Laminin-8 (α4β1γ1) is synthesized by lymphoid cells, promotes lymphocyte migration and costimulates T cell proliferation

Laminins are large heterotrimeric and multidomain glycoproteins with adhesive and signalling functions. They are major components of basement membranes but can also be found in other tissue compartments (Ekblom and Timpl, 1996; Miner et al., 1997; Colognato and Yurchenko, 2000). 12 laminin isoforms (laminin-1 to-12), each one assembled by a combination of one α, one β and one γ chain from five α chains, three β chains and three γ chains, have been identified so far (Miner et al., 1997; Colognato and Yurchenko, 2000). Laminins are synthesized by numerous cell types, and expression of laminin isoforms, particularly their α chain, is cell- and tissue-specific. The prototype laminin-1 (α1β1γ1), originally isolated from a mouse tumor, has been well characterized biochemically, and much of the in vitro functional data ascribed to laminins are based on studies performed with laminin-1 (Ekblom and Timpl, 1996). However, expression of this laminin isoform in adult tissues is highly restricted to a limited subpopulation of epithelial cells (Falk et al., 1999; Virtanen et al., 2000). In contrast, laminins containing α4 and α5 chains have a much wider tissue distribution, but their actions on cells are poorly known (Miner et al., 1996; Colognato and Yurchenco, 2000).

Laminins affect cell behaviour, including cell adhesion, migration and differentiation, through integrins and other cell surface receptors (Mercurio, 1995; Ekblom and Timpl, 1996; Colognato and Yurchenco, 2000). Integrins recognize laminin α chains and hence determine cell adhesion to laminin isoforms. Although some functions might be common to all laminin variants, others may be unique and isoform-specific, depending on the tissue or organ in which they are abundant. The physiological relevance of laminin α chains is illustrated by congenital muscular dystrophy and junctional epidermolysis bullosa, two genetic diseases of muscle and skin caused by mutations in laminin α2 and α3 chains, respectively (Ekblom and Timpl, 1996; Colognato and Yurchenco, 2000). The α4 chain is a relatively new member of the laminin family (Iivanainen et al., 1995; Richards et al., 1996; Iivanainen et al., 1997; Frieser et al., 1997). It has a size of 180-220 kDa and associates to either β1 and γ1 chains, to constitute laminin-8, or to β2 and γ1 to form laminin-9 (Miner et al., 1997; Iivanainen et al., 1997; Frieser et al., 1997). Laminin-8 (α4β1γ1), originally described in 1997 (Miner et al., 1997), is synthesized by vascular endothelial cells and adipocytes (Frieser et al., 1997; Niimi et al., 1997), and laminin α4 mRNA is also detected in heart, skeletal muscle, lung, fibroblasts and long-term bone marrow cultures (Miner et al., 1997; Iivanainen et al., 1995; Richards et al., 1996; Iivanainen et al., 1997; Gu et al., 1999). Recently, the presence of laminin-8 in blood platelets and its synthesis by erythromegakaryocytic cells were demonstrated (Geberhiwot et al., 1999; Geberhiwot et al., 2000a).

Lymphoid cells arise from stem cells in bone marrow and differentiate in thymus (T cells) or bone marrow (B cells) (Janeway et al., 1999). From these central lymphoid organs, lymphocytes migrate to lymph nodes, spleen and other peripheral lymphoid tissues, where they encounter antigens and become activated. Lymphocytes patrol the body by circulating between blood and lymph, and enter lymphoid tissues through high endothelial venules, specialized blood vessels with high endothelial cells (Janeway et al., 1999; Girard and Springer, 1995). Lymphocytes play fundamental roles in innate and adaptive immunity. T cells differentiate into either cytotoxic T cells or helper T cells that activate B cells and macrophages, whereas B cells differentiate into plasma cells that secrete antibodies. NK cells, the third type of lymphocytes, lack antigen-specific receptors but can detect and attack certain virus-infected and tumor cells. Induction of proliferation of resting T cells, either CD4 or CD8 subpopulations, by monocyte/macrophages or other antigen presenting cells requires not only engagement of the antigen receptor (CD3/TCR) but additional costimuli via accessory molecules (Janeway et al., 1999).

Synthesis, recognition and use of laminin isoforms by lymphoid cells are poorly understood. Expression of a laminin-like substance on the cell surface of mouse, rat and human NK cells, as detected by indirect immunofluorescence with a rabbit antiserum to mouse laminin-1, has been described (Hiserodt et al., 1985a; Vujanovic et al., 1988; Schwarz et al., 1988). However, molecular characterization of the antigen, or whether the structure represented de novo synthesis by the NK cells or uptake of exogenous laminin, was not established. Morrone et al. (Morrone et al., 1989) reported immunoprecipitation of 200 and 400 kDa proteins from metabolically radiolabeled rat NK cells with rabbit antibodies to laminin-1. However, identification of the immunoprecipitated polypeptides was not reported.

Although poorly characterized, laminin components are found in hematopoietic and lymphoid tissues. Immunostaining of bone marrow with antiserum to laminin-1 has demonstrated widespread distribution of laminins in the vascular basement membranes, and also in the intersinusoidal interstitial tissue (Gu et al., 1999; Nilsson et al., 1998). In thymus, a similar antiserum reacts with basement membranes bordering the capsule, septae and perivascular spaces (Berrih et al., 1985), whereas in lymph nodes, in addition to the vessel walls, a layer underlying the subcapsular sinus was stained (van den Berg et al., 1993; Jaspars et al., 1995). Interestingly, laminin is also detected in reticular fibers, an extensive array of extracellular matrix fibers of lymph nodes and other lymphoid tissues (Kramer et al., 1988; van den Berg et al., 1993; Jaspars et al., 1995). Since laminin α1 mRNA and polypeptide are not detected in hematopoietic/lymphoid tissues and most blood vessels (Chang et al., 1993; Falk et al., 1999; Gu et al., 1999), staining with the antiserum to laminin-1 may be due to recognition of laminin β1 and/or γ1 chains of other laminin isoforms.

