Article |
Address correspondence to David D. Roberts, National Institutes of Health, Building 10, Room 2A33, 10 Center Dr. MSC 1500, Bethesda, MD 20892-1500. Tel.: (301) 496-6264. Fax: (301) 402-0043. E-mail: droberts{at}helix.nih.gov
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: thrombospondins; integrins; chemotaxis; T cell antigen receptor; matrix metalloproteinases
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
TSP1 influences cell behavior by interacting with other extracellular matrix components, including latent TGFß1, and with specific cell surface receptors. TSP1 receptors include vß3,
4ß1,
5ß1, and
3ß1 integrins; CD36; CD47; low-density lipoprotein receptorrelated protein; and heparan sulfate proteoglycans (HSPGs) (Roberts, 1996). Therefore, responses of cells to TSP1 probably reflect the integration of simultaneous signals from multiple TSP1 receptors as well as receptors for some extracellular ligands of TSP1. The expression levels and activation states of these receptors must be considered to understand the function of each receptor in mediating specific TSP1 responses.
Several TSP1 receptors are expressed in hematopoietic cells and have been implicated in the regulation of immune functions by TSP1. 5ß1 and
4ß1 integrins contributed to PMA-stimulated adhesion of CD4+ peripheral T lymphocytes on TSP1 (Yabkowitz et al., 1993). Based on global analysis of gene expression in T cells exposed to TSP1, however, a primary effect of TSP1 is to inhibit T cell antigen receptor (TCR) signaling (Li et al., 2001). Studies using a T cell line demonstrated roles for CD47 and HSPGs in mediating the inhibitory effects of TSP1 on T cell activation (Li et al., 2001). TSP1 also inhibits T cell proliferation (Beppu et al., 2001). Furthermore, a CD47-binding peptide from TSP1 inhibited the development of naive T cells into Th1 effectors (Avice et al., 2000). In contrast, interactions of TSP1 or TSP1 peptides with HSPG and ß1 integrin receptors stimulated RasMAP kinase signaling in T cells (Wilson et al., 1999). Stimulatory effects of TSP1 or CD47-binding peptides derived from TSP1 have also been reported for the activation, infiltration, and clonal expansion of T cells (Reinhold et al., 1997; Ticchioni et al., 1997, 2001; Vallejo et al., 2000).
Taken together, these data indicate that TSP1 can both stimulate and inhibit specific signal transduction pathways in T cells. We have examined whether differential signaling through specific T cell TSP1 receptors could reconcile these apparently conflicting observations. We report here that the TSP1 receptors CD47 and 4ß1 integrin are differentially required for modulation of TCR signaling, T cell proliferation, matrix metalloproteinase (MMP) expression, and T cell motility by TSP1. Furthermore, we demonstrate that the
4ß1 integrindependent activities of TSP1 are replicated by recombinant NH2-terminal portions of TSP1 and TSP2 and identify peptide sequences in the NH2-terminal regions of both TSPs that are recognized by
4ß1 integrin.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Integrin-dependent adhesion of peripheral T cells to TSP1 is induced by phorbol esters (Yabkowitz et al., 1993), and we observed a similar induction of Jurkat cell adhesion on trimeric human thrombospondin-1 residues 1356 (NoC1) using PMA (Fig. 1 C). The dose dependence for PMA-activated cells was similar to that for TS2/16-activated cells. Remarkably, the corresponding recombinant trimeric NH2-terminal region of TSP2 was even more active for promoting adhesion of Jurkat cells activated using either PMA or TS2/16 (Fig. 1 C). Therefore, the NH2-terminal regions of both TSP1 and TSP2 contain binding sites for ß1 integrins.
Adhesion of CD4+ peripheral T cells showed a similar preference for the 4ß1 integrin binding sites in TSPs (Fig. 1 D). PMA activation stimulated adhesion to NoC1 and thrombospondin-2 residues 1359 (NoC2) to a similar extent as to TSP1, whereas adhesion to a fusion protein expressing the
5ß1 integrin binding domain of TSP1 was not enhanced by PMA.
