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© The Rockefeller University Press,
0021-9525/1999//777 $5.00
The Journal of Cell Biology, Volume 144, Number 4,
, 1999 777-788
Regular Articles |
Matrix Valency Regulates Integrin-mediated Lymphoid Adhesion via Syk Kinase
Department of Cell Biology, and || Department of Vascular Biology, The Scripps Research Institute, La Jolla, California 92037; and Department of Medicinal Chemistry and Pharmacology, Purdue University, West Lafayette, Indiana 47907
Lymphocytes accumulate within the extracellular matrix (ECM) of tumor, wound, or inflammatory tissues. These tissues are largely comprised of polymerized adhesion proteins such as fibrin and fibronectin or their fragments. Nonactivated lymphoid cells attach preferentially to polymerized ECM proteins yet are unable to attach to monomeric forms or fragments of these proteins without previous activation. This adhesion event depends on the appropriate spacing of integrin adhesion sites. Adhesion of nonactivated lymphoid cells to polymeric ECM components results in activation of the antigen receptor-associated Syk kinase that accumulates in adhesion-promoting podosomes. In fact, activation of Syk by antigen or agonists, as well as expression of an activated Syk mutant in lymphoid cells, facilitates their adhesion to monomeric ECM proteins or their fragments. These results reveal a cooperative interaction between signals emanating from integrins and antigen receptors that can serve to regulate stable lymphoid cell adhesion and retention within a remodeling ECM.
Key Words: integrin • lymphocyte • extracellular matrix • protein tyrosine kinase • cell adhesion
Abbreviations used in this paper: CTL, cytotoxic lymphocyte; ECM, extracellular matrix; LCL, lymphoblastoid cell line; LCMV, lymphocytic choriomenginitis virus; LIBS, ligand-induced binding site; MAP kinase, mitogen-activated protein kinase; PB, penton base; PE, phycoerythrin; PI(3)K, phosphoinositide-3-kinase.
Address correspondence to D. Cheresh, The Scripps Research Institute, Department of Immunology, IMM24, 10550 Torrey Pines Rd., La Jolla, CA 92037. Tel.: (619) 784-8281. Fax: (619) 784-8926. E-mail: cheresh{at}scripps.edu
LYMPHOCYTES traverse between the tissue and circulatory compartments in a regulated manner requiring the expression of cell adhesion receptors and their ligands (Springer, 1994; Butcher and Picker, 1996). Chronic inflammatory diseases are marked by both the deposition of provisional extracellular matrix (ECM),1 such as fibrin, and the accumulation of lymphocytes within affected tissues (Postigo, 1993). Lymphoid adhesion and migration on purified ECM components is largely dependent on integrins, heterodimeric (
/β) proteins that integrate extracellular interactions of cells with internal signal-transducing elements, including the cytoskeleton (Aplin et al., 1998). Lymphoid integrins, like those on other hematopoietic cells, are regulated at both the level of expression and function (Springer, 1994), suggesting that lymphoid cell interaction with, and adhesion to, ECM proteins is itself dependent upon initiating signaling events.
The induction of lymphoid adhesion in vitro has been associated with the activation of a variety of signaling molecules, yet in most cases these stimuli result in transient adhesion. However, it remains unclear how sustained lymphoid interaction with the ECM is maintained in vivo. Although conformational changes in integrin structure may occur in response to some activational protocols, these are often associated with ligand binding itself (Bazzoni and Hemler, 1998), and the precise relationship between affinity modulation and adhesion is still open to interpretation (Stewart and Hogg, 1996).
During wound repair, cancer, and inflammation, the ECM is comprised of provisional matrix proteins, including fibrinogen, fibronectin, and vitronectin. Each is capable of self-assembly and polymerization after vascular leakage (Dvorak et al., 1995). The rapid, organized deposition of these molecules results in binding sites for adhesion molecules, including integrins, being separated by only nanometers (Mosesson et al., 1995), which provides a mechanism for local avidity modulation within affected tissues. During tissue remodeling, proteolytic activity provides a complementary mechanism to digest the polymeric ECM. Provisional ECM, when intact, is immunopotentiating (Postigo et al., 1991; Halvorson et al., 1996), whereas proteolytic fragments are immunosuppressive (Robson et al., 1993; Edgington et al., 1985), suggesting an important role for ECM deposition and remodeling in lymphocyte responses (Ratner et al., 1992).