According to their tissue distribution, laminin isoforms may regulate proliferation, survival, differentiation, extravasation/migration and other activities of lymphoid cells. In vitro, laminin-1 inhibited both NK-mediated cytotoxicity and proliferation of spleen lymphocytes (Hiserodt et al., 1985b; Li and Cheung, 1992), and enhanced migration of the latter cells (Li and Cheung, 1992). Thymocytes and/or blood lymphocytes adhered minimally to laminin-1, but avidly to merosin (laminin-2/4, α2β1γ1/α2β2γ1) and bulk laminin isolated from placenta (Cardarelli and Pierschbacher, 1986; Shimizu et al., 1990a; Chang et al., 1993; Weeks et al., 1994; Chang et al., 1995). The latter laminin preparation presumably contained laminin-10/11 (α5β1γ1/α5β2γ1) (Ferletta and Ekblom, 1999). This preparation and merosin, but not laminin-1, were found to be comitogenic for human lymphoid cells (Matsuyama et al., 1989; Shimizu et al., 1990b; Chang et al., 1995). Moreover, accumulation of adoptively transferred lymphocytes into peripheral lymph nodes and cardiac allografts of recipients pretreated with rabbit antibodies to laminin-1 was significantly decreased compared to controls (Kupiec-Weglinski and de Sousa, 1991). In accordance with these findings, thymocytes and blood lymphocytes widely express α6β1 integrin as a major laminin receptor (Shimizu et al., 1990a; Chang et al., 1995). In addition, α3β1 integrin mediates adhesion of certain lymphoid cells to laminin-5 (α3β3γ2) and to other laminin preparations (Wayner et al., 1993; Chang et al., 1995).

To further analyse the expression, recognition and use of laminin isoforms by lymphoid cells, we have investigated in the present study synthesis of laminin-8 (α4β1γ1) by T, B and NK cells, and blood lymphocyte adhesion to and migration on laminin-8 and laminin-10 (α5β1γ1), the laminin isoforms of vascular endothelium (Frieser et al., 1997; Sorokin et al., 1997). As monocytic cells were recently found to express laminin-8 (Pedraza et al., 2000), we have also investigated whether laminin-8 has any costimulatory effect on T cell proliferation. We describe, for the first time, synthesis and secretion of a complete laminin molecule by lymphoid cells, promotion of lymphocyte migration by laminin-8, but not by laminin-10/11, and activation-dependent α6β1 integrin-mediated lymphocyte adhesion to laminin-8 and-10/11. These laminin isoforms, but not laminin-1, strongly costimulated T cell proliferation via the same integrin.

Jurkat (T cell leukemia) (Gillis and Watson, 1980), NAD-20 (lymphoblastoid B cell) (Patarroyo et al., 1988) and NKL (NK leukemia) (Robertson et al., 1996) cell lines were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (Life Technologies, Täby, Sweden) and antibiotics. In addition, NKL cell line was supplemented with 180 U/ml of recombinant IL-2. Peripheral blood mononuclear cells (PBMC) were obtained from healthy donors after Ficoll-Hypaque gradient centrifugation (Amersham Pharmacia AB, Uppsala, Sweden). Lymphocytes were subsequently isolated by collecting nonadherent cells to tissue culture flasks. CD4 T cells were obtained from PBMC by positive selection using magnetic beads coated with anti-CD4 antibody according to instructions from the manufacturer (Dynal AS, Oslo, Norway).

Monoclonal antibodies (mAb) DG10, 1928 (Chemicon International, Temecula, CA, USA) and LN-26 (Takara Shuzo Co., Kyoto, Japan) to human laminin β1 chain, and 22 (Transduction Labs, Lexington, KY, USA), 2E8, LN-41 (Takara) and LN-82 (Takara) to human laminin γ1 chain, were used. The chain specificity of these antibodies was confirmed by reactivity with recombinant human laminin β1 and γ1 chains (Geberhiwot et al., 2000b). Rabbit antibodies to recombinant human laminin α4 I/II domains were produced and immunoaffinity purified as previously described (Iivanainen et al., 1997). Their specificity was confirmed by reactivity against the recombinant protein (Kortesmaa et al., 2000) and by microsequencing of the immunoreactive band from platelets (Geberhiwot et al., 1999). Moreover, these antibodies did not stain muscle tissue sections or cell extracts from laminin α4 chain knock out (unpublished data). The rabbit antibodies were not used for immunoprecipitation or immunofluorescence, since they recognize denaturated antigen. Blocking mAbs to integrin chains included H12 (αL or CD11a, kindly provided by Prof. Hans Wigzell, Karolinska Institutet), 4B4 (β1 or CD29, Coulter, Hialeah, HI, USA) and GoH3 (α6 or CD49f, Immunotech, Marseille, France). mAb UCHT1 (Immunotech) to CD3 was used for T cell proliferation studies. Purified mouse (Coulter), rat (Serotec, Oxford, UK) and rabbit (Dakopatts, Copenhagen, Denmark) IgG were used as negative controls.

