INTRODUCTION

Laminins are large heterotrimeric and multidomainglycoproteins with adhesive and signalling functions. Theyare major components of basement membranes but can alsobe 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 assembledby a combination of one α, one β and one γ chain from fiveα chains, three β chains and three γ chains, have beenidentified so far (Miner et al., 1997; Colognato andYurchenko, 2000). Laminins are synthesized by numerouscell types, and expression of laminin isoforms, particularlytheir α chain, is cell- and tissue-specific. The prototypelaminin-1 (α1β1γ1), originally isolated from a mouse tumor,has been well characterized biochemically, and much of thein vitro functional data ascribed to laminins are basedon studies performed with laminin-1 (Ekblom and Timpl,1996). However, expression of this laminin isoform inadult tissues is highly restricted to a limited subpopulationof epithelial cells (Falk et al., 1999; Virtanen et al., 2000).

In contrast, laminins containing α4 and α5 chains have amuch wider tissue distribution, but their actions on cells arepoorly known (Miner et al., 1996; Colognato and Yurchenco,2000).

Laminins affect cell behaviour, including cell adhesion,migration and differentiation, through integrins and other cellsurface receptors (Mercurio, 1995; Ekblom and Timpl, 1996;Colognato and Yurchenco, 2000). Integrins recognize lamininα chains and hence determine cell adhesion to lamininisoforms. Although some functions might be common toall laminin variants, others may be unique and isoform-specific, depending on the tissue or organ in which they areabundant. The physiological relevance of laminin α chains isillustrated by congenital muscular dystrophy and junctionalepidermolysis bullosa, two genetic diseases of muscle and skincaused 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 andassociates to either β1 and γ1 chains, to constitute laminin-8,

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Laminins are a growing family of large heterotrimericproteins with cell adhesive and signalling functions. Theyare major components of basement membranes and arefound in many organs, including the vasculature and othercompartments of bone marrow, thymus, lymph nodes andspleen. However, expression, recognition and use of lamininisoforms by lymphoid cells are poorly understood. In thepresent study, lymphoid T cells (Jurkat) were found tosynthesize laminin α4, β1 and γ1 mRNAs and polypeptidesand to assemble the chains into laminin-8. LymphoblastoidB (NAD-20) cells, lymphoid NK (NKL) cells and bloodlymphocytes also contained laminin-8 and, after cellpermeabilization, practically all blood lymphocytes reactedwith mAbs to laminin β1 and γ1 chains. Followingstimulation, blood lymphocytes secreted laminin-8, and this

laminin isoform, but not laminin-10/11(α5β1γ1/α5β2γ1),promoted chemokine-induced migration of the cells. In anactivation-dependent manner, purified blood CD4 T cellsadhered to immobilized laminin-8 and laminin-10/11 byusing α6β1 integrin, but minimally to laminin-1 (α1β1γ1).Accordingly, laminin-8 and laminin-10/11, but not laminin-1, strongly costimulated proliferation of the T cells via thesame integrin. Thus, lymphoid cells are able to synthesizeand secrete complete laminin molecules. In addition,synthesis of laminin-8 and recognition of laminin-8 and -10/11 by lymphocytes indicate relevance of these lamininisoforms in lymphocyte physiology.

Key words: Lymphocyte, Laminin, Integrin, Migration, Proliferation

SUMMARY

Laminin-8 ( α4β1γ1) is synthesized by lymphoid cells,promotes lymphocyte migration and costimulates Tcell proliferationTarekegn Geberhiwot 1, Daniel Assefa 1, Jarkko Kortesmaa 2, Sulev Ingerpuu 3, Claudio Pedraza 1,Zenebech Wondimu 1, Jehad Charo 4, Rolf Kiessling 4, Ismo Virtanen 5, Karl Tryggvason 2

and Manuel Patarroyo 1,*1Microbiology and Tumor Biology Center and 2Division of Matrix Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institute,S 171 77 Stockholm, Sweden3Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia4Cancer Center Karolinska, Karolinska Hospital, Stockholm, Sweden5Institute of Biomedicine, Department of Anatomy, University of Helsinki, Helsinki, Finland*Author for correspondence (e-mail: Manuel.Patarroyo@mtc.ki.se)

Accepted 9 November 2000 Journal of Cell Science 114, 423-433 © The Company of Biologists Ltd