To investigate whether the induction of a polymeric status in ECM components could facilitate lymphoid adhesion and attachment, we examined the capacity of lymphocytes and lymphoid lines to interact with polymerized or unpolymerized ECM proteins or with structurally defined integrin-specific ligands. Lymphoid cells required activation to adhere to unpolymerized matrix components, but readily attached to multimeric matrix components. Lymphoid adhesion did not require affinity modulation, but rather was dependent upon the appropriate spacing of integrin adhesive sites. Engagement of integrin by polymeric adhesive sites initiated signaling through the hematopoietic kinase Syk. Activation of this kinase by mutagenesis or through antigenic stimulation resulted in cellular adherence to monomeric ligand. These results provide a mechanism for cooperation between antigen receptors and integrins during the activation of lymphocyte adhesion to the ECM.
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T cells were established from lymphocytic choriomeningitis virus (LCMV) preimmunized animals cocultured with LCMV-infected, irradiated helper cell populations as described (Hahn et al., 1994). In brief, splenic lymphocytes were harvested from infected mice, and cytotoxic T cells (CTLs) established by outgrowth in the presence of 10 U/ml murine interleukin (IL)-2 and irradiated LCMV-infected autologous helper cells. Cells were used in the assay after 5 d in culture. Sorted human lymphocytes expressing integrin vβ3 were isolated from PBMC after ficoll-hypaque differential gradient purification followed by flow cytometry (FACStarTM; Becton Dickinson) using murine monoclonal P4C10 (5 µg/ ml) purified from the hybridoma and rabbit antisera to integrin vβ3 (1: 1,200), detected with secondary goat anti–mouse (PE-conjugated) or goat anti–rabbit (FITC-conjugated) preadsorbed antisera, respectively (Southern Biotechnology). Populations expressing high levels of both integrins were principally monocytes and were identified and excluded by forward scatter, side scatter, and fluorescence (phycoerythrin [PE]) gating. Analysis of these populations with direct-labeled (FITC-conjugated) reagents 24 h later confirmed these cells were a mixed lymphocyte population (
65% CD19+, 20% CD3+) without monocytes (CD14–) or progenitor cells (CD38–). Antibodies were purchased from PharMingen.
Cell Adhesion
Cell adhesion to 48-well plates precoated with substrate ligand was performed as previously described (Filardo et al., 1995). In brief, cells were suspended at 106/ml in RPMI 1640, 1.0% BSA. 200-µl aliquots of cells were added to wells which had been precoated overnight with the specified ligands in PBS, pH 8.0, at 4°C and then blocked for 1 h at room temperature with RPMI, 3% BSA (Sigma). Fibronectin (GIBCO-BRL) and pronectin (Stratagene) were purchased; superfibronectin and monomeric vitronectin were gifts of E. Ruoslahti (The Burnham Institute, La Jolla, CA) and D. Sieffert (The Scripps Research Institute, La Jolla, CA). Recombinant, pentameric penton base (PB) was expressed in insect cells as described previously (Wickham et al., 1993). Monomeric PB integrin-binding domain (Mathias et al., 1994) was the gift of T. Muir (The Rockefeller University, New York, NY). Adhesion was allowed to proceed for 30 min at 37°C followed by washing to remove nonadherent cells and then quantitation of adherence by staining adherent fractions with crystal violet (0.1% in 0.15 M NaCl, 20% ethanol). Cell-bound stain was resolubilized in methanol and quantitated at 600 nm. Soluble fibrin was formed by the addition of 0.2 U thrombin in DME (50 U/ml stock) (Calbiochem-Novabiochem) to the specified fibrinogen (American Diagnostica) concentrations during coating. The addition of thrombin to BSA- or fibrinogen-coated wells served as a control and did not differ from thrombin-free coated surfaces. Molar concentrations of pentameric PB were calculated based upon the five individual molecules within each pentamer (therefore, five integrin-binding sites). To express PB concentration as an aggregate unit (pentamer), concentration may be reduced fivefold. Purified lymphocyte adhesion to substrate-coated chamber slides was quantitated by direct counting of six fields at 100x after sorting on a FACStarTM (Becton Dickinson). Three independent sorts were performed. Adhesion to cryosections from three different sources was performed with Cell Tracker Green-labeled LCL (5 µM for 15 min; Molecular Probes) in binding buffer (DME, 3% BSA, 100 U/ml heparin) as previously described (Stamper and Woodruff, 1976) and was quantitated similarly.
