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The Journal of Immunology, 2004, 172: 1397-1406.
Copyright © 2004 by The American Association of Immunologists

Mature Human Thymocytes Migrate on Laminin-5 with Activation of Metalloproteinase-14 and Cleavage of CD441

Mylène Vivinus-Nebot*,{dagger}, Patricia Rousselle{ddagger}, Jean-Philippe Breittmayer*, Claire Cenciarini*, Sonia Berrih-Aknin§, Suzanne Spong, Pasi Nokelainen, Françoise Cottrez*, M. Peter Marinkovich|| and Alain Bernard2,*,{dagger}

* Institut National de la Santé et de la Recherche Médicale, Unité 576, Nice, France; {dagger} Laboratoire d’Immunologie, Centre Hospitalier Universitaire, Nice, France; {ddagger} IBCP, Unité Mixte de Recherche 5086, Lyon, France; § Centre National de la Recherche Scientifique, Le Plessis Robinson, France; FibroGen, South San Francisco, CA 94080; and || Program in Epithelial Biology, Stanford University School of Medicine, Stanford, CA 94305


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have previously shown that laminin-5 is expressed in the human thymic medulla, in which mature thymocytes are located. We now report that laminin-5 promotes migration of mature medullary thymocytes, whereas it has no effect on cortical immature thymocytes. Migration was inhibited by blocking mAbs directed against laminin-5 integrin receptors and by inhibitors of metalloproteinases. Interactions of thymocytes with laminin-5 induced a strong up-regulation of active metalloproteinase-14. However, we found that thymocytes did not cleave the laminin-5 {gamma}2 chain, suggesting that they do not use the same pathway as epithelial cells to migrate on laminin-5. Interactions of thymocytes with laminin-5 also induced the release of a soluble fragment of CD44 cell surface molecule. Moreover, CD44-rich supernatants induced thymocyte migration in contrast with supernatants depleted in CD44 by immunoadsorption. CD44 cleavage was recently reported to be due to metalloproteinase-14 activation and led to increased migration in cancer cells. Thus, in this study, we show that laminin-5 promotes human mature thymocyte migration in vitro via a multimolecular mechanism involving laminin-5 integrin receptors, metalloproteinase-14 and CD44. These data suggest that, in vivo, laminin-5 may function in the migration of mature thymocytes within the medulla and be part of the thymic emigration process.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Tcell differentiation is a complex cellular process (1) that occurs as thymocytes migrate within different compartments of the thymus. Then, from the seeding by precursors to the export of newly mature T cells, migration is crucial (2, 3, 4, 5, 6, 7, 8) to allow developing T cells to interact sequentially with various specific tissue microenvironments, which provide decisive signals (3, 9, 10). Immature thymocytes are located in the cortex, and the most abundant subset expresses low levels of the CD3/TCR complex and both CD4 and CD8 molecules (double-positive (DP)3 cells). The heavily selected mature thymocytes are found in the medulla. They express high levels of the CD3/TCR complex and either CD4 or CD8 molecule (single-positive (SP) cells). The mechanisms that control mature thymocyte migration in the medulla and finally outside the thymus are still poorly understood, although they seem to be important events necessary to maintain a peripheral pool of T lymphocytes with a diverse repertoire (11, 12, 13, 14, 15). Recent studies have provided clues about the chemotactic signals that drive thymocytes toward the periphery (8, 16). However, it is likely that interactions with the thymic stroma, including extracellular matrix (ECM), could also play an important role in this process (10, 17). Laminin isoforms are major constituents of ECM and basement membranes (18, 19). Laminin-5 (Lam-5) is secreted by epithelial cells and supports cell migration during morphogenesis, wound healing, and tumor invasion. It is a major component of the skin basement membrane secreted by epidermal keratinocytes that have similarities to thymic epithelial cells (20). Lam-5 is expressed in the thymus (21, 22, 23). We previously reported that it is expressed in the thymic medullary stroma, at the level of medullary thymic epithelial cells and of the basement membrane of blood vessels, whereas its expression in the cortex is restricted to the subcapsular basement membrane (23). Lam-5 was reported to exert a protective role against apoptosis of immature thymocytes in mice (24), and we have previously shown that soluble Lam-5 can modulate human thymocyte proliferation (23), but the function of deposited Lam-5 in the thymic medulla is to date unknown. Lam-5 is a heterotrimer ({alpha}3{beta}3{gamma}2) secreted by epithelial cells as a precursor that undergoes processing of the {alpha}3 and {gamma}2 chains and has been reported to mediate epithelial cell migration (25, 26, 27, 28). Lam-5 can be cleaved by various proteolytic enzymes, including the metalloproteinases MMP-2 (27); MMP-14, also called MT1-MMP (28); and bone morphogenetic protein (BMP) -1 (29, 30, 31), an endopeptidase with a MMP domain. MMP-14 is anchored in the cell membrane (32), whereas MMP-2 is secreted. Activation of MMP-2 is regulated by the formation of MMP-2/tissue inhibitor of MMP-2 (TIMP-2)/MMP-14 complexes at the cell surface (33, 34). TIMP-2 is also a natural inhibitor of MMP-2 and MMP-14 activity (35, 36), and thus, MMP activity is tightly regulated. MMPs can process ECM components (37, 38, 39, 40), but MMP-14 was also reported to act as a processing enzyme for CD44, which was decisive to induce tumor cell migration (41, 42, 43). CD44 is a family of multifunctional cell adhesion molecules, the hemopoietic or standard type being the most abundant form (44). CD44 expression in thymocytes is much higher in mature medullary SP than in immature cortical DP (45, 46), and it was reported to play an important role in cell migration (44, 47, 48).