Mouse laminin-1 (α1β1γ1, isolated from EHS tumor) was obtained from Life Technologies, as well as human placenta laminin, which was purified with mAb 4C7 to laminin α5 chain and recently classified as laminin-10/11 (α5β1γ1/α5β2γ1) (Ferletta and Ekblom, 1999; Geberhiwot et al., 2000b). Full-length recombinant laminin-8 (α4β1γ1) was produced in a mammalian expression system and purified by affinity chromatography as described (Kortesmaa et al., 2000). The recombinant molecule was a Y-shaped heterotrimer, as expected for the native protein.

Total RNA was purified from cultured cells by using RNazol B (AMS Biotechnology, Täby, Sweden). cDNA synthesis and PCR were carried out using the Advantage RT-for-PCR kit (Clontech Laboratories Inc., Palo Alto, CA, USA). Briefly, after oligo(dT)₁₈ priming, mRNA was reverse-transcribed into cDNA by incubating for 60 minutes at 42°C with 200 units of M-MLV reverse transcriptase in 20 μl of reaction buffer containing 20 units of recombinant RNase inhibitor, and 10 mM of each dNTP. The reactions were terminated by heating the samples at 94°C for 5 minutes and the mixture was diluted to 100 μl with DEPC-treated water. Single-stranded cDNAs in 20 μl of reaction buffer containing 1.5 mM MgCl₂, 10 μM of each primer and dNTP mix (10 mM each) were amplified by 30 cycles of PCR using 1.25 units of Ampli Taq DNA polymerase (Perkin Elmer/Roche Molecular Systems, Inc., Branchburg, NJ, USA). The conditions for PCR were 94°C, 1 minute; 60°C, 1 minute; 72°C, 1 minute. For PCR of human laminin α4 cDNA, paired primers 5′-TGCCTACTTTACCAGGGTGG-3′ (sense strand) and 5′-AAACATGTAAACCAAGCGGC-3′ (antisense strand) were used to direct synthesis of a 481-bp product (nucleotides 4287-4767) (Geberhiwot et al., 2000a). For human laminin β1 cDNA, paired primers 5′-TTGGACCAAGATGTCCTGAG-3′ (sense strand) and 5′-CAATATATTCTGCCTCCCCG-3′ (antisense strand) were used and a 679-bp product was expected (nucleotides 955-1633) (Geberhiwot et al., 2000a). For human laminin γ1 cDNA, paired primers 5′-GCAAGACTGAACAGCAGACC-3′ (sense strand) and 5′-TCCTATCAAGATCGCTGACC-3′ (antisense strand) were used and a 687-bp product was expected (nucleotides 4241-4927) (Geberhiwot et al., 2000a). PCR products were analysed by electrophoresis using 2% agarose gels.

Following incubation at 37°C for 30 minutes in methionine- and cysteine-free medium, 30 million cells were labelled in 10 ml of methionine- and cysteine-free RPMI 1640 medium with 10% dialyzed fetal bovine serum containing 0.20 mCi/ml Trans³⁵S-label (ICN Radiochemical Inc.) for 4 hours at 37°C. After washing three times with cold PBS, the cells were lysed by adding 1 ml of lysis buffer (1% Triton X-100 in phosphate-buffered saline (PBS) with 1 μg/ml of aprotinin, 2 μM leupeptin, 2 μM pepstatin, 1 mM PMSF and 2 mM EDTA as protease inhibitors). The soluble fraction (cell lysate) was precleared with protein G-Sepharose beads (Amersham Pharmacia AB) and immunoprecipitation was performed by adding to 100 μl of cell lysate 5 μg of mAb followed by addition of 50 μl of protein G-Sepharose beads previously coated with 5 μl of rabbit anti-mouse Ig (Dakopatts).

Protein samples were analysed by SDS-PAGE. In western blots, filters were blocked with 0.1% Tween 20/5% dry milk in PBS. Peroxidase-linked anti-mouse or anti-rabbit immunoglobulin (Dakopatts) was used as secondary antibody, and ECL (Amersham, UK) was used as developer. Laminin was purified from cell lysates by immunoaffinity chromatography as previously described (Geberhiwot et al., 1999). Briefly, Sepharose CL-4B preadsorbed cell lysate was applied to laminin β1 antibody column (mAb DG10 coupled to CNBr-activated Sepharose 4B), which had been equilibrated with lysis buffer, and cycled twice. Following extensive washing, the proteins bound to the column were eluted by using high pH and the samples were collected in neutralizing buffer. After concentration, the purified material was analysed by SDS-PAGE and western blotting.

Isolated blood mononuclear leukocytes were washed with PBS and used non-permeabilized and permeabilized for immunofluorescence. Cells were permeabilized by using IntraStain kit (Dakopatts) according to instructions from the manufacturer. Indirect immunofluorescence was performed by incubating cells, after blocking with 2 mg/ml of human IgG (Sigma) for 30 minutes at 4°C, with saturating amounts of monoclonal antibodies, followed by fluorescein-conjugated F(ab)₂ fragments of rabbit anti-mouse immunoglobulin (Dakopatts) at 1:20 dilution. After staining, lymphocytes were gated, according to forward and side scatter, and analysed in a FACScan flow cytometer (Beckton Dickinson, Mountain View, USA).

To study laminin secretion, isolated lymphocytes were resuspended at 5×10⁷ cells/ml in plain RPMI. After preincubation for 15 minutes, the cells were stimulated with 200 nM tetradecanoyl phorbol acetate (TPA, also known as PMA; Sigma) for 20 minutes at 37°C. Following addition of protease inhibitors and centrifugation of the cells, supernatant was collected and analysed for secreted laminin.