Identification of an 4ß1 integrin recognition sequence in TSP1 and TSP2
The smallest portion of TSP1 tested that supported 4ß1 integrindependent adhesion of T cells contained amino acid residues 1175. Comparison of this sequence with known
4ß1 binding sequences in FN and vascular cell adhesion molecule-1 (VCAM-1) (Vonderheide et al., 1994; Moyano et al., 1997) using MACAW version 2.0.5 (Schuler et al., 1991) identified a potential recognition site at residues 159164, containing the sequence AELDVP. A synthetic peptide with this sequence inhibited Jurkat cell adhesion on substrates coated with NoC1 or NoC2 (Fig. 2 A). TSP2 contains a similar sequence at the same position (VALDEP) that conserves the Asp residue typically required for
4ß1 integrin ligands (Wang and Springer, 1998). A synthetic peptide, VALDEP, inhibited adhesion on NoC1 and NoC2 (Fig. 2 A). Substitution of the Asp residue of this peptide with Ala (VALAEP) markedly diminished its inhibitory activity, indicating that this residue is important for binding of the TSP2 peptide to
4ß1 integrin. Adhesion to TSP1(1175) was also specifically inhibited by the TSP2 peptide VALDEP but not by the control peptide VALAEP (Fig. 2 B). The
4ß1 integrin specificity of T cell adhesion on TSP1(1175) and NoC1 was further confirmed using the function-blocking
4ß1 antibody P4C2 (Fig. 2 B).
|
CD47 and HSPG are not sufficient to mediate T cell adhesion on TSP1
Although the preceding data establish that an 4ß1 integrin binding site is required for activation-dependent adhesion to the NH2-terminal region of TSP1, they do not exclude roles of the two additional T cell receptors, HSPG and CD47, in mediating adhesion on intact TSP1. We therefore compared activation-dependent adhesion of Jurkat mutants deficient in ß1 integrins (Romzek et al., 1998) or CD47 (Ticchioni et al., 2001). Flow cytometry confirmed deficiencies in the respective TSP1 receptors without a significant loss of the complementary receptor (Fig. 3 A). Neither the ß1 integrinactivating antibody nor PMA stimulation significantly stimulated adhesion of ß1 integrindeficient T cells on intact TSP1 or its NH2-terminal region (Fig. 3 B). Therefore, the heparin and CD47 binding sites of intact TSP1 were not sufficient to mediate adhesion in the absence of ß1 integrins. Conversely, adhesion of CD47-deficient T cells to the three proteins was induced by the ß1 integrinactivating antibody and by PMA (Fig. 3 B). Although adhesion of the CD47-deficient mutant was slightly less than that of wild-type cells, the comparable decrease in adhesion to TSP1, which contains the CD47 binding domain, and two recombinant portions of TSP1 lacking this domain indicates that any contribution of CD47 to adhesion on TSP1 is indirect.
|
|
4ß1 integrin mediates chemotaxis of T cells to TSP1
A soluble form of VCAM-1 induced chemotaxis of Jurkat and synovial T cells by binding to 4ß1 integrin (Kitani et al., 1998), suggesting that TSP1 might also be chemotactic for these cells. TSP1 induced a biphasic concentration-dependent chemotactic response in Jurkat cells that was maximal at 30 nM (Fig. 5 A). NoC1 and NoC2 also stimulated chemotaxis with similar dose response curves to platelet TSP1, indicating that the NH2-terminal region of TSP1 or TSP2 is sufficient to promote T cell chemotaxis. The same region was sufficient to stimulate chemotaxis of human primary CD4+ T cells, based on their responses to TSP1, NoC1, and NoC2 (Fig. 5 B).
|
We could not directly evaluate the role of CD47 in TSP1-stimulated chemotaxis using this approach, because CD47-deficient JinB8 cells did not migrate to any attractant tested (unpublished data). However, a function-blocking CD47 antibody (B6H12) inhibited motility stimulated by intact TSP1, NoC1, or NoC2 (Fig. 5 D). The latter do not contain the known TSP CD47 binding site, indicating that CD47 plays an essential but indirect role in these 4ß1-mediated chemotactic responses.
TSP1 induces MMP expression through 4ß1 integrin binding
Two previously known 4ß1 integrin ligands, VCAM-1 and FN, induce MMP-2 and MMP-9 mRNA and protein expression in T cells (Esparza et al., 1999; Yakubenko et al., 2000). TSP1 also induces MMP-9 in endothelial cells, but this induction was attributed to a different TSP1 receptor (Qian et al., 1997). We used reverse transcriptase (RT)-PCR to examine expression of mRNAs for several MMPs in Jurkat cells exposed to soluble or immobilized TSP1 (Fig. 6 A). Both forms of TSP1 induced MMP-2, but the response was greater using immobilized TSP1. Therefore, all subsequent experiments were performed using immobilized proteins. NoC1 and NoC2 also induced MMP-2 expression. The responses were time dependent and maximal for both proteins at 4 h (Fig. 6 A). Similar results were observed using CD4+ T cells (Fig. 6 A). Both immobilized and soluble TSP1 induced MMP-2 mRNA, and induction was also observed using NoC2.
|
MMP induction at the protein level was confirmed by gelatin zymography (Fig. 6 D). TSP1, NoC1, and NoC2 increased MMP-9 activity. MMP-2 activity was induced by NoC2 and, apparently, was processed to a smaller activated form in cells exposed to TSP1 (Fig. 6 D).