Consequently, it was of interest to determine whether Lam-5 could influence thymocyte migration. We have found that purified human Lam-5 induces SP, but not DP thymocytes to migrate. Concomitantly, MMP-14 is up-regulated and activated and surface CD44 is cleaved. Thus, we propose a model whereby immobilized Lam-5 and CD44 cleavage by MMPs have critical roles in the induction of migration in mature thymocytes.


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

Human Lam-5 was obtained by immunopurification of the conditioned medium of SCC25 cells using the 6F12 mAb, specific for {beta}3 chain of human Lam-5 (49), as previously described (50, 51). BSA and gelatin were purchased from Sigma-Aldrich (St. Louis, MO); chemicals for PAGE were from Bio-Rad (Richmond, CA). The BB94 synthetic inhibitor of MMPs was from British Biotech (Annapolis, MD). TIMP-2 was from R&D Systems (Abingdon, U.K.). MMP-2 was from Clinisciences (Montrouge, France). The BMP-1 inhibitor BI-1 (31) was a gift from Fibrogen (South San Francisco, CA).

Antibodies

Mouse mAb BM165 against {alpha}3 chain of human Lam-5 (51) and rabbit polyclonal pKa against human Lam-5 (52) were produced, as previously described. Mouse mAbs 4B4 against CD29 ({beta}1 integrin chain) and P1B5 against CD49c ({alpha}3 integrin chain) were from BD PharMingen (San Jose, CA). Mouse mAb GOH3 against CD49f ({alpha}6 integrin chain) was from Immunotech (Marseille, France). Mouse mAb D4B5 against {gamma}2 chain of human Lam-5, mouse mAb ASC-3 against CD104 ({beta}4 integrin chain), and rat mAb A020 against CD44 were from Chemicon International (Temecula, CA). Mouse mAb 5H2 against MMP-14 was from R&D Systems. Mouse mAbs O516 against CD4 (53) and L533 against CD8 (54) were generated in our laboratory. Secondary Abs, conjugated with fluorochromes, were purchased from DAKO (DAKOPATTS S/A, Copenhagen, Denmark). Goat anti-rabbit Ig conjugated with peroxidase was from Jackson ImmunoResearch Laboratories (West Grove, PA), and rabbit anti-mouse Ig conjugated with peroxidase was from Sigma-Aldrich.

Cells

Normal thymuses were obtained from children (<2 years) undergoing cardiac surgery. Thymocytes were prepared by physical disruption of the tissue and washes in RPMI 1640 and 10% FCS. Mature and immature thymocyte subsets were sorted out according to the surface density of CD4 and CD8 molecules as SP mature medullary cells and DP immature cortical cells with a FACStar cell sorter (BD Biosciences, Mountain View, CA).

Cytofluorimetry

Cells were washed three times with PBS and incubated at 4°C for 30 min in the dark in 100 µl PBS, 0.1% NaN3, and 0.1% BSA, with saturating concentration of FITC- or PE-conjugated mAbs, washed three times, and resuspended in PBS. For double-staining experiments, cells were first incubated with an anti-integrin Ab, next with a PE-conjugated mAb, and finally with FITC-conjugated CD3 mAb. Analysis was performed on a FACScan (BD Biosciences).

Microscopy

Thymocytes were added to Permanox Lab-Tek chamber slides (Nunc, Rochester, NY) 0.8 cm2/well, coated with human purified Lam-5 (2 µg/ml) or BSA as a control. Cells were incubated in serum-free medium for 3 h at 37°C, 5% CO2. Cells were fixed with paraformaldehyde, 1% final concentration, and morphology was then examined with a laser-scanning confocal microscope (Ultima Meridian; DGL Bioscience, Longjumeau, France).

Cell migration assays

Purified human Lam-5 was coated on 2.5-cm-diameter Transwell culture inserts, 5 µm pore size (Costar, Acton, MA), on both sides of the membrane, overnight at 4°C. We tested Lam-5 concentrations from 0.01 to 10 µg/ml with sorted thymocytes and mostly used a 2 µg/ml concentration. Cells (150,000 sorted thymocytes or 106 whole thymocytes) were settled in the upper chamber in HBSS medium supplemented with CaCl2 and MgCl2, both 1 mM, and incubated for 4 h at 37°C, 5% CO2. When Abs were used, cells were preincubated with the mAbs at 25 µg/ml (integrin mAbs) and in migration medium for 30 min at 4°C and then settled into inserts without washing. BM165 was added at concentrations ranging from 0.01 to 50 µg/ml. Inhibitors of MMPs were first preincubated overnight with thymocytes in serum-free medium. Thymocytes were then allowed to migrate in the presence of the inhibitors that were added in both compartments of the Transwell chamber. Experiments were performed in duplicates or triplicates. Transmigrated cells were collected in the lower chamber, and migration was quantified by using beads (Flow Count; Beckman Coulter, Fullerton, CA) and flow cytometry.