RESEARCH ARTICLE

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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), issynthesized by vascular endothelial cells and adipocytes(Frieser et al., 1997; Niimi et al., 1997), and laminin α4 mRNAis also detected in heart, skeletal muscle, lung, fibroblasts andlong-term bone marrow cultures (Miner et al., 1997; Iivanainenet al., 1995; Richards et al., 1996; Iivanainen et al., 1997; Guet al., 1999). Recently, the presence of laminin-8 in bloodplatelets and its synthesis by erythromegakaryocytic cells weredemonstrated (Geberhiwot et al., 1999; Geberhiwot et al.,2000a).

Lymphoid cells arise from stem cells in bone marrow anddifferentiate 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 otherperipheral lymphoid tissues, where they encounter antigensand become activated. Lymphocytes patrol the body bycirculating between blood and lymph, and enter lymphoidtissues through high endothelial venules, specialized bloodvessels with high endothelial cells (Janeway et al., 1999; Girardand Springer, 1995). Lymphocytes play fundamental roles ininnate and adaptive immunity. T cells differentiate into eithercytotoxic T cells or helper T cells that activate B cells andmacrophages, whereas B cells differentiate into plasma cellsthat secrete antibodies. NK cells, the third type of lymphocytes,lack antigen-specific receptors but can detect and attack certainvirus-infected and tumor cells. Induction of proliferation ofresting T cells, either CD4 or CD8 subpopulations, bymonocyte/macrophages or other antigen presenting cellsrequires 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 bylymphoid cells are poorly understood. Expression of a laminin-like substance on the cell surface of mouse, rat and human NKcells, as detected by indirect immunofluorescence with a rabbitantiserum to mouse laminin-1, has been described (Hiserodtet al., 1985a; Vujanovic et al., 1988; Schwarz et al., 1988).However, molecular characterization of the antigen, or whetherthe structure represented de novo synthesis by the NK cells oruptake of exogenous laminin, was not established. Morrone etal. (Morrone et al., 1989) reported immunoprecipitation of 200and 400 kDa proteins from metabolically radiolabeled ratNK cells with rabbit antibodies to laminin-1. However,identification of the immunoprecipitated polypeptides was notreported.

Although poorly characterized, laminin components arefound in hematopoietic and lymphoid tissues. Immunostainingof bone marrow with antiserum to laminin-1 has demonstratedwidespread distribution of laminins in the vascular basementmembranes, and also in the intersinusoidal interstitial tissue(Gu et al., 1999; Nilsson et al., 1998). In thymus, a similarantiserum reacts with basement membranes bordering thecapsule, septae and perivascular spaces (Berrih et al., 1985),whereas in lymph nodes, in addition to the vessel walls, a layerunderlying the subcapsular sinus was stained (van den Berg etal., 1993; Jaspars et al., 1995). Interestingly, laminin is alsodetected in reticular fibers, an extensive array of extracellularmatrix 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 notdetected in hematopoietic/lymphoid tissues and most bloodvessels (Chang et al., 1993; Falk et al., 1999; Gu et al., 1999),staining with the antiserum to laminin-1 may be due torecognition of laminin β1 and/or γ1 chains of other lamininisoforms.

According to their tissue distribution, laminin isoforms mayregulate proliferation, survival, differentiation, extravasation/migration and other activities of lymphoid cells. In vitro,laminin-1 inhibited both NK-mediated cytotoxicity andproliferation of spleen lymphocytes (Hiserodt et al., 1985b; Liand Cheung, 1992), and enhanced migration of the latter cells(Li and Cheung, 1992). Thymocytes and/or blood lymphocytesadhered minimally to laminin-1, but avidly to merosin(laminin-2/4, α2β1γ1/α2β2γ1) and bulk laminin isolated fromplacenta (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 containedlaminin-10/11 (α5β1γ1/α5β2γ1) (Ferletta and Ekblom, 1999).This preparation and merosin, but not laminin-1, were foundto be comitogenic for human lymphoid cells (Matsuyama etal., 1989; Shimizu et al., 1990b; Chang et al., 1995). Moreover,accumulation of adoptively transferred lymphocytes intoperipheral lymph nodes and cardiac allografts of recipientspretreated with rabbit antibodies to laminin-1 was significantlydecreased compared to controls (Kupiec-Weglinski and deSousa, 1991). In accordance with these findings, thymocytesand blood lymphocytes widely express α6β1 integrin as amajor laminin receptor (Shimizu et al., 1990a; Chang et al.,1995). In addition, α3β1 integrin mediates adhesion of certainlymphoid cells to laminin-5 (α3β3γ2) and to other lamininpreparations (Wayner et al., 1993; Chang et al., 1995).