4ß1 integrin binding is not sufficient for inhibition of TCR signaling by TSP1
We previously demonstrated that TSP1 inhibits TCR-mediated T cell activation (Li et al., 2001). To determine whether 4ß1 integrin is involved in this activity, we tested NoC1 and NoC2 for inhibition of TCR-mediated T cell activation (Fig. 7 A). TSP1 inhibited TCR-mediated T cell activation measured by induction of cell surface CD69 expression (90% inhibition in mean fluorescent intensity relative to the anti-CD3stimulated positive control), but NoC1 (6% inhibition) and NoC2 (-12% inhibition) showed minimal or no inhibitory effects even using a fourfold higher molar concentration. Thus, the NH2-terminal regions of these proteins are not sufficient to mediate the inhibitory activity of TSP1 for T cell activation. TSP1 comparably inhibited CD69 expression in the wild-type and ß1 integrindeficient Jurkat cell lines (Fig. 7 B), demonstrating that ß1 integrins are not required for the inhibitory effect of TSP1 on TCR signaling. Therefore binding of TSP1 to
4ß1 integrin is neither necessary nor sufficient for the antagonist activity of TSP1 on TCR signaling. We previously demonstrated that two other T cell receptors for TSP1, CD47 and HSPG, mediate this inhibitory activity (Li et al., 2001).
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cellular responses to TSP1 can be modulated by altering the expression of specific TSP1 receptors, the activation state of these receptors, or conformation states of TSP1 that regulate its binding to specific receptors (Rodrigues et al., 2001). T cells express at least three TSP1 receptors, and of these, 4ß1 is known to be regulated in its affinity and avidity for binding to ligands.
4ß1 integrin is inactive in resting T cells but has low activity in resting Jurkat cells (Jakubowski et al., 1995). Activation of protein kinase C by phorbol esters, which increases
4ß1 avidity but not its affinity for ligands (for review see Woods and Shimizu, 2001), induced adhesion of T cells on immobilized TSP1 and the NH2-terminal region of TSP2. Soluble TSP1 also inhibited interactions of activated
4ß1 integrin with its high-affinity ligand VCAM-1. Therefore both stimulatory and inhibitory activities of TSPs may be modulated by signals that alter
4ß1 integrin activation.
4ß1 integrin activation is modulated by signals resulting from ligation of the TCR (Jakubowski et al., 1995), CD47 (Ticchioni et al., 2001), and some chemokine receptors (Jakubowski et al., 1995). Notably, CD47 itself is a TSP1 receptor, and binding of TSP1 peptides to CD47 activates several integrins (for review see Brown and Frazier, 2001). Therefore, binding of soluble TSP1 to CD47 could potentially enhance adhesion of T cells to immobilized TSP1 mediated by
4ß1 integrin. This cross-talk between CD47 and
4ß1 integrin may regulate arrest of T cells on inflammatory endothelium (Ticchioni et al., 2001).
Our data define a new 4ß1 integrin recognition sequence in the NH2-terminal pentraxin-related domain of TSP1. This site is conserved in TSP2 and is distinct from the previously identified
3ß1 integrin binding site in the same domain of TSP1 (Krutzsch et al., 1999). Thus, the NH2-terminal domains of TSPs functionally resemble the paralogous G domains of laminins (Beckmann et al., 1998) in that they contain multiple ß1 integrin recognition sites. Although the LDVP sequence we identified in TSP1 is conserved at the corresponding position in some laminin G domain modules (unpublished data), recognition of laminins by
4ß1 integrin has not been reported. Based on alignment with other members of the pentraxin family of known structure (Beckmann et al., 1998), the
4ß1 integrin binding sites of TSP1 and TSP2 are located in a loop connecting the predicted ß strands J and K. This is consistent with the location of the
4ß1 integrin binding sequence IDSP of VCAM-1 in a loop between its C and D ß strands (Jones et al., 1995).
TSP1 regulates the expression of MMP-9 in endothelial cells (Qian et al., 1997), and TSP2 also regulates extracellular MMP-2 through direct interactions (Yang et al., 2001). Previously identified ligands of 4ß1 integrin, including VCAM1 and FN, induced expression of MMPs in T cells and fibroblasts (Huhtala et al., 1995; Xia et al., 1996b; Yakubenko et al., 2000). Our data demonstrate that recombinant portions of TSP1 and TSP2 containing
4ß1 integrin binding sites are sufficient to induce MMP-2, MMP-7, and MMP-9 expression in T cells. Notably, these recombinant proteins lack the TSP type 1 repeat sequence implicated in MMP-9 induction by TSP1 in endothelial cells (Qian et al., 1997). Our data suggest that MMP induction is a general T cell response to all
4ß1 integrin ligands and that this integrin may mediate the observed effects of TSPs on MMP expression in other cell types.