Real-time PCR

Total RNA was extracted from cells by RNAzol (Invitrogen, Friendswood, TX) and treated with DNase (DNase amplification grade I; Invitrogen, San Diego, CA) incubated for 4 h on BSA or Lam-5. First-strand cDNA was prepared from 1 µg total RNA (Superscript II kit; Invitrogen). The real-time quantitative PCR was performed with the SYBR Green PCR core reagents kitin, an ABI PRISM 5700 sequence detection system (Applied Biosystems, Foster City, CA), according to the manufacturer’s instructions. DNA templates were then amplified with specific primers designed to span exon-intron junctions to prevent amplification of genomic DNA. The sequences of the primers for MMP-2 were 5'-CCCTCGCAAGCCCAAGT-3' and 5'-GCACGAGCAAAGGCATCAT-3'. The sequences of the primers for MMP-14 were 5'-CAGTGGATGGACACGGAGAA-3' and 5'-GGTTTTTGGGTTTATCAGGAACAG-3'. Expression of target genes was measured after normalization of RNA concentration with four different housekeeping genes, and values were expressed as fold increased expression above a theoretical negative sample, as previously described (55).

Zymography

Thymocytes were cultivated for 24 h in serum-free medium supplemented with 1 mM CaCl2 and MgCl2 on immobilized Lam-5 or on BSA as a control. Gelatin zymography was performed as previously described (36, 56). Briefly, the samples were mixed with SDS-PAGE sample buffer without reducing agent and were subjected to electrophoresis with an SDS-polyacrylamide gel containing 10% polyacrylamide copolymerized with 1 mg/ml gelatin. The in-gel proteolytic reactions were performed by incubating the gel at 37°C for 24 h, and gelatinolytic activity was visualized as negative staining with Coomassie blue.

Immunoblots

To analyze MMP-14 expression and activation, 5 x 106 cells were incubated overnight on adsorbed Lam-5 or BSA as a control, at 37°C, 5% CO2 in HBSS supplemented with 1 mM CaCl2 and MgCl2. Cells were then removed, washed in PBS, and lysed in 1% Triton X-100 on ice for 30 min, and Laemmli sample buffer was added to lysates after centrifugation.

To detect CD44 in the culture supernatants, 5 x 106 sorted thymocytes were incubated on Lam-5 for 24 h at 37°C, 5% CO2 in HBSS supplemented with 1 mM CaCl2 and MgCl2. The medium was then collected and treated with 10% TCA for 1 h on ice. After centrifugation, the precipitate was washed in 70% ethanol, briefly dried, and resuspended in Laemmli sample buffer.

After separation by SDS-PAGE, samples were transferred to ECL nitrocellulose membrane (Millipore, Bedford, MA). The blotted membrane was saturated 1 h at 37°C in PBS and 5% nonfat milk, and next incubated overnight at 4°C with primary Abs diluted in PBS and 5% nonfat milk. After four washes in PBS containing 0.4% Tween, the membrane was incubated with HRP-conjugated secondary Abs diluted in PBS and 5% nonfat milk. After four washes, immunolabeling was revealed by chemiluminescence (ECL+; Amersham, Saclay, France).

CD44 depletion of supernatants

To deplete CD44 in the supernatants of thymocytes incubated on Lam-5, we performed two successive immunoprecipitations with the CD44 mAb P245 (10 µg/ml). To verify CD44 depletion, immunoprecipitates were separated by SDS-PAGE, transferred to nitrocellulose, and blotted with the CD44 mAb A020 (2 µg/ml). Migration assays were next performed with 106 thymocytes suspended in HBSS medium supplemented with 1 mM CaCl2 and MgCl2 or in the different supernatants in transwells coated with BSA or Lam-5.

Analysis of Lam-5 processing by thymocytes

A cellular matrix enriched in unprocessed Lam-5 was produced by incubating SCC25 cells in a 96-well culture plate in the presence of BI-1 (31) at 40 µM. Cells were removed by washing plates twice with PBS and incubating with 20 mM ammonium hydroxide for 5 min. Wells were next washed twice with water and twice with PBS. Processed Lam-5 produced by SCC25 cells cultivated without the inhibitor was used as a control. The matrix deposited in culture wells was finally incubated 24 h at 37°C with 2 x 106 thymocytes/well or with culture medium only as a negative control. After incubation of thymocytes was completed, culture wells were washed twice with PBS and incubated 5 min with 20 mM ammonium hydroxide. Wells were next washed twice with water, twice with PBS, and incubated with 15 µl of lysis buffer (0.122 g Tris HCl adjusted to pH 6.8 with 48 g urea and 5 ml of 2-ME/100 ml of buffer) for 30 min at room temperature. Finally, lysis buffer from four identical wells was collected in one tube with sample buffer and loaded onto a 6% acrylamide gel. Lam-5 chains were then transferred onto nitrocellulose and analyzed by Western blot with anti-human Lam-5 Abs.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Lam-5 induces a migratory phenotype of human mature thymocytes

Mature and immature thymocyte subsets were sorted out according to the surface density of CD4 and CD8 molecules (Fig. 1) as SP mature medullary cells and DP immature cortical cells. Sorted thymocytes were incubated on Lam-5 coatings or on BSA as a control, in cation-containing medium for 4 h at 37°C. Incubation on Lam-5 induced SP thymocytes to adopt a morphology very similar to the aspect of mobile lymphocytes (Fig. 1, SP/Lam-5). By contrast, immature DP thymocytes kept a round shape under the same conditions (Fig. 1, DP/Lam-5). When incubated on BSA as a negative control, SP kept a round shape (Fig. 1, SP/BSA).