Transmigration of lymphocytes through protein-coated filters was measured microscopically and by flow cytometry. Briefly, human blood mononuclear cells (5×10⁵, containing 55% lymphocytes) in 100 μl RPMI-1640 medium were added to the top chamber of a 6.5 mm diameter, 5 μm pore polycarbonate Transwell culture insert (Costar, Cambridge, MA, USA) and incubated at 37°C for 3 hours in the absence (spontaneous migration) or presence (chemokine-stimulated migration) of 500 ng/ml stromal cell derived factor (SDF-1α, R&D Systems, Abingdon, UK) in the lower chamber. The filters had been previously coated overnight with 20 μg/ml of various laminin isoforms or human serum albumin (HSA) (Sigma), and blocked with 0.5% HSA for 1 hour. To efficiently remove all transmigrated cells from the lower chamber, a final concentration of 10 mM EDTA was added to each well and cells were resuspended vigorously. Thereafter, the resuspended cells were fixed with 1% formaldehyde and the cell number was determined microscopically by counting five fields with a 40× objective. The percentage of lymphocytes in the cell population was determined in a FACScan (Beckton Dickinson, San Jose, CA, USA) by gating on their forward and side scatter. In statistical analysis (Student’s t-test), mean and standard deviation (s.d.) were calculated as well as level of significance (^*P<0.05), comparing the various laminin isoforms to HSA.

In studies of cell adhesion to immobilized proteins, isolated CD4 T cells resuspended in RPMI (2×10⁶/ml) with 0.5% HSA were used. 96-well plates (MaxiSorp, Nunc, Roskilde, Denmark) were coated overnight at room temperature with HSA, mouse laminin-1, recombinant laminin-8 or placenta laminin-10/11 at 20 μg/ml. After blocking with bovine serum albumin, 2×10⁵ cells/well (100 μl) were incubated in absence or presence of 200 nM TPA (Sigma) for 1 hour at 37°C. Following five washes with plain medium, adherent cells were fixed by adding 50 μl of fixative solution (paraformaldehyde 40 g/l, NaH₂PO₄.H₂O, 16.97 g/l; NaOH, 3.86 g/l; and D-glucose, 5.4 g/l; prepared at 65°C, pH 7.4) for 15 minutes and thereafter 50 μl of filtered Toluidine Blue dye (Sigma, 0.5% w/v in PBS) was added overnight at room temperature. The plate was then washed with copious amounts of distilled water. Adherent cells were quantified in a microplate reader (Multiskan Bichromatic, Labsystems, Helsinki, Finland) by measuring absorbance at 690 nm after releasing the blue dye with 100 μl of 2% sodium dodecyl sulfate (Bio-Rad Laboratories, Richmond, CA, USA). The effect of blocking mAbs to integrin α6 and β1 chains on the laminin-specific cell adhesion was tested by pretreating the cell suspension with antibodies (20 μg/ml) for 15 minutes before adding the cells to the laminin-coated wells. Cell adhesion in the presence of control IgG was defined as 100% adherence. In statistical analysis (Student’s t-test), mean and s.d. were calculated as well as the level of significance (^*P<0.05; ^**P<0.01;^***P<0.001), comparing the various laminin isoforms to HSA or the blocking antibodies to the IgG control.

Cell proliferation, measured as ³H-thymidine incorporation, was performed in triplicate in 96-well microtiter plates (Costar Corp., Cambridge, MA, USA) coated overnight with 0.5 μg/ml of anti-CD3 mAb. Following three washes with PBS, wells were coated with 2.5 μg/ml of HSA or various laminin isoforms for 4 hours at 37°C. Optimal antibody and laminin concentrations were determined in early dose-response studies (data not shown). Thereafter, purified CD4 T cells resuspended in AIM-V serum-free medium (Life Technologies, Inc.) were cultured in the plates (4×10⁴ cells/well) for 4 days at 37°C in 5% CO₂ humidified air, and pulsed with 1 μCi/well of (methyl-³H) thymidine (Amersham, UK) for the final 12 hours. Cells were then harvested onto filter paper using a semiautomatic cell harvester (Tomtec, Inc.) and ³H-thymidine incorporation was measured in a liquid scintillation counter (Beckman Instruments, Inc). The effect of blocking mAbs to integrin α6 and β1 chains on the costimulatory effect of laminins was tested by incubating the cells with the antibodies (20 μg/ml) during the assay. In statistical analysis (Student’s t-test), mean and s.d. were calculated as well as level of significance (^*P<0.05; ^**P<0.01), comparing the various laminin isoforms to HSA or the blocking antibodies to the IgG control, in presence of the mAb to CD3.

Expression of mRNAs encoding for laminin-8 chains in lymphoid cells was investigated by RT-PCR using pairs of primers that were designed on the basis of the reported cDNA sequences of human laminin α4, β1 and γ1 chains. Amplified products with the expected size were detected with primers for α4 (481 bp), β1 (679 bp) and γ1 (687) after 30 cycles of PCR with reverse transcribed Jurkat mRNA (Fig. 1). Similar results were obtained with NAD-20 cells (data not shown).

The lysate of metabolically radiolabeled Jurkat cells was immunoprecipitated with mAbs to either β1 or γ1 chains to analyse synthesis and assembly of laminin components (Fig. 2). All four antibodies immunoprecipitated polypeptides of 220 and 200 kDa. Similar results were obtained with NAD-20 cells (data not shown).