Inhibition of TCR signaling by TSP1 in Jurkat cells is independent of 4ß1 integrin ligation, but this result does not eliminate the possibility that TSP1 and TSP2 may positively modulate T cell activation through this receptor. Integrins are well-documented costimulators of TCR signaling in peripheral T cells (for review see Epler et al., 2000). However, Jurkat cells lack CasL, which is required for ß1 integrin costimulation of TCR signaling (Kamiguchi et al., 1999). Therefore, we can observe only an inhibitory effect of TSP1 on TCR signaling in Jurkat T cells. In normal T cells, a second positive signal from TSP1 interacting with
4ß1 integrin may offset this inhibition. Further investigation is needed to define the net effect of TSP1 on TCR signaling and T cell activation in various physiological and pathological contexts.
Both TSP1 and FN recognize 4ß1 and
5ß1 integrins on T cells. Our data show that the relative contributions of these two integrins differ, however, for mediating T cell adhesion to each protein.
5ß1 integrin is the predominant FN receptor, whereas
4ß1 is the major TSP1 receptor. FN is a constitutive component of extracellular matrix, but in most tissues TSP1 is only present in matrix at sites of injury and tissue remodeling (Adams et al., 1995). The different relative strengths of TSP1 and FN interactions with these two integrins may therefore indicate to a T cell whether the tissue microenvironment it transverses is undergoing these processes, and this may in turn modulate each of the T cell behaviors we have examined here.
Although FN and TSP1 share these two integrin receptors on T cells, their effects on T cell behavior differ. FN stimulates proliferation of T cells and TCR signaling (Davis et al., 1990; Shimizu et al., 1990), whereas TSP1 inhibits these responses. 4ß1 and
5ß1 were implicated in the stimulation of T cell proliferation by FN (Davis et al., 1990; Shimizu et al., 1990), which is consistent with our evidence that these integrins mediate some positive effects of TSP1 on T cells but not its antiproliferative activity. CD47, which is required for the inhibitory activities, is a receptor for TSP1 but not for FN.
Our data suggest that TSP1 and TSP2 could modulate recirculation of T cells and diapedesis of other leukocytes. We demonstrated that TSPs inhibit VCAM-1 binding to T cells and T cell adhesion mediated by 4ß1 integrin binding to VCAM-1. Because
4 integrin function is essential for lymphocyte recruitment to sites of inflammation (Arroyo et al., 2000), TSP1 and TSP2 could potentially inhibit this process. Conversely, we found that TSP1 and the NH2-terminal portion of TSP2 stimulate chemotaxis of T cells by engagement of the same integrin and stimulate expression of several MMPs that may be necessary for passage of T cells through basement membranes (Xia et al., 1996a; Faveeuw et al., 2001) or tissue invasion (Lynch and McDonnell, 2000). Thus, expression of TSP1 or TSP2 in tissues could enhance recruitment of T cells to these sites by facilitating cell motility and invasion. Combined with the higher potency we observed for stimulating chemotaxis versus inhibiting
4ß1 integrinVCAM-1 binding, the net effect of TSP1 and TSP2 expression in tissue may be to enhance recruitment. In this context, it is notable that increased leukocyte recruitment was observed in breast tumors overexpressing TSP1 (Weinstat-Saslow et al., 1994).
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Recombinant soluble 7 domain VCAM-1 (S7DVCAM-1, residues 1674 of the mature protein; GenBank/EMBL/DDBJ accession no. X53051) was expressed in insect cells after insertion into pAcGP67.coco and generation of recombinant baculovirus. S7DVCAM-1 was purified on a Ni+-chelate resin as previously described for NoC1 (Misenheimer et al., 2000).
Site-directed mutagenesis of Asp(162) to Ala in TSP1(1175) was performed using the Stratagene QuickChange mutagenesis kit. The forward and reverse primer sequences were 5'-GAG AAT GCT GAG TTG GCC GTC CCC ATC CAA AGC G-3' and 5'-GCT TTG GAT GGG GAC GGC CAA CTC AGC ATT CTC C-3', respectively. After transformation into Escherichia coli XL1- blue, mutant clones were verified by DNA sequencing. The mutated plasmid was then transformed into E. coli A4255F-. E. coli A4255F- transformants were grown overnight at 28°C in Superbroth plus 50 µg/ml carbenicillin and induced by adding 10 g/liter of glucose and incubating at 42°C for 2 h. Inclusion bodies were isolated, and the mutant recombinant protein was purified as previously described for the wild-type recombinant protein (Vogel et al., 1993).