Radioimmunoassay
Adhesion plates (48 well) were coated exactly as described for the adhesion assay and blocked with 1.0% BSA in DME (assay buffer) for 1 h at 37°C. Wells were probed by the addition of with 0.5 µg of [125I] radiolabeled mAb DAV-1, which recognizes an epitope (IRGDTFAT) within the integrin-binding domain (Stewart et al., 1997). DAV-1 was radiolabeled with [125I] (ICN) using Iodobeads (Pierce) according to the manufacturer's instructions. The specific binding of DAV-1 (< 1%) was assessed after 1 h. Specific binding was resolubilized by the addition of 1% SDS, 50 mM Tris, pH 6.8, 10% betamercaptoethanol (100°C) (Sigma) after extensive washing (five times) in assay buffer.
DAV-1 binding to BSA alone was not significantly different from background, and was subtracted from all values to get specific binding. Radioactivity bound per unit area was used to calculate sites present after normalizing specific activity per mol of DAV-1 in each experiment.
Integrin-binding Assay
Adhesion plates (96 well) were coated exactly as described for the adhesion assay (0.5 µg/ml of fibrinogen/fibrin) and blocked with 1.0% BSA in DME (assay buffer) for 1 h at 37°C. Wells were probed by the addition of soluble, biotinylated integrin generously provided by S.L. Goodman (Merck KGaA; Mehta et al., 1998) for 2 h at 37°C, washed with PBS five times, and then bound integrin was detected by the addition of secondary horseradish peroxidase-conjugated anti-biotin secondary antibody (Sigma). After four further washes, the chromogenic substrate 3,3', 5,5'-tetramethyl benzidine (Bio-Rad) was added and absorbance quantitated at 450 nm after a 10-min incubation.
Immunoprecipitation and Kinase Activity
LCL were washed twice in PBS, then aliquots (5 x 106 in 4,000 µl) were plated for 15 min in DME on surfaces coated with different substrate as indicated. Excess media was then aspirated, leaving the bulk of the cells (adherent and nonadherent) in the final 800 µl. Cells were lysed by the addition of 700 µl 1.0% Nonidet P-40, 50 mM Tris-buffered lysates, pH 7.4, containing 2x protease inhibitor cocktail (Boehringer Mannheim) 2 mM PMSF, 2 mM EGTA, and 2 mM sodium vanadate (Sigma). Specific kinases were immunoprecipitated after preclearing lysates with protein A–Sepharose (Pierce) by the addition of monoclonal antibody 4D10 (Syk) (Santa Cruz Biotechnology), or polyclonal antisera against phosphoinositide-3-kinase (PI[3]K)–p85 (Grb-1) (Upstate Biotechnology) or Syk (Santa Cruz Biotechnology) (2–4 µg/ml) and the addition of protein A– or protein G–Sepharose, as appropriate. Alternatively, immunoprecipitations were performed with agarose-coupled polyclonal antisera to Syk or Lyn (Santa Cruz Biotechnology). Immunoprecipitated proteins were resolved by 10–12% SDS-PAGE and transferred to polyvinylene difluoride membranes to facilitate phosphotyrosine analysis via immunodetection with monoclonal antibodies 4G10 (Upstate Biotechnology) and PY72 (prepared from the hybridoma). Similarly, Syk or Lyn autophosphorylation activity was determined from the immunoprecipitates as described by the addition of exogenous 32P-ATP (5 µCi; ICN) in kinase buffer (20 mM Hepes, pH 7.4, 10 mM MnCl2, 150 mM NaCl) as described (Burg et al., 1994). All activity was normalized by reprobing to account for small variations in protein loading of individual lanes. The p85-associated PI(3)K activity was determined after immunoprecipitation by the addition of exogenous 32P-ATP and (4,5) PIP2 substrate (Calbiochem-Novabiochem) followed by silica gel thin layer chromatography (1:1:1, CHCl3, CH3OH, HCl [1 M]) as previously described (Li et al., 1998) and subsequent visualization via autoradiography or ImageQuant phosphorimager analysis (Molecular Dynamics).