In further studies, western blot analysis of Jurkat cell lysate was performed. Under reducing conditions, mAb DG10 to laminin β1 chain and mAb 22 to laminin γ1 chain recognized polypeptides with the expected size of 220 and 200 kDa, respectively. Since rabbit antibodies to laminin α4 chain reacted with several polypeptides of smaller size than the expected one in the total cell lysate, the results concerning the presence of this chain were inconclusive (data not shown).

Laminin was isolated from Jurkat (T) cell lysates by immunoaffinity chromatography on a laminin β1 (DG10) antibody column and then analysed by western blotting to identify the α chain and to study the physical association of the components (Fig. 3). Under reducing conditions, mAbs to β1 and γ1 chains strongly reacted with 220 and 200 kDa polypeptides, respectively. Affinity-purified rabbit antibodies to laminin α4 chain were strongly reactive with polypeptides of 200 and 180 kDa. Similar results were obtained with the material isolated from NAD-20 (B) and NKL (NK) cell lines, though certain degradation of laminin α4 chain was apparent in the latter cells (Fig. 3). Under nonreducing conditions, laminin β1 and γ1 chains comigrated with a slower electrophoretic mobility (approximately 420 kDa), whereas the α4 chain migration was faster (approximately 200 kDa).

Jurkat and NKL are cell lines of malignant origin (Gillis and Watson, 1980; Robertson et al., 1996), whereas NAD-20 was established by transformation of B lymphocytes with Epstein Barr virus (Patarroyo et al., 1988). To determine the presence of laminin in normal lymphocytes, immunofluorescence flow cytometry of purified blood mononuclear cells was used (Fig. 4). Lymphocytes, gated by forward and side scatter, displayed reactivity with mAb H12 to CD11a (αL integrin chain), with the characteristic pattern of two positive populations. Monoclonal antibodies LN-26 and LN-41 to laminin β1 and γ1 chains, respectively, bound minimally to intact lymphocytes, whereas practically all lymphocytes were reactive with both antibodies after cell permeabilization.

To determine whether blood lymphocytes contained laminin-8, the cell lysate of lymphocytes isolated from blood mononuclear cells was immunopurified on the laminin β1 antibody column, and the purified material was then analysed by western blotting (Fig. 5A). Similar to the findings with lymphoid cell lines, mAbs to β1 and γ1 chains recognized polypeptides of 230 and 220 kDa, respectively, whereas rabbit antibodies to α4 reacted with a broad band of 180-200 kDa, under reducing conditions.

In further experiments, the supernatant of isolated blood lymphocytes stimulated with phorbol ester for 30 minutes was analysed by western blotting to investigate secretion of laminin-8 by lymphoid cells (Fig. 5B). As in the cell lysate, laminin α4, β1 and γ1 chains were detected in the secreted material. Cell-associated and secreted laminins were indistinguishable.

Lymphocytes secrete laminin-8 and may interact with the endogenous laminin. Alternatively, the cells may recognize exogenous laminin-8 in blood vessels and other tissue compartments. To determine the effect of laminin-8 on lymphocyte migration, transmigration of blood lymphocytes through recombinant laminin-8-coated filters was studied in insert assays. Compared to HSA, laminin-8 enhanced lymphocyte migration in the presence of chemoattractant by 57%, and this effect was statistically significant (P<0.05) (Fig. 6). Laminin-1 also enhanced cell migration (P<0.05), but to a lower extent (20%), whereas laminin-10/11 tended to be inhibitory (−35%). For all substrates, SDF-1α stimulated a major increase in lymphocyte migration compared to cells without chemoattractant. Approximately 15% of the lymphocyte population transmigrated on the laminin-8 substrate in the presence of SDF-1α in 3 hours. The opposing effects of laminin-8 and-10/11 on cell migration were more evident with blood monocytes (Pedraza et al., 2000).

To investigate whether laminin-8 was adhesive for lymphocytes, adhesion of isolated CD4 T cells to immobilized recombinant laminin-8 was studied (Fig. 7A). Plastic surfaces were coated with HSA, mouse laminin-1, placenta laminin-10/11, or recombinant laminin-8. After incubating the lymphocytes for 1 hour at 37°C on the protein-coated surfaces in the absence or presence of phorbol ester, nonadherent cells were removed by extensive washing. The constitutive cell adhesion to laminin-8 and-10/11 was modest. However, following stimulation of the cells, these laminin isoforms were most adhesive, laminin-10/11 being more active than laminin-8 (P<0.001 and P<0.05, respectively). In contrast, cell adhesion, either constitutive or stimulated, to laminin-1 was low. Higher amounts of laminin-1 (50 μg/ml) did not increase the level of cell adhesion (data not shown).

α6β1 integrin has been recently identified as a laminin-8 receptor in platelets, vascular endothelial and monocytic cells (Geberhiwot et al., 1999; Kortesmaa et al., 2000; Pedraza et al., 2000). Likewise, mAbs to integrin β1 and α6 chains inhibited almost completely the stimulated adhesion of CD4 T cells to recombinant laminin-8 (Fig. 7B). Similar results were obtained when laminin-10/11 was used as substrate. In accordance with these findings, CD4 T cells from peripheral blood are known to express α6β1 integrin (Shimizu et al., 1990a).