Cell culture
Jurkat T cells were maintained in RPMI 1640 medium supplemented with 10% FBS, 2 mM glutamine, penicillin, and streptomycin. ß1 integrindeficient (A1) and CD47-deficient (JinB8) T cell lines derived from Jurkat T cells (Romzek et al., 1998; Ticchioni et al., 2001) were provided by Yoji Shimizu (University of Minnesota Medical School, Minneapolis, MN) and Eric Brown (University of California San Francisco, San Francisco, CA), respectively. All cell cultures were grown at 37°C with 5% CO2.
Human primary CD4+ T cells were isolated by magnetic sorting using CD4 positive selection MicroBeads and autoMACS magnetic isolation system (Miltenyi Biotec). Human peripheral blood mononuclear cells were isolated by Ficoll gradient centrifugation from buffy coats obtained from normal donors (protocol no. 99-CC-0168), and CD4+ cells were purified according to the manufacturer's instructions. Cells were resuspended in complete RPMI medium and rested at 4°C overnight. FACS® analysis demonstrated >95% purity of the isolated CD4+ T cells (unpublished data).
Antibodies and reagents
Anti-CD3 antibody (clone HIT3a) was obtained from BD PharMingen. Anti-CD47 antibody (clone C1Km) was from ICN Biomedicals. A function-blocking antibody for 4ß1 integrin (clone P4C2) was from GIBCO BRL. A CD47 functionblocking antibody, B6H12, and the ß1 integrin functionstimulating antibody, TS2/16 (Hemler et al., 1984), were produced from hybridoma cell lines (American Type Culture Collection). A human integrin ß1 blocking antibody (mAb13) was provided by Ken Yamada (National Institute of Dental and Craniofacial Research, Bethesda, MD). An
5ß1 integrin peptide antagonist (GRGDNP; Pierschbacher and Ruoslahti, 1987) and an
4ß1 integrin antagonist (4-((2-methylphenyl)aminocarbonyl)aminophenyl)acetyl-LDVP (Lin et al., 1999) were obtained from Bachem. SDF1
was obtained from Sigma-Aldrich.
Cell adhesion assays
Matrix proteinmediated cell adhesion was measured as previously described (Wilson et al., 1999). TSP1 and the NoC proteins were coated in PBS, and the other recombinant portions of TSP1 were coated in 25 mM NaHCO3 buffer, pH 8.2. Overnight cell cultures (<5 x 105 cells/ml) were resuspended in RPMI with 0.1% BSA (Sigma-Aldrich) at 2 x 105 cells/ml. The plates were chilled in a 4°C bath, and 100 µl of cell suspension with the indicated treatment was added into each well. The plates were then incubated in a 37°C bath for 15 min. Unbound cells were removed by washing, and adherent cells were quantified by hexosaminidase assay (Wilson et al., 1999).
Adhesion was also assessed using a microscopic assay. TSP1, recombinant proteins, or S7DVCAM-1 diluted in Dulbecco's PBS or NaHCO3 buffer were adsorbed on bacteriological polystyrene dishes by incubation overnight at 4°C. After blocking with 1% BSA in Dulbecco's PBS, adhesion assays were performed by adding cells to prewarmed dishes containing RPMI with 1 mg/ml BSA. Cell attachment and spreading were quantified after 15 min by washing to remove nonadherent cells, fixing the adherent cells with 1% glutaraldehyde in PBS, and staining with Diff-Quik (Dade International).
Cell migration assay
A 48-well microwell modified Boyden chamber was used with 8-µm (Jurkat) or 5-µm (CD4+ T cells) pore polyvinylpyrrolidone-free polycarbonate membranes (Neuro Probe, Inc.). Wells in the lower chamber contained assay medium (RPMI with 0.1% BSA) and attractants as indicated. Polycarbonate membranes were coated overnight at 4°C with either 100 µg/ml of gelatin in 0.1% acetic acid or 20 µg/ml of polylysine in PBS and air dried. Jurkat cultures (<5 x 105 cells/ml) were resuspended in assay medium at 2 x 106 cells/ml with the indicated additions, added into wells of the upper chamber, and incubated at 37°C for 25 h. The membranes were fixed and stained, cells on the upper face were removed, and cells migrated to the lower face of the membrane were counted microscopically.
Flow cytometry analysis
To analyze T cell activation, expression of CD69 was measured by flow cytometry as previously described (Li et al., 2001). Cells were activated by incubating with surface-bound anti-CD3 antibody (BD PharMingen) in RPMI plus 0.1% BSA. After a 24-h stimulation at 37°C, the cells were washed and stained with PE-conjugated anti-CD69 antibody (BD PharMingen). CD69 expression was quantified using a FACS®Caliber flow cytometer (Becton Dickinson).