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vβ3 expressed on a subpopulation of lymphoid cells (Halvorson et al., 1996). To examine lymphoid interaction with a tumor-associated ECM, nonactivated lymphoblastoid B cells (LCL) were assessed for their attachment to fibrin-rich cryosections of human breast tumors (Fig. 1, A and B). LCL attachment localized to regions of fibrin deposition (Fig. 1 C), and the observed adhesion was significantly blocked (Fig. 1 D) with monoclonal (mAb) LM609 directed to integrin vβ3 (65%, Fig. 1 E), a known receptor for fibrinogen/fibrin (Cheresh, 1987) (and other provisional ECM components) expressed on LCL. However, these tissues also contain other adhesive ligands for integrin vβ3. Thus, fibrin does not account for all the adhesive interactions observed.
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vβ3 and β2 integrins, as determined using function-blocking mAbs LM609 and TS1/ 18, respectively (Fig. 2 B). In contrast, adherent M21 melanoma cells readily attached to fibrinogen or its polymeric form, fibrin, in a dose-dependent manner (Fig. 2 A) and this was blocked by mAb LM609 (data not shown), as previously reported (Felding-Habermann et al., 1992).
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15–20 nM. These results suggested that polymerization of fibrinogen to fibrin did not result in exposure of adhesive sites with increased affinity for integrin. Importantly, preactivation of LCL by phorbol ester (Shimizu, 1996), which has been shown to promote integrin clustering in lymphoid cells (Haverstack et al., 1992; van Kooyk et al., 1994), activated lymphoid attachment to fibrinogen, demonstrating that this ligand supported lymphoid adhesion (Fig. 2 D). In contrast, attachment to fibrinogen (or BSA) was not rescued by pretreating either the plates or the LCL with thrombin, suggesting that thrombin was not itself an activator of LCL attachment (data not shown). LCL also gained the ability to bind unpolymerized fibronectin or vitronectin after exposure to PMA (Fig. 2 D). However, in all cases examined, multivalent and polymerized forms of fibronectin and vitronectin, like fibrin, supported LCL adhesion without previous stimulation (Fig. 2 D) suggesting that LCL adhesion was inducible by polymeric integrin ligands, and that this may be a general property of lymphoid cell integrins.
Multivalent Integrin Ligands Mimic Polymeric ECM and Facilitate Lymphoid Adhesion
To further establish the structural basis of lymphoid-mediated integrin function, LCL were allowed to attach to one of two structurally-defined vβ3 ligands. In this case, microtiter wells were coated with a substrate comprised of either a monovalent or pentavalent form of the adenovirus PB protein (Mathias et al., 1994). The pentavalent form of PB contains five available vβ3-binding sites equally separated by 60 Å (Stewart et al., 1997), whereas the monomeric construct contains only a single vβ3-binding site (Mathias et al., 1994). As shown in Fig. 3 A, the adherent M21 cell line attached to either form of PB, whereas LCL selectively attached to the pentamer. Adhesion to multimeric PB was entirely dependent on integrin vβ3, since either LM609 or a peptide antagonist of this integrin completely blocked adhesion while antibodies to other integrins showed no effect (Fig. 3 B). The addition of soluble monomeric or multimeric PB also disrupted adhesion (Fig. 3 B), indicating that either form of PB was an effective soluble inhibitor of integrin vβ3.
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Integrin organization on the cell surface may be an important factor contributing to adhesion, since the activation of LCL attachment to monomeric PB (Fig. 3 B) did not influence vβ3 expression (our unpublished data). Lymphoid integrins can be activated by conformational (affinity) changes by a variety of reagents, including manganese (Bazzoni and Hemler, 1998). Manganese activated the attachment of LCL to monomeric PB similarly to PMA (Fig. 4 A), and caused conformational changes in integrin vβ3 which were detected with monoclonal antibody ligand-induced binding site (LIBS)-1 (Fig. 4 B) (Frelinger et al., 1990). Although LIBS-1 detects the ligand-occupied form of vβ3, the presence of manganese elicited conformational changes which exposed this epitope on LCL even in the absence of ligand (Fig. 4 B, top). In contrast, LCL activated with PMA did not differ from basal LIBS-1 expression (Fig. 4 B, bottom). Further, PMA did not stabilize integrin binding of soluble ligand (a relative measurement of affinity) (Frelinger et al., 1990; Filardo et al., 1995). Nonactivated or PMA-activated LCL were allowed to bind PB monomer, and LIBS-1 was used to detect ligand binding as a function of ligand concentration. No difference in half-maximal ligand occupancy was evident between PMA-activated and untreated LCL for soluble monomeric PB (1.5 µM) (Fig. 4 C). We observed that LCL displayed the same relative affinity for ligand as determined for M21 cells (Fig. 4 C) and other adherent cells (Filardo et al., 1995). Thus, activation of LCL by PMA leading to stable adhesion on monomer does not appear to result from preexisting affinity changes.