In addition to CD3/TCR engagement, induction of proliferation of resting T cells is known to require additional costimuli (Janeway et al., 1999). Fibronectin, bulk placenta laminin (presumably containing laminin-10/11) and merosin (laminin-2/4) have been reported to be comitogenic for lymphoid cells through integrin receptors (Matsuyama et al., 1989; Shimizu et al., 1990a; Chang et al., 1995). To investigate whether laminin-8, which is expressed by monocytic and B cells (Pedraza et al., 2000 and present study), provides a similar costimulatory signal, recombinant laminin-8 and mAb UCHT1 to CD3 were coimmobilized, and isolated CD4 T cells were incubated on the coated wells for 4 days. Under these experimental conditions, extensive cell proliferation was induced, measured as ³H-thymidine incorporation (Fig. 8A). Recombinant laminin-8 or the CD3 mAb on their own failed to induce any significant cell proliferation. Interestingly, laminin-1 was inactive, whereas laminin-10/11 was as effective as laminin-8. The costimulatory signal by laminin-8 and laminin-10/11 was mediated via α6β1 integrin, as indicated by almost complete inhibition of the response by blocking antibodies to β1 and α6 integrin chains (Fig. 8B). When coimmobilized with mAb to CD3, mAb GoH3 to α6 integrin chain (0.5-10 μg/ml), but not rat IgG control, similarly induced a strong proliferative response in the lymphocytes (data not shown).

In the present study we demonstrate, for the first time, that T, B and NK cells synthesize laminin-8 and that blood lymphocytes contain and secrete this laminin isoform. We show expression of laminin α, β and γ chains, and their assembly into a complete laminin molecule in lymphoid cells. We also demonstrate promotion of lymphocyte migration by laminin-8, but not by laminin-10/11, activation-dependent adhesion of blood lymphocytes to laminin-8 and-10/11, and costimulation of T cell proliferation by these laminin isoforms via α6β1 integrin.

Jurkat, NAD-20 and NKL are relatively mature lymphoid cells of T, B and NK cell origin, respectively, and represent the three major types of lymphocytes (Gillis and Watson, 1980; Patarroyo et al., 1988; Robertson et al., 1996). Expression of mRNAs for laminin α4, β1 and γ1 chains in the lymphoid cells, as detected by RT-PCR, strongly suggested biosynthesis of laminin-8. Metabolic labelling followed by immunoprecipitation with mAbs to laminin β1 and γ1 chains revealed two bands of 220 and 200 kDa, suggesting comigration of the α chain with either the β or the γ chains. In contrast to laminin β1 (220 kDa) and γ1 (200 kDa) chains, laminin α4 chain could not be detected by western blotting of total cell lysate, probably due to small amounts of the chain and/or masking of the polypeptide by other proteins. However, this chain (200/180 kDa) was readily detected following immunoaffinity purification on the laminin β1 antibody column. As in other laminin isoforms (Ekblom and Timpl, 1996), the laminin α chain appeared to be rate-limiting in laminin synthesis. The presence of laminin-8 was unambiguously demonstrated by identification of physically associated laminin α4, β1 and γ1 chains. In contrast to laminin-8 from lung and kidney (Miner et al., 1997), but similarly to laminin-8 from other hematopoietic cells (Geberhiwot et al., 1999; Geberhiwot et al., 2000a; Pedraza et al., 2000), the α4 chain was noncovalently associated to disulfide-bonded β1γ1 chains in lymphoid cells. This was concluded from the disparate electrophoretic mobility of, on the one hand, β1 and γ1 chains (slow) and, on the other hand, the α4 chain (fast), under nonreducing conditions.

Practically 100% of blood lymphocytes reacted with mAbs to laminin β1 and γ1 chains by immunofluorescence flow cytometry, suggesting that all lymphocytes, and not just a subpopulation, contained laminin. Since cell permeabilization was required for detection, laminin appears to have an intracellular localization. Similarly to the lymphoid cell lines, isolated blood lymphocytes contained laminin-8. Since RNA and protein synthesis is very low in these resting cells, their laminin-8 originated most likely from biosynthesis in precursor cells in thymus, bone marrow and/or other lymphoid tissues. Stimulation of blood lymphocytes with phorbol ester, an activator of protein kinase C, followed by analysis of the supernatant identified laminin-8 as a novel secretory product of lymphoid cells. This prompt secretion may be relevant to the function of the laminin molecule.

The relationship between the laminin-like substance on the cell surface of mouse, rat and human NK cells reported by another research group (Hiserodt et al., 1985a; Vujanovic et al., 1988; Schwarz et al., 1988) and the laminin-8 of lymphocytes is presently unknown. The antigen, detected by immunofluorescence with a rabbit antiserum to mouse laminin-1, was not molecularly characterized. In preliminary studies, we have been unable to demonstrate staining of intact blood NK cells (CD3−, CD56+) with mAbs to laminin β1 and γ1 chains (unpublished data). The identity of the 200 and 400 kDa proteins from metabolically radiolabeled rat NK cells immunoprecipitated with rabbit antibodies to laminin-1 reported by Morrone et al. (Morrone et al., 1989) is also unknown. Although the 200 kDa band may correspond to laminin α4, β1 and/or γ1 chains, it is unlikely that the 400 kDa polypeptide is laminin α1 since expression of this chain is mostly restricted to epithelial cells (Falk et al., 1999; Virtanen et al., 2000). Moreover, we have been unable to detect laminin α1 mRNA or full-length (300-400 kDa) laminin α chains in lymphoid cells (unpublished data). Similar to our results, Chang et al. (Chang et al., 1993) could not detect transcripts for laminin α1 or α2 chains in isolated mouse thymocytes by RT-PCR. It is noteworthy that the studies on NK cell laminin by the other groups were performed during the 1980s, when only one laminin α chain, namely α1, had been identified.