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

MATERIALS AND METHODS

Cells, antibodies and purified proteinsJurkat (T cell leukemia) (Gillis and Watson, 1980), NAD-20(lymphoblastoid B cell) (Patarroyo et al., 1988) and NKL (NKleukemia) (Robertson et al., 1996) cell lines were cultured in RPMI1640 supplemented with 10% fetal bovine serum (Life Technologies,Täby, Sweden) and antibiotics. In addition, NKL cell line wassupplemented with 180 U/ml of recombinant IL-2. Peripheral bloodmononuclear cells (PBMC) were obtained from healthy donors afterFicoll-Hypaque gradient centrifugation (Amersham Pharmacia AB,Uppsala, Sweden). Lymphocytes were subsequently isolated by

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collecting nonadherent cells to tissue culture flasks. CD4 T cells wereobtained from PBMC by positive selection using magnetic beadscoated with anti-CD4 antibody according to instructions from themanufacturer (Dynal AS, Oslo, Norway).

Monoclonal antibodies (mAb) DG10, 1928 (Chemicon International,Temecula, CA, USA) and LN-26 (Takara Shuzo Co., Kyoto, Japan) tohuman laminin β1 chain, and 22 (Transduction Labs, Lexington, KY,USA), 2E8, LN-41 (Takara) and LN-82 (Takara) to human laminin γ1chain, were used. The chain specificity of these antibodies wasconfirmed by reactivity with recombinant human laminin β1 and γ1chains (Geberhiwot et al., 2000b). Rabbit antibodies to recombinanthuman laminin α4 I/II domains were produced and immunoaffinitypurified as previously described (Iivanainen et al., 1997). Theirspecificity was confirmed by reactivity against the recombinant protein(Kortesmaa et al., 2000) and by microsequencing of the immunoreactiveband from platelets (Geberhiwot et al., 1999). Moreover, theseantibodies did not stain muscle tissue sections or cell extracts fromlaminin α4 chain knock out (unpublished data). The rabbit antibodieswere not used for immunoprecipitation or immunofluorescence, sincethey recognize denaturated antigen. Blocking mAbs to integrin chainsincluded 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). mAbUCHT1 (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 negativecontrols.

Mouse laminin-1 (α1β1γ1, isolated from EHS tumor) was obtainedfrom Life Technologies, as well as human placenta laminin, whichwas purified with mAb 4C7 to laminin α5 chain and recently classifiedas 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 andpurified by affinity chromatography as described (Kortesmaa et al.,2000). The recombinant molecule was a Y-shaped heterotrimer, asexpected for the native protein.

Reverse transcription-polymerase chain reactionTotal RNA was purified from cultured cells by using RNazol B (AMSBiotechnology, Täby, Sweden). cDNA synthesis and PCR werecarried out using the Advantage RT-for-PCR kit (ClontechLaboratories Inc., Palo Alto, CA, USA). Briefly, after oligo(dT)18priming, mRNA was reverse-transcribed into cDNA by incubating for60 minutes at 42°C with 200 units of M-MLV reverse transcriptase in20 µl of reaction buffer containing 20 units of recombinant RNaseinhibitor, and 10 mM of each dNTP. The reactions were terminatedby heating the samples at 94°C for 5 minutes and the mixture wasdiluted to 100 µl with DEPC-treated water. Single-stranded cDNAsin 20 µl of reaction buffer containing 1.5 mM MgCl2, 10 µM of eachprimer and dNTP mix (10 mM each) were amplified by 30 cycles ofPCR using 1.25 units of Ampli Taq DNA polymerase (PerkinElmer/Roche Molecular Systems, Inc., Branchburg, NJ, USA). Theconditions for PCR were 94°C, 1 minute; 60°C, 1 minute; 72°C, 1minute. For PCR of human laminin α4 cDNA, paired primers 5′-TGCCTACTTTACCAGGGTGG-3′ (sense strand) and 5′-AAACATGTAAACCAAGCGGC-3′ (antisense strand) were used todirect synthesis of a 481-bp product (nucleotides 4287-4767)(Geberhiwot et al., 2000a). For human laminin β1 cDNA, pairedprimers 5′-TTGGACCAAGATGTCCTGAG-3′ (sense strand) and 5′-CAATATATTCTGCCTCCCCG-3′ (antisense strand) were used and a679-bp product was expected (nucleotides 955-1633) (Geberhiwot etal., 2000a). For human laminin γ1 cDNA, paired primers 5′-GCAAGACTGAACAGCAGACC-3′ (sense strand) and 5′-TCCTATCAAGATCGCTGACC-3′ (antisense strand) were used anda 687-bp product was expected (nucleotides 4241-4927) (Geberhiwotet al., 2000a). PCR products were analysed by electrophoresis using2% agarose gels.