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vβ3 ligands (Fig. 5 A). In each case, attachment could be inhibited with monoclonal antibody LM609 (data not shown). In contrast, B cell tumor lines which do not express vβ3 such as DT40 and Ramos, failed to attach to either form of PB (Fig. 5 A). Although integrin vβ3 has been described in tissue-associated lymphocyte populations (Halvorson et al., 1996), it is only minimally expressed on circulating lymphocytes (Hemler, 1990). However, when the subpopulation of lymphocytes expressing vβ3 were isolated from peripheral blood (< 2%) (Fig. 5 B, inset), these cells also demonstrated selective adhesion to both multimeric PB and fibrin relative to monomeric PB and fibrinogen, respectively (Fig. 5 B). Thus, lymphocytes which express appropriate receptors can attach to multivalent ligands without a requirement for exogenous activation.
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75%) during the initial 5–15 min (data not shown). Therefore, to investigate possible mechanisms leading to adhesion, lysates from LCL plated on monomeric or multimeric ligands for 15 min were subjected to immunoprecipitation and phosphotyrosine analysis. Although LCL failed to express detectable levels of FAK (data not shown), they expressed both Syk and PI(3)K. Attachment of LCL to pentavalent PB resulted in enhancement of Syk phosphorylation compared with Syk isolated from cells plated on the monomeric substrate or on BSA (4.5 ± 1.7 fold) (Fig. 6 A). Importantly, there was a corresponding two- to threefold increase in Syk kinase activity in cells adherent to the pentamer compared with cells plated on the monomer or the nonadherent substrate protein BSA (Fig. 6 B). In contrast, an increase in phosphorylation of the p85 subunit of PI(3)K (Fig. 6 C) and PI(3)K activity (Fig. 6 D) was observed in cells interacting with either pentamer or monomer relative to cells plated on BSA. These data suggest that lymphoid cell PI(3)K becomes activated with a low threshold of integrin ligation in general, whereas integrin ligation leading to (stable) adhesion is specifically associated with activation of Syk.
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5 mM), but was only slightly impacted by the PI(3)K inhibitor LY294002 (Fig. 7). Although LY294002 treated cells attached, they exhibited reduced PI(3)K activity (Fig. 7, inset), failed to spread (data not shown), and were more susceptible to shear force during washing, resulting in an apparent slight inhibition of adhesion. Similar results were obtained with an alternative inhibitor of PI(3)K, wortmannin (increasing effect from 70–80 nM, data not shown). Although selective for the kinase Syk, piceatannol has pleiotropic effects on cells, including the blockade of src family kinases at increased concentrations. However, no significant inhibition of the related src family kinase Lyn (the major src kinase in LCL) was found in these studies at the concentrations of piceatannol used (Fig. 7, inset).