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  		FIGURE 1. Microscopic analysis of cell shape modifications of sorted thymocytes incubated on immobilized Lam-5. Mature and immature thymocytes were sorted out according to the surface density of CD4 and CD8 molecules as SP mature medullary cells and DP immature cortical cells, with at least 95% purity. The percentage of cells is indicated in the corner of the corresponding quadrant. Cells were next incubated on immobilized Lam-5 (2 µg/ml) for 4 h at 37°C, in medium supplemented with 1 mM CaCl2 and MgCl2, without FCS. SP/Lam-5, SP mature thymocytes incubated on Lam-5. Induction of cells with a polarized morphology, displaying uropodia and lamellipodia. DP/Lam-5, DP incubated on Lam-5. SP/BSA, SP incubated on BSA. Scale bar, 10 µm.

 
Lam-5 supports migration of mature human thymocytes

To measure the motile behavior of thymocytes on immobilized Lam-5, we performed migration assays in Transwell chambers. Thymocytes were sorted as immature DP or mature SP cells. They were incubated in the upper compartment of Transwell chambers whose membrane had been coated on both sides either with various amounts of purified human Lam-5 ranging from 0.01 to 10 µg/ml or BSA as a control. Cells were allowed to migrate for 4 h at 37°C. Transmigrated cells were next harvested in the lower chamber and counted with fluorescent beads. From Fig. 2A, it can be seen that only mature SP-sorted thymocytes migrated on Lam-5, whereas immature DP did not, as compared with migration on BSA-coated filters. From Fig. 2B, it can be seen that Lam-5 added in a soluble rather than coated form either in the upper or in the lower chamber did not induce significant thymocyte transmigration to the lower chamber. In the presence of the anti-Lam-5 mAb BM165, which recognizes a Lam-5 epitope involved in cell adhesion and migration (51), thymocyte migration on Lam-5 was inhibited (Fig. 2C). We also compared sorted CD4+CD8- and CD4-CD8+ SP and found that both subsets were able to migrate on Lam-5 (Fig. 2D). We finally analyzed the expression of the different Lam-5 receptors on thymocytes by performing a double staining with mAbs specific for the integrin chains {alpha}3, {alpha}6, {beta}1, and {beta}4, and a CD3 mAb. We found that expressions of {alpha}3, {alpha}6, and {beta}4 chains were much higher on CD3high most mature thymocytes (Fig. 3) than on CD3intermediate and CD3low immature thymocytes.



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  		FIGURE 2. SP, but not DP thymocytes migrate in Transwell chambers coated with purified human Lam-5. Sorted thymocytes were settled in the upper chamber in serum-free medium supplemented with cations, and cells were allowed to migrate for 4 h at 37°C. Transmigrated cells were collected in the lower chamber and counted with fluorescent beads on a FACScan. A, Differential effect of immobilized Lam-5 on transmigration of mature SP cells (•) vs immature DP cells ({blacksquare}). SP and DP sorted thymocytes were submitted to migrate on various concentrations of immobilized Lam-5 expressed in micrograms per milliliter, as indicated. B, Differential effect of immobilized Lam-5 (XL Lam-5) and soluble Lam-5 added either in the upper (up) or lower (down) compartment on mature SP thymocyte migration. C, BM165, a blocking anti-Lam-5 mAb, was added in the upper chamber at indicated concentrations and efficiently blocked thymocyte migration on immobilized Lam-5, by contrast with identical amounts of control mouse IgG1 (MIgG1). D, Migration of CD4+CD8- and CD4-CD8+ sorted SP on Lam-5.

 


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  		FIGURE 3. Expression of Lam-5 receptor integrin chains on thymocyte subsets analyzed by cytofluorimetry. Thymocytes were doubly stained with mAbs specific for the {alpha}3 (A), {alpha}6 (B), {beta}1 (C), and {beta}4 (D) chains of integrins, namely P1B5, GOH3, 4B4, and 3E1F6, respectively, and a CD3 mAb to visualize the expression of these integrin chains on the most mature CD3high thymocytes or on immature CD3intermediate and CD3low thymocytes.

 
{alpha}3{beta}1 and {alpha}6{beta}4 receptors are both involved in human thymocyte migration on Lam-5

We performed migration assays in Transwell chambers with sorted thymocytes, as described above, but cells were preincubated for 30 min at 4°C with mAbs directed against the Lam-5 receptors on thymocytes: {alpha}6{beta}4, {alpha}6{beta}1, and {alpha}3{beta}1. All mAbs were adhesion-blocking mAbs used at saturating concentrations: 4B4 anti-{beta}1 mAb (57), ASC-3 anti-{beta}4 mAb (58), GOH3 anti-{alpha}6 mAb (59), and P1B5 anti-{alpha}3 mAb (60). Cells were then deposited in the upper compartment without washing and allowed to migrate for 4 h at 37°C. From Fig. 4, it can be seen that a complete inhibition of migration was obtained with the anti-{beta}1 mAb 4B4 and the anti-{alpha}3 mAb P1B5. Because it has previously been reported that the anti-{alpha}3 mAb P1B5 induces keratinocyte intercellular adhesion (61), we verified that the inhibition of migration obtained with this mAb was not due to thymocyte aggregation. We found that P1B5 did not induce thymocyte aggregation in the conditions of our migration assay that are serum free (data not shown). A significant, but incomplete inhibition was also observed with the anti-{beta}4 mAb ASC-3 and the anti-{alpha}6 mAb GOH3. These data suggest that {alpha}3{beta}1 and {alpha}6{beta}4 are both involved in thymocyte migration on Lam-5. The role of {alpha}6{beta}1 remains unclear because both chains of this integrin are shared with the other two receptors.