Metabolic labelling of the cells with [35S]methionine and[35S]cysteine and immunoprecipitationFollowing incubation at 37°C for 30 minutes in methionine- andcysteine-free medium, 30 million cells were labelled in 10 ml ofmethionine- and cysteine-free RPMI 1640 medium with 10% dialyzedfetal bovine serum containing 0.20 mCi/ml Trans35S-label (ICNRadiochemical Inc.) for 4 hours at 37°C. After washing three timeswith 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/mlof aprotinin, 2 µM leupeptin, 2 µM pepstatin, 1 mM PMSF and 2 mMEDTA as protease inhibitors). The soluble fraction (cell lysate) wasprecleared with protein G-Sepharose beads (Amersham PharmaciaAB) and immunoprecipitation was performed by adding to 100 µl ofcell 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).

Gel electrophoresis, immunoblotting, enhancedchemiluminescence and purification of laminin byimmunoaffinity chromatographyProtein samples were analysed by SDS-PAGE. In western blots, filterswere blocked with 0.1% Tween 20/5% dry milk in PBS. Peroxidase-linked anti-mouse or anti-rabbit immunoglobulin (Dakopatts) wasused as secondary antibody, and ECL (Amersham, UK) was used asdeveloper. Laminin was purified from cell lysates by immunoaffinitychromatography as previously described (Geberhiwot et al., 1999).Briefly, Sepharose CL-4B preadsorbed cell lysate was applied tolaminin β1 antibody column (mAb DG10 coupled to CNBr-activatedSepharose 4B), which had been equilibrated with lysis buffer, andcycled twice. Following extensive washing, the proteins bound to thecolumn were eluted by using high pH and the samples were collectedin neutralizing buffer. After concentration, the purified material wasanalysed by SDS-PAGE and western blotting.

Immunofluorescence flow cytometryIsolated blood mononuclear leukocytes were washed with PBS andused non-permeabilized and permeabilized for immunofluorescence.Cells were permeabilized by using IntraStain kit (Dakopatts)according to instructions from the manufacturer. Indirectimmunofluorescence was performed by incubating cells, afterblocking with 2 mg/ml of human IgG (Sigma) for 30 minutes at 4°C,with saturating amounts of monoclonal antibodies, followed byfluorescein-conjugated F(ab)2 fragments of rabbit anti-mouseimmunoglobulin (Dakopatts) at 1:20 dilution. After staining,lymphocytes were gated, according to forward and side scatter, andanalysed in a FACScan flow cytometer (Beckton Dickinson, MountainView, USA).

Laminin secretion and cell migration and adhesion assaysTo study laminin secretion, isolated lymphocytes were resuspended at5×107 cells/ml in plain RPMI. After preincubation for 15 minutes, thecells were stimulated with 200 nM tetradecanoyl phorbol acetate(TPA, also known as PMA; Sigma) for 20 minutes at 37°C. Followingaddition of protease inhibitors and centrifugation of the cells,supernatant was collected and analysed for secreted laminin.