VCAM-1 cell binding assay
S7DVCAM-1 was labeled with 125I using Iodogen (Pierce Chemical Co.). Jurkat T cells were washed with 4°C chilled Dulbecco's PBS (without Ca2+ or Mg2+) and resuspended in chilled binding buffer (RPMI with 0.1% BSA) at 3 x 106 cells/ml. On ice, 100 µl of the cell suspension was premixed with the indicated concentrations of TSP1, NoC1, or NoC2. 125I-S7DVCAM-1, diluted in chilled binding buffer, was added into each tube to bring the final volume to 200 µl. The tubes were mixed by vortexing and transferred to a 37°C water bath for 15 min. The cell suspensions were then transferred to plastic tubes containing 100 µl of Nyosil oil (William F. Nye, Inc.), centrifuged for 1 min, and washed with 200 µl of cell binding buffer. The pellets were collected, and the bound radioactivity was quantified.
Proliferation
Effects of soluble or immobilized TSP1 reagents on Jurkat cell proliferation were quantified using a tetrazolium dye proliferation assay (Cell Titer Assay; Promega). The indicated cell lines (7.5 x 103 cells/well) were seeded in 96-well Nunc tissue culture plates for treatment with soluble proteins or in 96-well Nunc Maxisorp plates precoated with TSP1 or NoC1 and blocked with 1% BSA. Proliferation was assessed after growth for 72 h in RPMI medium containing 2% FCS.
MMP expression
Semiquantitative RT-PCR was used to analyze effects of TSPs on expression of mRNAs for MMP-2, MMP-7, MMP-9, and MMP-14 by Jurkat and CD4+ T cells. Cells were placed in untreated Falcon 1008 dishes or dishes previously coated overnight with TSP1, NoC1, or NoC2. Total RNA was isolated using Trizol reagent (GIBCO BRL) according to the instructions of the manufacturer. First strand cDNA synthesis was performed with Superscript II reverse transcriptase (GIBCO BRL) and 16 µg/ml oligo(dT) using 2 µg of total RNA. The enzyme was inactivated at 70°C for 15 min. The cDNA was amplified using Platinum Taq DNA polymerase (GIBCO BRL) and specific primer pairs to amplify glyceraldehyde phosphate dehydrogenase, MMP-2, MMP-7, MMP-9, or MMP-14 sequences (Table I). Amplification was conducted using two cycles of 1 min at 95°C and 4 min at 55°C followed by the indicated number of cycles of 1 min at 95°C, 2.5 min at 55°C, and 10 min at 70°C.
|
![]() |
Footnotes |
---|
![]() |
Acknowledgments |
---|
This work was supported in part by NIH grants HL54462 and 56396 (to D.F. Mosher).
Submitted: 26 September 2001
Revised: 20 March 2002
Accepted: 20 March 2002
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adams, J.C., R.P. Tucker, and J. Lawler. 1995. The Thrombospondin Gene Family. R.G. Landes Company, Austin, TX. 200 pp.
Arroyo, A.G., D. Taverna, C.A. Whittaker, U.G. Strauch, B.L. Bader, H. Rayburn, D. Crowley, C.M. Parker, and R.O. Hynes. 2000. In vivo roles of integrins during leukocyte development and traffic: insights from the analysis of mice chimeric for alpha 5, alpha v, and alpha 4 integrins. J. Immunol. 165:46674675.
Avice, M.N., M. Rubio, M. Sergerie, G. Delespesse, and M. Sarfati. 2000. CD47 ligation selectively inhibits the development of human naive T cells into Th1 effectors. J. Immunol. 165:46244631.
Beppu, R., K. Nakamura, H. Miyajima-Uchida, M. Kuroki, P.D. Khare, Y. Yamauchi, Y. Yamashita, and T. Shirakusa. 2001. Soluble thrombospondin-1 suppresses T cell proliferation and enhances IL-10 secretion by antigen presenting cells stimulated with phytohemagglutinin. Immunol. Invest. 30:143156.[CrossRef][Medline]
Brown, E.J., and W.A. Frazier. 2001. Integrin-associated protein (CD47) and its ligands. Trends Cell Biol. 11:130135.[CrossRef][Medline]
Davis, L.S., N. Oppenheimer-Marks, J.L. Bednarczyk, B.W. McIntyre, and P.E. Lipsky. 1990. Fibronectin promotes proliferation of naive and memory T cells by signaling through both the VLA-4 and VLA-5 integrin molecules. J. Immunol. 145:785793.
Esparza, J., C. Vilardell, J. Calvo, M. Juan, J. Vives, A. Urbano-Marquez, J. Yague, and M.C. Cid. 1999. Fibronectin upregulates gelatinase B (MMP-9) and induces coordinated expression of gelatinase A (MMP-2) and its activator MT1-MMP (MMP-14) by human T lymphocyte cell lines. A process repressed through RAS/MAP kinase signaling pathways. Blood. 94:27542766.