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  		FIGURE 4. Inhibition of migration by adhesion-blocking mAbs directed against Lam-5 receptors. Sorted mature SP thymocytes were preincubated with blocking mAbs directed against the integrin chains of Lam-5 receptors, anti-{alpha}6 mAb GOH3, anti-{alpha}3 mAb P1B5, anti-{beta}4 mAb ASC-3, and anti-{beta}1 mAb 4B4, or with mouse IgG1 (MIg) as a negative control, and settled into inserts without washing. Migration was then performed in Transwell chambers, as described in Fig. 2. Abs were used at 25 µg/ml.

 
MMPs are involved in thymocyte migration on Lam-5

Cell migration through tissues may also require ECM degradation by various enzymes, including MMPs. MMPs can also process ECM substrates to modify their functional properties, as has been described for Lam-5 processing, which promotes epithelial cell migration (27, 28). MMP-2 (27) and MMP-14 (28) have been reported to play a role in cleavage of the Lam-5 {gamma}2 chain and subsequent functional activity (27, 28). Thus, we first tested the effect of the synthetic inhibitor of MMPs BB94. BB94 significantly inhibited thymocyte migration on Lam-5 (Fig. 5). By contrast, the DMSO vehicle at the same dilution had no effect. Inhibition of migration was not related to thymocyte mortality or inhibition of adhesion to Lam-5 in the presence of BB94 (data not shown). Moreover, migration was also fully inhibited with the recombinant form of the natural tissue inhibitor of MMPs, TIMP-2.



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  		FIGURE 5. Inhibition of mature thymocyte migration on Lam-5 by inhibitors of MMPs. Sorted mature SP thymocytes were preincubated overnight at 37°C with the synthetic inhibitor of MMPs, BB94 (10 µM), or recombinant TIMP-2 (10 µg/ml), in serum-free medium, and next settled into inserts without washing. Inhibitors were also added in the lower chamber at the same concentration, and migration was performed in Transwell chambers, as described in Fig. 2. A DMSO solution was used as a control for BB94.

 
The membrane-localized MMP-14 activation status was analyzed on blots performed with lysates of thymocytes cultured on immobilized Lam-5 or on BSA as a control, and we found that the 60-kDa active form of MMP-14 was present in much greater amount in cells cultured on Lam-5 (Fig. 6B). The cytosolic tyrosine kinase Zap-70 was used as a protein-loading control (Fig. 6B). This augmentation was at least in part due to an increased transcription of MMP-14 after thymocytes interacted with Lam-5, as shown by real-time PCR results displayed in Fig. 6A. By contrast, we observed no increase of MMP-2 transcription. We also performed zymography assays with supernatants from unsorted thymocytes cultured on immobilized Lam-5 or BSA to investigate the activation status of secreted MMP-2 (Fig. 6C), and we detected only the 72-kDa proform of MMP-2 in both cases. By contrast, active MMP-2 was found in supernatants of cells cultured in Matrigel, showing that active MMP-2 could be detected from these cells when they were cultivated in different conditions. The experiment was repeated with sorted thymocytes, but we still could not detect active MMP-2 in the supernatants (Fig. 6D), and we neither detected active MMP-2 associated to thymocytes (Fig. 6E).



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  		FIGURE 6. MMP-14 and MMP-2 expression and activation in thymocytes incubated on Lam-5. Thymocytes were cultured for 24 h at 37°C on immobilized Lam-5 or BSA as a control, in serum-free medium supplemented with 1 mM CaCl2 and MgCl2. A, Real-time PCR realized with thymocytes incubated on Lam-5 or BSA, as indicated. B, MMP-14 expression analyzed by Western blot performed with the anti-MMP-14 mAb 5H2 on lysates obtained from thymocytes incubated on BSA or Lam-5, as indicated. The 66-kDa form and the intermediate 63-kDa form of MMP-14 are not fully activated by contrast with the 60-kDa form. The membrane was reprobed with an anti-Zap 70 Ab to confirm equal loading of the gel. C and D, MMP-2 activity analyzed by zymography. C, Supernatants of whole thymocytes cultivated on BSA, Lam-5, or Matrigel were analyzed. Culture medium and Matrigel were loaded as negative controls, whereas purified active MMP-2 was loaded as a positive control. D, Supernatants of sorted mature SP thymocytes or immature DP thymocytes cultured on Lam-5 were analyzed. Culture medium and purified active MMP-2 were loaded as negative and positive controls. E, Membrane-bound MMP-2 expression analyzed by Western blot performed with the anti-MMP-2 mAb VB3 on lysates obtained from thymocytes incubated on BSA or Lam-5, as indicated. Active MMP-2 was loaded as a positive control. Only the 72-kDa proform was detected in cell lysates.

 
BMP-1 is another MMP that can cleave Lam-5, and thus may influence its functional properties (29, 31). We had previously found that BMP-1 RNA was also constitutively expressed in human thymocytes (data not shown), and we investigated its role in thymocyte migration on Lam-5 by performing migration assays in Transwell chambers in the presence of BI-1, an inhibitor specific for BMP-1 (31). BI-1 did not inhibit thymocyte migration on Lam-5 (Fig. 7), even when preincubated overnight with cells and used at the highest nontoxic concentration for these cells (40 µM). BI-1 was, however, able to fully inhibit Lam-5 {gamma}2 chain processing in SCC25 human epithelial cell line, showing that the inhibitor was active (Fig. 8, lanes 1). Therefore, we first conclude that at least one MMP is involved in migration of thymocytes on Lam-5, and second, that among MMPs that were previously shown to be able to process Lam-5 and change its functions, only MMP-14 seems to be involved.