Transmigration of lymphocytes through protein-coated filters wasmeasured microscopically and by flow cytometry. Briefly, humanblood mononuclear cells (5×105, containing 55% lymphocytes) in 100µl RPMI-1640 medium were added to the top chamber of a 6.5 mmdiameter, 5 µm pore polycarbonate Transwell culture insert (Costar,Cambridge, MA, USA) and incubated at 37°C for 3 hours in theabsence (spontaneous migration) or presence (chemokine-stimulatedmigration) of 500 ng/ml stromal cell derived factor (SDF-1α, R&DSystems, Abingdon, UK) in the lower chamber. The filters hadbeen previously coated overnight with 20 µg/ml of various lamininisoforms or human serum albumin (HSA) (Sigma), and blocked with0.5% HSA for 1 hour. To efficiently remove all transmigrated cells

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from the lower chamber, a final concentration of 10 mM EDTA wasadded to each well and cells were resuspended vigorously. Thereafter,the resuspended cells were fixed with 1% formaldehyde and the cellnumber was determined microscopically by counting five fields witha 40× objective. The percentage of lymphocytes in the cell populationwas 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 calculatedas well as level of significance (*P<0.05), comparing the variouslaminin isoforms to HSA.

In studies of cell adhesion to immobilized proteins, isolated CD4T cells resuspended in RPMI (2×106/ml) with 0.5% HSA were used.96-well plates (MaxiSorp, Nunc, Roskilde, Denmark) were coatedovernight at room temperature with HSA, mouse laminin-1,recombinant laminin-8 or placenta laminin-10/11 at 20 µg/ml. Afterblocking with bovine serum albumin, 2×105 cells/well (100 µl) wereincubated in absence or presence of 200 nM TPA (Sigma) for 1 hourat 37°C. Following five washes with plain medium, adherent cellswere fixed by adding 50 µl of fixative solution (paraformaldehyde 40g/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 offiltered Toluidine Blue dye (Sigma, 0.5% w/v in PBS) was addedovernight at room temperature. The plate was then washed withcopious amounts of distilled water. Adherent cells were quantified ina microplate reader (Multiskan Bichromatic, Labsystems, Helsinki,Finland) by measuring absorbance at 690 nm after releasing the bluedye with 100 µl of 2% sodium dodecyl sulfate (Bio-Rad Laboratories,Richmond, CA, USA). The effect of blocking mAbs to integrin α6and β1 chains on the laminin-specific cell adhesion was tested bypretreating the cell suspension with antibodies (20 µg/ml) for 15minutes before adding the cells to the laminin-coated wells. Celladhesion in the presence of control IgG was defined as 100%adherence. In statistical analysis (Student’s t-test), mean and s.d. werecalculated as well as the level of significance (*P<0.05; **P<0.01;*** P<0.001), comparing the various laminin isoforms to HSA or theblocking antibodies to the IgG control.

Cell proliferation assayCell proliferation, measured as 3H-thymidine incorporation, wasperformed in triplicate in 96-well microtiter plates (Costar Corp.,Cambridge, MA, USA) coated overnight with 0.5 µg/ml of anti-CD3mAb. 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 inearly dose-response studies (data not shown). Thereafter, purifiedCD4 T cells resuspended in AIM-V serum-free medium (LifeTechnologies, Inc.) were cultured in the plates (4×104 cells/well) for4 days at 37°C in 5% CO2 humidified air, and pulsed with 1 µCi/wellof (methyl-3H) thymidine (Amersham, UK) for the final 12 hours.Cells were then harvested onto filter paper using a semiautomaticcell harvester (Tomtec, Inc.) and 3H-thymidine incorporation wasmeasured in a liquid scintillation counter (Beckman Instruments, Inc).The effect of blocking mAbs to integrin α6 and β1 chains on thecostimulatory effect of laminins was tested by incubating the cellswith 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 ofsignificance (*P<0.05; **P<0.01), comparing the various lamininisoforms to HSA or the blocking antibodies to the IgG control, inpresence of the mAb to CD3.

RESULTS

Expression of laminin α4, β1 and γ1 mRNAs inlymphoid cellsExpression of mRNAs encoding for laminin-8 chains inlymphoid cells was investigated by RT-PCR using pairs ofprimers that were designed on the basis of the reported cDNAsequences of human laminin α4, β1 and γ1 chains. Amplifiedproducts with the expected size were detected with primers forα4 (481 bp), β1 (679 bp) and γ1 (687) after 30 cycles of PCRwith reverse transcribed Jurkat mRNA (Fig. 1). Similar resultswere obtained with NAD-20 cells (data not shown).