Vascular laminin-8 may contribute to extravasation of blood lymphocytes, whereas endogenous laminin-8 may mediate lymphocyte migration and chemotaxis in extravascular loci. Compared to HSA, exogenous laminin-8 and, to a lower extent, laminin-1, reproducibly enhanced lymphocyte migration in presence of SDF-1, a potent chemoattractant for mononuclear leukocytes, which binds to the chemokine receptor CXCR4. In contrast, laminin-10/11 tended to inhibit cell migration. As only a fraction (15%) of the lymphocytes transmigrated, a particular lymphocyte subpopulation appears to be motile. The promoting and inhibitory effects of laminin-8 and laminin-10/11, respectively, on leukocyte migration are more evident when blood monocytes are used (Pedraza et al., 2000). The opposite effects of these laminin isoforms on lymphocyte migration suggest that laminin-8 promotes migration of lymphoid cells in tissues, whereas laminin-10/11 determines their tissue localization (arrest). Interestingly, considerable lymphocyte migration was also observed on the albumin-substrate, suggesting that endogenous laminin-8 participates in the process. It has been reported that IL-8-induced locomotion of T lymphocytes in a collagen matrix is abolished by a mAb to α6 integrin (Friedl et al., 1995). Since laminin was not present in the matrix and α6 integrins do not bind collagen, endogenous laminin-8 could mediate the lymphocyte migration. Keratinocyte migration is known to depend on endogenously secreted laminin-5 (α3β3γ2) (Zhang and Kramer, 1996).

In other studies, laminin-1 promoted in vitro migration of spleen lymphocytes and NK cells (Li and Cheung, 1992; Somersalo and Saksela, 1991), and bulk placenta laminin facilitated chemotaxis of malignant plasma cells (Shibayama et al., 1995). Compared to type 2 T helper (Th2) cells, Th1 cells preferentially migrated on laminin-1 by using α6β1 integrin (Colontonio et al., 1999), whereas replacement of the α6A integrin splice variant by α6B in lymph node T cells reduced migration of the cells on laminin-1, but not on fibronectin (Gimond et al., 1998). Notably, vascular α6 integrin mediated homing of pro-T cells to the thymus (Dunon and Imhof, 1993). Modulation in vivo of lymphocyte traffic, particularly inhibition of lymphocyte accumulation in peripheral lymph nodes, by anti-laminin-1 antibodies (Kupiec-Weglinski and de Sousa, 1991) indicated the role of laminins in lymphocyte migration.

In spite of several immunohistochemical studies (Berrih et al., 1985; van den Berg et al., 1993; Wayner et al., 1993; Castanos-Velez et al., 1995; Jaspars et al., 1995; Jaspars et al., 1996; Virtanen et al., 1996; Nilsson et al., 1998; Gu et al., 1999; Vivinus-Nebot et al., 1999), distribution of laminin isoforms in lymphoid tissue is poorly understood. Vessels, including high endothelial venules (HEVs), are immunoreactive with antibodies to laminin-1 and laminin β1 and γ1 chains (van den Berg, 1993; Jaspars et al., 1995; Castanos-Velez et al., 1995; Jaspars et al., 1996). Since in lymphoid tissues laminin α1 chain is not detected and laminin α2 chain expression appears to be rather limited (Chang et al., 1993; Virtanen et al., 1996; Falk et al., 1999), other laminin α chains could be present. Indeed, laminin α5 chain, as detected by mAb 4C7, is found in the subepithelial basement membrane of the capsule and in the basal laminae of stromal and parenchymal vessels in thymus (Virtanen et al., 1996). This laminin chain is also found in basement membranes of capillaries and HEVs and, most likely, in the reticular fibers of lymphoid tissue (Sorokin et al., 1997). These laminin-containing fibers, which radiate from the basement membrane of HEVs and are more abundant in the interfollicular area, could form a scaffold for lymphocyte localization and/or a pathway for lymphocyte migration within the lymphoid tissue (Kramer et al., 1988). In addition, a granular staining of lymphoid follicles, probably corresponding to follicular dendritic cells, was revealed with antibodies to laminin-1 (Jaspars et al., 1995). Antibodies to laminin-5 (α3β3γ2) also reacted with certain components of the lymphoid tissue, including vessels (Wayner et al., 1993; Jaspars et al., 1996; Virtanen et al., 1996; Vivinus-Nebot et al., 1999). Interestingly, immunohistochemical studies have shown that laminin antigen can be also found on the luminal surface of HEVs and lung capillaries (Girard and Springer, 1995; Hilario et al., 1996), suggesting a role of vascular laminins in early leukocyte-endothelium interactions. Immunolocalization of laminin α4 chain in lymphoid tissue is presently unknown. Since laminin-8 is a major endothelial laminin (Frieser et al., 1997), it could be expressed by HEVs. Moreover, upregulation of laminin α4 mRNA in vascular endothelial cells by IL-1 (Frieser et al., 1997) suggests participation of laminin-8 in inflammatory responses. Considering the cell specific expression, tissue distribution and biological effects of laminin-8 and-10/11, these laminin isoforms appear to be most relevant in lymphocyte physiology.