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  		FIGURE 7. BMP-1 is not involved in thymocyte migration on Lam-5. The specific inhibitor for BMP-1, BI–1, does not inhibit thymocyte migration on Lam-5. Unsorted thymocytes were preincubated overnight at 37°C with BI–1 at indicated concentrations in serum-free medium, and then settled into inserts without washing, and migration was performed in Transwell chambers, as described in Fig. 4. The same dilutions of DMSO were used as negative controls, and the effect was compared with the inhibition induced by the synthetic inhibitor of MMPs, BB94 (10 µM). Inhibitors were also added in the lower chamber at the same concentration.

 


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  		FIGURE 8. Western blot analysis of Lam-5 processing by thymocytes: thymocytes can process the {alpha}3, but not the {gamma}2 chain. We generated a cellular matrix enriched in unprocessed Lam-5 secreted by SCC25 cell cultured in the presence of the processing inhibitor BI-1 used at 40µM (lane 1). Next, we incubated this matrix with serum-free culture medium alone (lane 1) or with thymocytes (lane 2) for 24 h at 37°C. Lane 3, The processed matrix secreted by SCC25 cells in absence of inhibitor. After incubation, thymocytes and culture medium were removed, and the matrix was collected from the wells and analyzed by immunoblotting with the polyclonal anti-Lam-5 pKa (A) or D4B5 mAb specific for the {gamma}2 chain (B).

 
Thymocytes are not able to process the Lam-5 {gamma}2 chain

To address the question of the function of MMPs in Lam-5-induced thymocyte migration, we first asked whether thymocytes were able to process Lam-5 {gamma}2 chain. For this purpose, we used a cellular matrix enriched in unprocessed Lam-5 secreted by SCC25 cells cultured in the presence of the BMP-1 inhibitor BI-1 (31). SCC25 cells were then removed, and the matrix deposited in culture wells was next incubated with culture medium only as a negative control (Fig. 8, lanes 1) or with thymocytes for 24 h at 37°C (Fig. 8, lanes 2). Lam-5 collected from SCC25 cells plated without inhibitor was used as a positive control for processing (Fig. 8, lanes 3). After incubation, thymocytes and culture medium were removed and the matrix was collected from the wells. The collected matrix was analyzed by immunoblotting with a polyclonal anti-Lam-5 Ab (Fig. 8A) or D4B5 mAb specific for the {gamma}2 chain (Fig. 8B). We observed that the matrix deposited by SCC-25 cultured with the inhibitor contained only the unprocessed form of the {gamma}2 chain (155 kDa), whether it had been incubated with medium only (Fig. 8, lanes 1) or with thymocytes (Fig. 8, lanes 2) for 24 h. The processed form of the {gamma}2 chain (105 kDa) was only detected in the control matrix deposited by SCC-25 cells cultured in the absence of the inhibitor (Fig. 8, lanes 3). Fig. 8 shows results obtained with unsorted thymocytes, but identical results were obtained with sorted cells, either SP or DP thymocytes (data not shown). These data show that human thymocytes are not able to process the {gamma}2 chain of Lam-5.

Interaction of mature thymocytes with Lam-5 induces the release of a soluble fragment of CD44, which depends on the activity of matrix MMPs

MMP-14 activation and coexpression with CD44 have been found to induce the processing of CD44 on tumor cells and to be critical for their migration (41, 43). We therefore attempted to determine a role for CD44 in thymocyte migration on Lam-5. CD44 is highly expressed on mature thymocytes, and, as described earlier, incubation of thymocytes on Lam-5 strongly increases expression of MMP-14. These mature thymocytes are induced to migrate on Lam-5, whereas cortical immature thymocytes, which display low expression of CD44, do not migrate. To determine whether activation of MMP-14 induces processing of CD44 in thymocytes, we incubated sorted thymocytes on immobilized Lam-5 and analyzed the supernatants for CD44 processing. The release of a soluble fragment of CD44 was tested by immunoblotting with the rat CD44 mAb A020. We found that incubation of SP thymocytes with Lam-5 induced the release of a soluble fragment of CD44, whereas none was visible in supernatants of DP cells incubated on Lam-5 or in supernatants of cells incubated on BSA (Fig. 9A). Thus, only the interaction of SP thymocytes with Lam-5 resulted in the release of a soluble fragment of CD44. MMPs were previously reported to induce CD44 shedding (41, 42, 62), and we have observed that BB94 and TIMP-2 inhibited thymocyte migration on Lam-5; therefore, we checked the ability of these MMP inhibitors to also influence the release of a soluble fragment of CD44. We found that incubation of mature SP thymocytes on Lam-5 in the presence of BB94 or TIMP-2 inhibited the cleavage of CD44, showing that it is MMP dependent (Fig. 9B).



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  		FIGURE 9. Detection of soluble CD44 in supernatants of thymocytes incubated on Lam-5 by immunoblot. A, Soluble CD44 is only detected in supernatants from SP cells. Sorted mature SP or immature DP thymocytes were incubated for 24 h on Lam-5 or BSA, as indicated, in migration medium. Supernatants were precipitated with 10% TCA and analyzed by immunoblotting with the CD44 mAb A020. B, The cleavage of CD44 is MMP dependent. Thymocytes were preincubated overnight at 37°C with the synthetic inhibitor of MMPs, BB94 (10 µM), or recombinant TIMP-2 (10 µg/ml), in serum-free medium, and then incubated on immobilized Lam-5 or BSA, as indicated, for 24 h at 37°C, in migration medium. Supernatants were precipitated with 10% TCA, and cells were lysed in 1% Triton X-100. Supernatants and cell lysates were analyzed by immunoblotting with the CD44 mAb A020.