Synthesis and expression of laminin β1 and γ1polypeptides in lymphoid cellsThe lysate of metabolically radiolabeled Jurkat cells wasimmunoprecipitated with mAbs to either β1 or γ1 chains toanalyse synthesis and assembly of laminin components (Fig.2). All four antibodies immunoprecipitated polypeptides of 220

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Fig. 1.RT-PCR of laminin-8 chains in Jurkat T cells. Amplificationof laminin α4, β1 and γ1 cDNA fragments by RT-PCR of total RNAfrom Jurkat cells. GAPDH and buffer were used as positive andnegative controls, respectively. The result after 30 cycles of RT-PCRis shown.

Fig. 2. Immunoprecipitation from 35S-methionine/cysteine-labeledJurkat cells with mAbs to laminin β1 and γ1 chains. Names of theantibodies and their specificity (in parentheses) are shown. mIgG,mouse IgG. Arrows indicate bands of 220 and 200 kDa,corresponding to laminin β1 and γ1 chains, respectively.Immunoprecipitated materials were separated by SDS-gelelectrophoresis under reducing conditions (6% acrylamide).

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and 200 kDa. Similar results were obtained with NAD-20 cells(data not shown).

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

Laminin α4 polypeptide is expressed andnoncovalently associated to disulfide-bonded β1γ1chains to form laminin-8 in T, B and NK cellsLaminin was isolated from Jurkat (T) cell lysates byimmunoaffinity chromatography on a laminin β1 (DG10)antibody column and then analysed by western blotting toidentify the α chain and to study the physical association ofthe components (Fig. 3). Under reducing conditions, mAbs toβ1 and γ1 chains strongly reacted with 220 and 200 kDapolypeptides, respectively. Affinity-purified rabbit antibodies

to laminin α4 chain were strongly reactive with polypeptidesof 200 and 180 kDa. Similar results were obtained withthe material isolated from NAD-20 (B) and NKL (NK) celllines, though certain degradation of laminin α4 chain wasapparent in the latter cells (Fig. 3). Under nonreducingconditions, laminin β1 and γ1 chains comigrated with a slowerelectrophoretic mobility (approximately 420 kDa), whereas theα4 chain migration was faster (approximately 200 kDa).

Blood lymphocytes contain and, followingstimulation, secrete laminin-8Jurkat and NKL are cell lines of malignant origin (Gillis andWatson, 1980; Robertson et al., 1996), whereas NAD-20 wasestablished by transformation of B lymphocytes with EpsteinBarr virus (Patarroyo et al., 1988). To determine the presenceof laminin in normal lymphocytes, immunofluorescence flowcytometry of purified blood mononuclear cells was used (Fig.4). Lymphocytes, gated by forward and side scatter, displayedreactivity with mAb H12 to CD11a (αL integrin chain), withthe characteristic pattern of two positive populations.Monoclonal antibodies LN-26 and LN-41 to laminin β1 and γ1chains, respectively, bound minimally to intact lymphocytes,whereas practically all lymphocytes were reactive with bothantibodies after cell permeabilization.

To determine whether blood lymphocytes containedlaminin-8, the cell lysate of lymphocytes isolated from bloodmononuclear cells was immunopurified on the laminin β1

Fig. 3.Western blot analysis of laminin β1 antibody-immunoaffinitypurified material from T (Jurkat), B (NAD-20) and NK (NKL) celllines. Mouse IgG (mIgG), rabbit IgG (rIgG), purified rabbitantibodies to laminin α4 chain (anti-LNα4), mAb to laminin β1chain (DG10) and mAbs to laminin γ1 chain (22 and 2E8) were used.Under reducing conditions (R, 6% acrylamide gels), arrows indicatebands of 220, 200 and 180 kDa corresponding to laminin β1, γ1 andα4 chains, respectively. Under nonreducing conditions (NR, 3%acrylamide gels), arrows indicate bands of approximately 420 and200 kDa, corresponding to disulfide-bonded laminin β1γ1 dimer andα4 monomer, respectively. Since mAb 22 reacts preferentially withreduced antigen, under nonreducing conditions this antibody wasreplaced by mAb 2E8.

