling properties of lymphocyte function-associated antigen (LFA-1) in human lymphocyte subsets. J. Immunol. 143:3157.[Abstract]
  47. Miyamoto, S., H. Teramoto, O. A. Coso, J. S. Gutkind, P. D. Burbelo, S. K. Akiyama, K. M. Yamada. 1995. Integrin function: molecular hierarchies of cytoskeletal and signaling molecules. J. Cell Biol. 131:791.[Abstract/Free Full Text]
  48. Hynes, R. O.. 1992. Integrins: versatility, modulation, and signaling in cell adhesion. Cell 69:11.[Medline]
  49. Stupack, D. G., E. Li, S. A. Silletti, J. A. Kehler, R. L. Geahlen, K. Hahn, G. R. Nemerov, D. A. Cheresh. 1999. Matrix valency regulates integrin-mediated lymphoid adhesion via Syk kinase. J. Cell Biol. 144:777.[Abstract/Free Full Text]
  50. Yablonski, D., M. R. Kuhne, T. Kadlecek, A. Weiss. 1998. Uncoupling of nonreceptor tyrosine kinases from PLC-{gamma}1 in an SLP-76-deficient T cell. Science 281:413.[Abstract/Free Full Text]
  51. Peter, K., T. E. O’Toole. 1995. Modulation of cell adhesion by changes in {alpha}Lß2 (LFA-1, CD11a/CD18) cytoplasmic domain/cytoskeleton interaction. J. Exp. Med. 181:315.[Abstract/Free Full Text]
  52. Stewart, M. P., C. Cabañas, N. Hogg. 1996. T cell adhesion to intercellular adhesion molecule-1 (ICAM-1) is controlled by cell spreading and the activation of integrin LFA-1. J. Immunol. 156:1810.[Abstract]
  53. Sampath, R., P. J. Gallagher, F. M. Pavalko. 1998. Cytoskeletal interactions with the leukocyte ß2 cytoplasic tail. J. Biol. Chem. 273:33588.[Abstract/Free Full Text]
  54. Potter, D. A., J. S. Tirnauer, R. Jansen, D. E. Croall, C. N. Hughes, K. A. Fiacco, J. W. Mier, M. Maki, I. M. Herman. 1998. Calpain regulates actin remodeling during cell spreading. J. Cell Biol. 141:647.[Abstract/Free Full Text]
  55. Clark, E. A., S. J. Shattil, M. H. Ginsberg, J. Bolen, J. S. Brugge. 1994. Regulation of the protein tyrosine kinase pp72syk by platelet agonists and the integrin {alpha}IIbß3. J. Biol. Chem. 269:28859.[Abstract/Free Full Text]
  56. Lin, T. H., C. Rosales, K. Mondal, J. B. Bolen, S. Haskill, R. L. Juliano. 1995. Integrin-mediated tyrosine phosphorylation and cytokine message induction in monocytic cells: a possible signaling role for the Syk tyrosine kinase. J. Biol. Chem. 270:16189.[Abstract/Free Full Text]
  57. Yan, S. R., M. Huang, G. Berton. 1997. Signaling by adhesion in human neutrophils. Activation of the p72syk tyrosine kinase and formation of protein complexes containing p72syk and Src family kinases in neutrophils spreading over fibrinogen. J. Immunol. 158:1902.[Abstract]
  58. Gotoh, A., H. Takahira, R. L. Geahlen, H. E. Broxmeyer. 1997. Cross-linking of integrins induces tyrosine phosphorylation of the proto-oncogene product Vav and the protein tyrosine kinase Syk in human factor-dependent myeloid cells. Cell Growth Differ. 8:721.[Abstract]
  59. Gao, J., K. E. Zoller, M. H. Ginsberg, S. J. Shattil. 1997. Regulation of the pp72syk protein tyrosine kinase by platelet integrin {alpha}IIbß3. EMBO J. 16:6414.[Medline]
  60. Isakov, N., R. L. Wange, W. H. Burgess, J. D. Watts, R. Aebersold, L. E. Samelson. 1995. ZAP-70 binding specificity to T cell receptor tyrosine-based activation motifs: the tandem SH2 domains of ZAP-70 bind distinct tyrosine-based activation motifs with varying affinity. J. Exp. Med. 181:375.[Abstract/Free Full Text]
  61. Ottinger, E. A., M. C. Botfield, S. E. Shoelson. 1998. Tandem SH2 domains confer high specificity in tyrosine kinase signaling. J. Biol. Chem. 273:729.[Abstract/Free Full Text]
  62. Chan, A. C., A. S. Shaw. 1995. Regulation of antigen receptor signal transduction by protein tyrosine kinases. Curr. Opin. Immunol. 8:394.
  63. Rabinowich, H., M. Manciulea, R. B. Herberman, T. L. Whiteside. 1996. ß1 integrin-mediated activation of focal adhesion kinase and its association with Fyn and ZAP-70 in human NK cells. J. Immunol. 157:3860.[Abstract]
  64. Bacon, K. B., M. C. Szabo, H. Yssel, J. B. Bolen, T. J. Schall. 1996. RANTES induces tyrosine kinase activity of stably complexed p125FAK and ZAP-70 in human T cells. J. Exp. Med. 184:873.[Abstract/Free Full Text]
  65. Salgia, R., S. Avraham, E. Pisick, J. L. Li, S. Raja, E. A. Greenfield, M. Sattler, H. Avraham, J. D. Griffin. 1996. The related adhesion focal tyrosine kinase forms a complex with paxillin in hematopoietic cells. J. Biol. Chem. 271:31222.[Abstract/Free Full Text]



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