 
Thymocyte motility is increased by soluble CD44

We observed that incubation of thymocytes on Lam-5 induced the release of soluble CD44. Because it was reported in other cell types that CD44 cleavage promoted migration, we investigated whether the supernatants of thymocytes cultivated on Lam-5 could enhance thymocyte migration in Transwell chambers. We incubated thymocytes on immobilized Lam-5, in cation-supplemented serum-free medium for 24 h at 37°C, and collected the supernatants by centrifugation. Next, we incubated other thymocytes with these supernatants for 30 min at 37°C before settling them into Transwell inserts coated with BSA. As a positive control for migration, we used Transwell chambers coated with Lam-5 and thymocytes suspended in migration medium, as previously described. We observed that resuspending cells in supernatants from thymocytes cultivated on Lam-5 instead of the migration medium induced a quick and significant migration (Fig. 10B). Indeed, after 30 min, the degree of migration was greater than in the positive control. Moreover, previous CD44 depletion of the supernatant by immunoadsorption inhibited this migration, suggesting that CD44 was involved (Fig. 10B). CD44 depletion was performed with the CD44 mAb P245 and assessed by performing a second immunoprecipitation with the immunoadsorbed supernatant and analyzing the resulting immunoprecipitates by Western blot with the CD44 mAb A020 (Fig. 10A). Interestingly, the migration induced by coated Lam-5 was slower (Fig. 10B; t = 30 min), suggesting that what was necessary for migration was immediately available in supernatants of thymocytes cultured on Lam-5, whereas it had to be generated by thymocytes reacting with coated Lam-5.



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  		FIGURE 10. Soluble CD44 increases thymocyte migration in Transwell chambers. A, CD44 Western blot of supernatants before and after immunoadsorption. Thymocytes were incubated on Lam-5 for 24 h in migration medium, and supernatants were collected by centrifugation. A part of these supernatants was depleted in CD44 by immunoprecipitation with the CD44 mAb P245. We assessed CD44 depletion by performing a second immunoprecipitation with the immunoadsorbed supernatant and analyzing the resulting imunoprecipitates by Western blotting with the CD44 mAb A020. B, Fresh thymocytes were suspended either in migration medium (0) or in the different supernatants, untreated (+ sup) or depleted in CD44 (+ dep sup). They were allowed to migrate in inserts coated with BSA or Lam-5, for 30 min or 4 h, as indicated.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We report in this study that human mature SP, but not immature DP thymocytes are induced to migrate in vitro on Lam-5. Interestingly, a recent study has also shown that only mature SP thymocytes are able to adhere to Lam-5 (63). In agreement with previous studies (23, 63, 64), we observed that expression of Lam-5 receptors was significantly higher on the most mature CD3high subset, which might explain at least in part the selective migration of mature thymocytes on Lam-5. The viability of both SP and DP remained excellent in the conditions of our migration assays, whether they were incubated on BSA or on Lam-5 (data not shown), which suggests that apoptosis or protection from apoptosis of one of these two subsets did not explain their differential migration on Lam-5. Not all SP adopted a migratory phenotype, which is most likely due to the heterogeneity of the mature thymocyte subset, as illustrated, for instance, by phenotypic heterogeneity (65, 66). Indeed, it has been reported that only a small proportion of SP present in the thymic medulla is ready to be exported (66). We have previously reported that Lam-5 is expressed in the thymic medullary parenchyma in which mature SP thymocytes are located (23), but Lam-5 functions in the thymus are poorly understood. It was reported to exert a protective role against apoptosis of immature thymocytes in mice (24), this function supported by the cortical location of Lam-5 as a component of basement membranes in contact with immature thymocytes. We have also previously shown that soluble Lam-5 could modulate human thymocyte proliferation (23). However, the function supported by deposited Lam-5 in the thymic medulla in which mature thymocytes are located has to date remained unknown. Our present data indicate that deposited Lam-5, which triggers nonlymphoid cell motility, could be used by mature thymocytes as a migratory substrate within the medulla, possibly as part of the mature cell exit phenomenon. Previously, we reported Lam-5 expression at the level of medullary epithelial cells and of basal laminae of vessels (23). These regions form the boundaries of the perivascular space of medullary vessels, a compartment in which thymocytes are packed before export (67, 68). Therefore, the anatomical distribution of Lam-5 in the medulla, taken together with the ability of Lam-5 to trigger mature thymocyte migration in vitro, suggests that Lam-5 may play a role in T cell migration within the thymic medulla and in exit from the thymus.

We found that blocking {alpha}3{beta}1 interactions with Lam-5 by adhesion-blocking mAbs totally inhibited thymocyte migration, whereas blocking {alpha}6{beta}4 only partially inhibited cell migration. These data emphasize the prominent role of {alpha}3{beta}1 in Lam-5-dependent migration of thymocytes, as has been reported for other cell types (25, 69, 70).