Faveeuw, C., G. Preece, and A. Ager. 2001. Transendothelial migration of lymphocytes across high endothelial venules into lymph nodes is affected by metalloproteinases. Blood. 98:688695.
Guo, N., V.S. Zabrenetzky, L. Chandrasekaran, J.M. Sipes, J. Lawler, H.C. Krutzsch, and D.D. Roberts. 1998. Differential roles of protein kinase C and pertussis toxin-sensitive G-binding proteins in modulation of melanoma cell proliferation and motility by thrombospondin-1. Cancer Res. 58:31543162.[Abstract]
Hemler, M.E., F. Sanchez-Madrid, T.J. Flotte, A.M. Krensky, S.J. Burakoff, A.K. Bhan, T.A. Springer, and J.L. Strominger. 1984. Glycoproteins of 210,000 and 130,000 m.w. on activated T cells: cell distribution and antigenic relation to components on resting cells and T cell lines. J. Immunol. 132:30113018.
Huhtala, P., M.J. Humphries, J.B. McCarthy, P.M. Tremble, Z. Werb, and C.H. Damsky. 1995. Cooperative signaling by alpha 5 beta 1 and alpha 4 beta 1 integrins regulates metalloproteinase gene expression in fibroblasts adhering to fibronectin. J. Cell Biol. 129:867879.[Abstract]
Jones, E.Y., K. Harlos, M.J. Bottomley, R.C. Robinson, P.C. Driscoll, R.M. Edwards, J.M. Clements, T.J. Dudgeon, and D.I. Stuart. 1995. Crystal structure of an integrin-binding fragment of vascular cell adhesion molecule-1 at 1.8 A resolution. Nature. 373:539544.[CrossRef][Medline]
Kamiguchi, K., K. Tachibana, S. Iwata, Y. Ohashi, and C. Morimoto. 1999. Cas-L is required for beta 1 integrin-mediated costimulation in human T cells. J. Immunol. 163:563568.
Kitani, A., N. Nakashima, T. Izumihara, M. Inagaki, X. Baoui, S. Yu, T. Matsuda, and T. Matsuyama. 1998. Soluble VCAM-1 induces chemotaxis of Jurkat and synovial fluid T cells bearing high affinity very late antigen-4. J. Immunol. 161:49314938.
Krutzsch, H.C., B. Choe, J.M. Sipes, N. Guo, and D.D. Roberts. 1999. Identification of an alpha(3)beta(1) integrin recognition sequence in thrombospondin-1. J. Biol. Chem. 274:2408024086.
Lawler, J., M. Sunday, V. Thibert, M. Duquette, E.L. George, H. Rayburn, and R.O. Hynes. 1998. Thrombospondin-1 is required for normal murine pulmonary homeostasis and its absence causes pneumonia. J. Clin. Invest. 101:982992.
Li, Z., L. He, K.E. Wilson, and D.D. Roberts. 2001. Thrombospondin-1 inhibits TCR-mediated T lymphocyte early activation. J. Immunol. 166:24272436.
Lynch, C.C., and S. McDonnell. 2000. The role of matrilysin (MMP-7) in leukaemia cell invasion. Clin. Exp. Metastasis. 18:401406.[CrossRef][Medline]
Mansfield, P.J., and S.J. Suchard. 1994. Thrombospondin promotes chemotaxis and haptotaxis of human peripheral blood monocytes. J. Immunol. 153:42194229.
Misenheimer, T.M., K.G. Huwiler, D.S. Annis, and D.F. Mosher. 2000. Physical characterization of the procollagen module of human thrombospondin 1 expressed in insect cells. J. Biol. Chem. 275:4093840945.
Moyano, J.V., B. Carnemolla, C. Dominguez-Jimenez, M. Garcia-Gila, J.P. Albar, P. Sanchez-Aparicio, A. Leprini, G. Querze, L. Zardi, and A. Garcia-Pardo. 1997. Fibronectin type III5 repeat contains a novel cell adhesion sequence, KLDAPT, which binds activated alpha4beta1 and alpha4beta7 integrins. J. Biol. Chem. 272:2483224836.
Pierschbacher, M.D., and E. Ruoslahti. 1987. Influence of stereochemistry of the sequence Arg-Gly-Asp-Xaa on binding specificity in cell adhesion. J. Biol. Chem. 262:1729417298.
Pierson, B.A., K. Gupta, W.-S. Hu, and J.S. Miller. 1996. Human natural killer cell expansion is regulated by thrombospondin-mediated activation of transforming growth factor-beta1 and independent accessory cell-derived contact and soluble factors. Blood. 87:180189.