Adhesion of lymphoid cells to laminin-8 and-10/11 has not been previously reported or recognized. Laminin-8 was not available for adhesion studies, and presence of laminin-10/11 in the commercial placenta laminin preparation was only recently established (Ferletta and Ekblom, 1999). Adherence of blood CD4 T cells to laminin-8 and-10/11 following stimulation with phorbol ester indicated that the adhesion was activation-dependent and suggested that these laminin isoforms could be relevant to adhesion-dependent lymphocyte function/activities. The low levels of adhesion of blood lymphocytes to mouse laminin-1 described by us and by others (Weeks et al., 1994) may be due to its α1 chain, which is absent in hematopoietic and lymphoid tissues and in most blood vessels (Chang et al., 1993; Falk et al., 1999; Gu et al., 1999; Virtanen et al., 2000). Alternatively, glycosylation or other modifications of this mouse tumor laminin may be responsible. The receptor for laminin-8 and-10/11 in the blood lymphocytes was identified as α6β1 integrin. Platelets and monocytic cells also use this integrin to adhere to laminin-8 (Geberhiwot et al., 1999; Pedraza et al., 2000). Thymocytes and blood lymphocytes express α6β1 integrin (Shimizu et al., 1990a; Chang et al., 1993; Chang et al., 1995), and phorbol esters are known to enhance avidity of leukocyte integrins for their ligands (Patarroyo et al., 1985; Shimizu et al., 1990).

In related studies by other groups, mouse and human thymocytes and human blood lymphocytes were found to adhere to bulk laminin isolated from human placenta, but at low levels or not at all to mouse laminin-1 (Cardarelli and Pierschbacher, 1986; Shimizu et al., 1990a; Chang et al., 1993; Weeks et al., 1994; Chang et al., 1995). Adhesion of thymocytes to the bulk laminin, which probably contained laminin-10/11, was mediated by α6β1 integrin, but α3β1 integrin also contributed (Chang et al., 1995), and the cells adhered to a similar extent to a merosin preparation (laminin-2/4, α2β1γ1/α2β2γ1) (Chang et al., 1995). Stimulated NK cells also adhered to laminin (unspecified isoform) via α6β1 integrin (Gismondi et al., 1992). Notably, lymphoid cells of B cell origin were also able to adhere to bulk placenta laminin (Segat et al., 1994), and blood leukocytes, including lymphocytes, were found to tether and arrest on laminin under physiological shear flow via α6β1 integrin (Kitayama et al., 2000).

Antigen-specific activation by T cells requires not only CD3/TCR engagement by antigen/major histocompatibility complex (Ag/MHC) but additional costimuli (Janeway et al., 1999). In the present study, laminin-8 or mAb to CD3 on their own failed to drive the CD4 T cells to proliferate, but together had a synergistic effect. Laminin-10/11 was also comitogenic, whereas laminin-1 was inactive. The costimulatory signal was transduced via α6β1 integrin, the same integrin that mediated cell adhesion. Our data confirm the inability of the mouse EHS laminin-1 to costimulate proliferation of human lymphoid cells described by other groups (Matsuyama et al., 1989; Shimizu et al., 1990b). However, the laminin-1 has been reported to costimulate mouse lymph node T cell proliferation (Gimond et al., 1998), and to inhibit, to some extent, growth of mouse spleen lymphocytes stimulated by Concanavalin A (Li and Cheung, 1992). Bulk placenta laminin (presumably containing laminin-10/11) and, to a lower extent, merosin (laminin-2/4) were also reported to be comitogenic, whereas laminin-5 (α3β3γ2) had a minimal costimulatory effect (Shimizu et al., 1990b; Chang et al., 1995; Vivinus-Nebot et al., 1999). Both laminin-2/4 and laminin-5 supported survival of early thymocytes (Iwao et al., 2000; Kim et al., 2000). Inasmuch as T cells normally recognize antigen present on cells (with MHC molecules) rather than on extracellular matrices, possibilities must be considered that laminin-8 and other laminin isoforms become important for T cell activation when presented on the surface of other cells, such as antigen-presenting cells. In support of this hypothesis, laminin epitopes can be detected on the surface of cells of the monocytic lineage, including activated macrophages (Wicha and Huard, 1983; Pedraza et al., 2000), and laminin-8 is also synthesized by B lymphocytes (present study).

Laminin recognition appears to contribute to other lymphocyte function/activities. Laminin-1 and-10/11 triggered Ca²⁺ signalling in Jurkat cells via α6β1 integrin (Weismann et al., 1997), and laminin (unspecified isoform) induced TNFα secretion by interacting CD4 T cells and macrophages, in the absence of antigenic stimulus (Hershkoviz et al., 1993). Laminin-1 inhibited the lysis of target cells by NK cells but had no effect on the cytotoxicity mediated by T lymphocytes (Hiserodt et al., 1985b), and laminin receptor expression of the target cells correlated with their NK sensitivity (Laybourn et al., 1989). Antibodies to laminin-1 inhibited cytotoxicity mediated by NK cells, but not by cytotoxic T lymphocytes (Hiserodt et al., 1985a), and α6β1 integrin was found to participate in natural killing (Lowdell et al., 1995). Based on these studies and our data, it is tempting to speculate that laminin-8 participates in NK recognition.

Synthesis, secretion, recognition and use of laminin-8 by lymphoid cells indicate the relevance of this laminin isoform in lymphocyte function/activities. In addition to stimulating cell migration and proliferation, recognition of exogenous and/or endogenous laminin-8 may contribute to lymphocyte survival, maturation, cytotoxicity and tissue localization. All these processes participate in the development of immune/inflammatory responses and in the pathophysiology of lymphoid malignancies.

This project was supported by Cancerfonden and the Karolinska Institute. The authors gratefully acknowledge Drs E. Engvall, D. Gigliotti and A. Iivanainen for providing antibodies, and Drs George Klein, Michael J. Robertson and Mikael Jondal for providing cell lines. We also thank Dr Timo Pikkarainen for comments on the manuscript and Dr B. Chambers for revising the English text.