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vβ3 and do not attach to PB (Fig. 5 A), but do express β1 integrins (Fig. 9 B, inset) which bind this region of fibronectin. Consistent with this, adhesion was blocked by a monoclonal antibody to β1 integrin (data not shown). These data show that β1 integrin-dependent adhesion to fibronectin, like that mediated by vβ3, can also be upregulated by Syk activation, and that Syk requires activation to promote lymphoid cell adhesion to integrin ligands. |
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Evidence is presented that a crucial aspect to initiating lymphoid adhesion is the functional multimerization of binding sites for integrins. The current study used the PB protein of adenovirus, a model ligand bound exclusively by integrins (Wickham et al., 1994) (Fig. 3 B), which provided a structurally defined ligand to examine lymphoid interactions with different substrate forms. In fact, the intact PB pentamer is known to have five available integrin-binding sites arranged in a pentagonal array with a separation of 60 Å (Stewart et al., 1997) allowing it to accommodate the binding of five integrins simultaneously. We contend that, like polymerized ECM proteins, PB acts as a clustered ligand and exploits local avidity as a means to initiate signaling events. It is clear that the overall (general) density of interaction is not as efficient in this respect, since much higher levels of monomeric penton base did not support adhesion. In fact, monomer coated surfaces containing 800 integrin sites/mm2 were unable to support lymphoid adhesion, whereas multimer-coated surfaces with less than 125 sites/mm2 available supported LCL adhesion. To demonstrate that the monomer could support adhesion, PMA, an agonist which promotes lymphoid integrin clustering via a PKC-dependent pathway (Haverstack et al., 1992; van Kooyk et al., 1994), was used to rescue attachment to monomer coated surfaces. Alternatively, it is possible that adhesion was observed on the multimeric proteins because the pentameric form of PB, and of ECM proteins in general, present higher affinity sites for integrin interactions upon multimerization. However, at least in the case of vβ3/fibrinogen interaction, this does not appear to be the case (Fig. 2 B). Competitive blocking studies indicate that soluble PB monomer and the pentamer have a similarly high affinity for integrin (IC50 18 and 5 nM, respectively) although pentameric PB is significantly more potent at inducing integrin-mediated signaling events (Li et al., 1998) (Li, E., and G. Nemerow, unpublished data). Therefore, these findings suggest that appropriately spaced clustering of integrin ligand provides a specific mechanism through which these postreceptor events promote stable adhesion. The fact that adherent M21 cells attached to either substrate suggests that lymphoid cells regulate postreceptor ligation events differently than adherent cell types.
By itself, affinity modulation of integrin function did not appear to explain the observed agonist-induced adhesion, since soluble integrins maintained the same affinity for either multimeric or monomeric ligand (Fig. 2 C). It remains possible that avidity changes in integrin (clustering) can result in higher affinity through local packing and lateral stabilization of ligated integrin. Such changes might not be detected by conformational/affinity measurements such as LIBS binding due to steric issues of accessibility. Further study will be necessary to differentiate pure affinity changes from local packing effects. However, in addition to activation of PKC and Rho-dependent integrin clustering, PMA activates a variety of cellular signaling pathways, and induction of integrin-mediated adhesion by this agonist has previously been suggested to occur as a result of postreceptor events (Danilov and Juliano, 1989). The current investigation similarly provides evidence for postreceptor signaling events inducing attachment, and suggests basic differences in the response of lymphoid cells and adherent cells to different ligand forms. These findings suggest that ligand polymerization may provide all cues necessary to facilitate stable lymphocyte attachment.
Based upon these results, we propose that integrin ligation-dependent signaling impacts the ability of lymphoid cells to attach to the ECM. Several morphological (e.g., cell spreading) and signaling events were observed to occur during adhesion, including the phosphorylation of both Syk and p85. Although p85-associated PI(3)K activity increased after interaction with PB pentamer, it was similarly observed after contact with monomeric PB where no stable adhesion was detected. Inhibitors of PI(3)K did not block attachment of LCL to pentameric PB, however, they did disrupt spreading and the extension of processes (Fig. 3 A and our unpublished observations). However, some low level of PI(3)K activity may be required for adhesion, and our data supports the possibility that activation of PI(3)K plays a crucial role in attachment under conditions of flow/high shear. Pretreatment with piceatannol blocked adhesion to pentamer at very low concentrations (5 µM), suggesting that activation of Syk was required to induce subsequent attachment (Fig. 7), but was not necessarily involved in spreading or maintenance of LCL adhesion. These results are supported by the colocalization of Syk with actin during initial cell polarization/podosome formation (Fig. 8). The activation of Syk in lymphoid cells by antigenic stimulation or by reconstitution with constitutively active mutants rescued lymphoid cell attachment to monomer-coated surfaces (Fig. 9). Together, these results indicate that integrin postreceptor signaling events are crucial in governing LCL adhesion, and that local clustering of ligand provides a means to elicit integrin-mediated signaling resulting in stable attachment. Parallel studies confirmed that attachment to PB occurred as a result of postreceptor signaling in other cell types. For example, we were unable to observe attachment to PB with melanoma cells bearing a mutant β3 integrin tail (N744A), which is deficient in signaling but not ligand binding (Filardo et al., 1995) (our unpublished observations).