Reinhold, M.I., F.P. Lindberg, G.J. Kersh, P.M. Allen, and E.J. Brown. 1997. Costimulation of T cell activation by integrin-associated protein (CD47) is an adhesion-dependent, CD28-independent signaling pathway. J. Exp. Med. 185:111.
Roberts, D.D. 1996. Regulation of tumor growth and metastasis by thrombospondin-1. FASEB J. 10:11831191.
Roberts, D.D., J. Cashel, and N. Guo. 1994. Purification of thrombospondin from human platelets. J. Tissue Cult. Methods. 16:217222.
Rodrigues, R.G., N. Guo, L. Zhou, J.M. Sipes, S.B. Williams, N.S. Templeton, H.R. Gralnick, and D.D. Roberts. 2001. Conformational regulation of the fibronectin binding and alpha 3beta 1 integrin-mediated adhesive activities of thrombospondin-1. J. Biol. Chem. 276:2791327922.
Romzek, N.C., E.S. Harris, C.L. Dell, J. Skronek, E. Hasse, P.J. Reynolds, S.W. Hunt III, and Y. Shimizu. 1998. Use of a beta1 integrin-deficient human T cell to identify beta1 integrin cytoplasmic domain sequences critical for integrin function. Mol. Biol. Cell. 9:27152727.
Schuler, G.D., S.F. Altschul, and D.J. Lipman. 1991. A workbench for multiple alignment construction and analysis. Proteins. 9:180190.[Medline]
Shimizu, Y., G.A. van Seventer, K.J. Horgan, and S. Shaw. 1990. Costimulation of proliferative responses of resting CD4+ T cells by the interaction of VLA-4 and VLA-5 with fibronectin or VLA-6 with laminin. J. Immunol. 145:5967.
Ticchioni, M., M. Deckert, F. Mary, G. Bernard, E.J. Brown, and A. Bernard. 1997. Integrin-associated protein (CD47) is a comitogenic molecule on CD3-activated human T cells. J. Immunol. 158:677684.[Abstract]
Ticchioni, M., V. Raimondi, L. Lamy, J. Wijdenes, F.P. Lindberg, E.J. Brown, and A. Bernard. 2001. Integrin-associated protein (CD47/IAP) contributes to T cell arrest on inflammatory vascular endothelium under flow. FASEB J. 15:341350.
Vallejo, A.N., L.O. Mugge, P.A. Klimiuk, C.M. Weyand, and J.J. Goronzy. 2000. Central role of thrombospondin-1 in the activation and clonal expansion of inflammatory T cells. J. Immunol. 164:29472954.
Vonderheide, R.H., T.F. Tedder, T.A. Springer, and D.E. Staunton. 1994. Residues within a conserved amino acid motif of domains 1 and 4 of VCAM-1 are required for binding to VLA-4. J. Cell Biol. 125:215222.[Abstract]
Weinstat-Saslow, D.L., V.S. Zabrenetzky, K. VanHoutte, W.A. Frazier, D.D. Roberts, and P.S. Steeg. 1994. Transfection of thrombospondin 1 complementary DNA into a human breast carcinoma cell line reduces primary tumor growth, metastatic potential, and angiogenesis. Cancer Res. 54:65046511.[Abstract]
Wilson, K.E., Z. Li, M. Kara, K.L. Gardner, and D.D. Roberts. 1999. Beta 1 integrin- and proteoglycan-mediated stimulation of T lymphoma cell adhesion and mitogen-activated protein kinase signaling by thrombospondin-1 and thrombospondin-1 peptides. J. Immunol. 163:36213628.
Woods, M.L., and Y. Shimizu. 2001. Signaling networks regulating beta 1 integrin-mediated adhesion of T lymphocytes to extracellular matrix. J. Leukoc. Biol. 69:874880.
Xia, M., D. Leppert, S.L. Hauser, S.P. Sreedharan, P.J. Nelson, A.M. Krensky, and E.J. Goetzl. 1996a. Stimulus specificity of matrix metalloproteinase dependence of human T cell migration through a model basement membrane. J. Immunol. 156:160167.[Abstract]
Yabkowitz, R., V.M. Dixit, N. Guo, D.D. Roberts, and Y. Shimizu. 1993. Activated T-cell adhesion to thrombospondin is mediated by the alpha 4 beta 1 (VLA-4) and alpha 5 beta 1 (VLA-5) integrins. J. Immunol. 151:149158.
Yang, Z., D.K. Strickland, and P. Bornstein. 2001. Extracellular matrix metalloproteinase 2 levels are regulated by the low density lipoprotein-related scavenger receptor and thrombospondin 2. J. Biol. Chem. 276:84038408.