Lam-5 is secreted by epithelial cells in a precursor form that is subsequently processed by specific enzymes. Although both {alpha}3 and {gamma}2 chains of Lam-5 can be processed, recent studies have suggested a link between epithelial cell motility and the Lam-5 {gamma}2 chain processing that can be induced by MMP-2 (27), MMP-14 (28), or BMP-1 (29, 31). We report in this study that incubation of thymocytes with synthetic and natural inhibitors of MMPs fully inhibited migration on Lam-5, showing that MMPs are involved in this process. We investigated the three MMPs reported to induce Lam-5 processing to date and, although they were all constitutively expressed, our data only suggested a role for MMP-14. Indeed, interaction of thymocytes with Lam-5 increased MMP-14 expression in its active form, due at least in part to increased transcription. In contrast, we found no change in MMP-2 expression or activation state. It was rather surprising that activation of MMP-14 did not induce the subsequent activation of MMP-2. We did find, however, that thymocytes were able to convert MMP-2 to its active form when cells were cultured in Matrigel, suggesting an exquisite regulation of MMP-2 activation in the presence of Lam-5. Interestingly, it has been previously reported that an increase in MMP-14 activity does not necessarily enhance the activation of pro-MMP-2 (36). Moreover, we found that CD44 was involved in thymocyte migration on Lam-5, and a recent study has shown that CD44 can bind to MMP-14 via its hemopexin-like (PEX) domain and trigger CD44 shedding and cell migration (43). Then CD44 ligation to the PEX domain of MMP-14 on thymocyte surface might prevent the formation of MMP-14 homodimers through the same PEX domain (71) and subsequent MMP-2 activation. With regard to BMP-1, we found that thymocyte migration on Lam-5 was not inhibited by a specific inhibitor of BMP-1 (31). Therefore, we conclude that at least one MMP, most likely MMP-14, is involved in thymocyte migration on Lam-5.

Consequently, we asked whether MMPs promoted thymocyte migration by processing Lam-5 or through some other mechanism. Using a cellular matrix enriched in unprocessed Lam-5 as a substrate, we observed that human thymocytes could not process the {gamma}2 chain. These data suggest that the mechanisms driving human thymocyte migration on Lam-5 differ from those reported for epithelial cells (27, 28). Yet, it is noteworthy that epithelial cells secrete Lam-5 in an unprocessed form they must cleave before migrating on it, whereas thymocytes migrate on Lam-5 that was previously processed and deposited in the ECM by medullary thymic epithelial cells.

MMPs were also reported to interact with cell adhesion molecules (42, 72, 73, 74), including the widely expressed hemopoietic isoform of CD44. It has been shown that CD44 can be cleaved by MMP-14, resulting in the release of a soluble fragment of CD44 and increased cell motility (42). We therefore attempted to determine a role for CD44 in thymocyte migration on Lam-5. We found that the interaction of mature thymocytes with Lam-5 induced the release of a soluble fragment of CD44. This release was inhibited by the MMP inhibitors BB94 or TIMP-2, demonstrating that cleavage was MMP dependent. This led us to hypothesize that MMPs induce CD44 processing in thymocytes incubated on Lam-5, releasing a soluble fragment of CD44 that promotes cell migration, as has been described for tumor cells. MMP-14 is most likely involved in this process because it is up-regulated in thymocytes interacting with Lam-5, but we do not exclude a role for other MMPs.

To directly assess the role of CD44 cleavage in Lam-5-induced thymocyte migration, we determined whether soluble CD44 could stimulate thymocyte migration. Supernatants from thymocytes cultivated on Lam-5 were used as a source of soluble CD44. Adding CD44-rich supernatants to thymocytes in Transwell chambers resulted in rapid cell migration, an effect that disappeared after CD44 immunoadsorption, suggesting that soluble CD44 plays a decisive role. These data show that soluble CD44 was generated by thymocytes incubated on Lam-5, which promoted their migration in Transwell chambers. The soluble fragment of CD44 most likely interacts with a cell surface molecule on thymocytes that remains to be identified. However, the persisting transmembrane fragment might also play a role because it has been shown that cleavage of the CD44 extracellular domain is followed by release of a cytoplasmic domain that translocates to the nucleus and acts as a transcription factor (75). A recent study has demonstrated a cooperation of MMP-14 and CD44 in tumor cell migration (43). The authors have shown that CD44 bound MMP-14 via its hemopexin domain and that mutations of either of those two molecules impeded migration and CD44 shedding. Our data suggest that MMP-14 function might be much the same in thymocyte migration on Lam-5. These studies, performed with tumor cells, and our own data obtained with normal primary cells emphasize that MMPs can modulate cell migration not only by modifying the microenvironment, but also by interacting with cell surface molecules.

Thus, in this study, we have shown that in vitro Lam-5 promotes human mature thymocyte migration via a multimolecular mechanism involving integrins; MMPs, most likely MMP-14; and the CD44 cell surface molecule. We hypothesize that mature thymocyte interactions with Lam-5 increase MMP-14 activity and CD44 cleavage, which in turn promotes cell migration.


   Acknowledgments
 
We thank the Centre Cardiothoracique de Monaco and Frédérique Truffault for their help regarding thymus supply.


   Footnotes
 
1 This work was supported by the Institut National de la Santé et de la Recherche Médicale and the Association pour la Recherche contre le Cancer (No. 9680 and No. 5977). Back

2 Address correspondence and reprint requests to Dr. Alain Bernard, Institut National de la Santé et de la Recherche Médicale Unité 576, Hôpital de l’ Archet 1, Route de Saint Antoine de Ginestière, BP3079, 06202 Nice, France. E-mail address: abernard{at}unice.fr Back

3 Abbreviations used in this paper: DP, double positive; BI-1, BMP-1 inhibitor; BMP, bone morphogenetic protein; ECM, extracellular matrix; Lam-5, laminin-5; MMP, metalloproteinase; PEX, hemopexin-like; SP, single positive; TIMP, tissue inhibitor of MMP. Back

Received for publication May 12, 2003. Accepted for publication November 13, 2003.


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