The activation of the hematopoietic kinase Syk occurs via the antigen receptor, soluble agonists, and integrin interactions with immobilized substrate (Burg et al., 1994; Lin et al., 1995; Gotoh et al., 1997; Okazaki et al., 1997). It is clear that qualitative differences exist in the outcome after interaction with different ligands. Activation of Syk through the antigen receptor leads to activation of MAP kinases and proliferation (Wan et al., 1996; Jacinto et al., 1998), whereas our results suggest a role for Syk in regulating adhesion. Adhesion alone was not sufficient to fully activate freshly isolated lymphocytes, since attachment to multimeric integrin ligands did not lead to significant proliferation of nonactivated, sorted peripheral blood lymphocytes (our unpublished observations). However, after initial antigenic stimulation, CTL adhesion to monomer and pentameric ligands was observed, and Syk phosphorylation was maintained throughout the course of the adhesion. Supporting this result, coexpression of integrin vβ3 and the 
subunit of the T cell antigen–receptor complex are known to be sufficient for continued CTL clone proliferation and cytokine secretion (Halvorson et al., 1996). The subunit consists of a transmembrane domain and an immunoreceptor tyrosine-based activation motif (ITAM)-containing docking motif which is recognized specifically by the dual SH2 domains of Syk and its homologue -associated protein (ZAP)-70 (Chan and Shaw, 1996), providing a mechanism by which integrin-mediated Syk activation pathways and antigen-stimulated pathways may couple.
Similarities do exist in the initial signaling pathways triggered by integrins or antigen receptors, since both integrins and antigen receptors activate specific members of the src kinase family identified as activators of Syk (Burg et al., 1994; Shattil et al., 1994; El Hillal et al., 1997; Hunter and Shimizu, 1997). Moreover, cross-linking either antigen receptor or integrins results in Syk activation (Rabinowich et al., 1996), suggesting that clustering of integrins by multimeric ligand leads to Syk activation. Syk, in turn, is known to phosphorylate cytosolic target proteins including Pyk-2 (Okazaki et al., 1997), -tubulin (Peters et al., 1996), and the guanine nucleotide exchange factor Vav (Deckert et al., 1996; Gotoh et al., 1997; Teramoto et al., 1997), supporting a mechanism for rapid cytoskeletal reorganization in lymphoid cells during both antigen presentation and adhesion to the ECM (Kupfer and Singer, 1989; Hahn et al., 1994). However, Syk is not always required for the activation of downstream targets by G protein– coupled receptors or other agonists (Wan et al., 1996). Therefore, modulation of integrin function by chemokines (Carr et al., 1996; Laudanna et al., 1996), multimeric ECM, and/or antigenic stimulation may potentiate adhesion via somewhat distinct signaling pathways.
In general, recognition of ECM by lymphocytes is critical to a variety of processes, including inflammation, lymphoid maturation, tissue retention, trafficking, and surveillance (Butcher and Picker, 1996). The implication of Syk as a crucial effector of lymphocyte–ECM interactions is interesting, since Syk-deficient lymphocytes are defective in tissue entry and distribution, as well as in subsequent maturation steps in vivo (Turner et al., 1994). Thymic maturation of T cells requires both integrins (Crisa et al., 1995) and Syk kinases (Cheng et al., 1995). Taken together, these observations reveal a mechanism whereby lymphoid responses are modulated by the local composition of the ECM, and specifically by the multimeric presentation of integrin binding sites. Integrin-mediated adhesion to polymeric ligands plays a key role in modulating lymphoid responses through the central kinase Syk, which in turn exerts pleiotropic effects upon lymphoid cells. This interplay may be important in the understanding of lymphoid cell behavior in vivo, and suggests a prominent role for the local ECM in governing lymphoid behavior in malignancy and inflammatory processes.
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Acknowledgments |
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This work was funded by grants from National Institutes of Health to D.A. Cheresh (CA 45726 and CA 50286), G.R. Nemerow (HL 54352 and EY 11431), K. Hahn (AI 39643), R.L. Geahlen (CA 37372) and the Joseph Drown Foundation to D.G. Stupack. D.G. Stupack was a recipient of a fellowship from the Canadian Arthritis Society. This is manuscript number 12080-IMM from The Scripps Research Institute.
Submitted: 23 July 1998
Revised: 20 January 1999
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