Fig. 4.Detection of laminin β1 and γ1 chains in blood lymphocytesby immunofluorescence flow cytometry. Reactivity of monoclonalantibodies with nonpermeabilized and permeabilized cells is shown.Mouse IgG control (dotted line) and mAbs (shaded peak) H12(CD11a, αL integrin), LN-26 (laminin β1 chain) and LN-41 (lamininγ1 chain). Decreased reactivity with CD11a in permeabilized cells isdue to the effect of fixation, required in the cell permeabilizationprocedure, on the epitope detected by mAb H12.

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antibody column, and the purified material was then analysedby western blotting (Fig. 5A). Similar to the findings withlymphoid cell lines, mAbs to β1 and γ1 chains recognizedpolypeptides of 230 and 220 kDa, respectively, whereas rabbitantibodies to α4 reacted with a broad band of 180-200 kDa,under reducing conditions.

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

Laminin-8, but not laminin-10/11, promotesmigration of blood lymphocytesLymphocytes secrete laminin-8 and may interact with theendogenous laminin. Alternatively, the cells may recognizeexogenous laminin-8 in blood vessels and other tissuecompartments. To determine the effect of laminin-8 onlymphocyte migration, transmigration of blood lymphocytesthrough recombinant laminin-8-coated filters was studied ininsert assays. Compared to HSA, laminin-8 enhancedlymphocyte migration in the presence of chemoattractant by57%, and this effect was statistically significant (P<0.05) (Fig.6). Laminin-1 also enhanced cell migration (P<0.05), but toa lower extent (20%), whereas laminin-10/11 tended to beinhibitory (−35%). For all substrates, SDF-1α stimulateda major increase in lymphocyte migration compared tocells without chemoattractant. Approximately 15% of thelymphocyte population transmigrated on the laminin-8substrate in the presence of SDF-1α in 3 hours. The opposingeffects of laminin-8 and -10/11 on cell migration were moreevident with blood monocytes (Pedraza et al., 2000).

Stimulated blood lymphocytes adhere to laminin-8and laminin-10/11 via α6β1 integrinTo investigate whether laminin-8 was adhesive forlymphocytes, adhesion of isolated CD4 T cells to immobilizedrecombinant laminin-8 was studied (Fig. 7A). Plastic surfaceswere coated with HSA, mouse laminin-1, placenta laminin-10/11, or recombinant laminin-8. After incubating thelymphocytes for 1 hour at 37°C on the protein-coated surfacesin the absence or presence of phorbol ester, nonadherent cells

were removed by extensive washing. The constitutive celladhesion to laminin-8 and -10/11 was modest. However,following stimulation of the cells, these laminin isoforms weremost adhesive, laminin-10/11 being more active than laminin-8 (P<0.001 and P<0.05, respectively). In contrast, celladhesion, either constitutive or stimulated, to laminin-1 waslow. Higher amounts of laminin-1 (50 µg/ml) did not increasethe level of cell adhesion (data not shown).

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

Laminin-8 and laminin-10/11, but not laminin-1,costimulate T cell proliferation via α6β1 integrinIn addition to CD3/TCR engagement, induction ofproliferation of resting T cells is known to require additionalcostimuli (Janeway et al., 1999). Fibronectin, bulk placentalaminin (presumably containing laminin-10/11) and merosin(laminin-2/4) have been reported to be comitogenic forlymphoid cells through integrin receptors (Matsuyama et al.,1989; Shimizu et al., 1990a; Chang et al., 1995). To investigatewhether laminin-8, which is expressed by monocytic and Bcells (Pedraza et al., 2000 and present study), provides a similarcostimulatory signal, recombinant laminin-8 and mAb UCHT1to CD3 were coimmobilized, and isolated CD4 T cells wereincubated on the coated wells for 4 days. Under theseexperimental conditions, extensive cell proliferation wasinduced, measured as 3H-thymidine incorporation (Fig. 8A).Recombinant laminin-8 or the CD3 mAb on their own failedto induce any significant cell proliferation. Interestingly,laminin-1 was inactive, whereas laminin-10/11 was as effectiveas laminin-8. The costimulatory signal by laminin-8 andlaminin-10/11 was mediated via α6β1 integrin, as indicated byalmost complete inhibition of the response by blockingantibodies to β1 and α6 integrin chains (Fig. 8B). Whencoimmobilized with mAb to CD3, mAb GoH3 to α6 integrinchain (0.5-10 µg/ml), but not rat IgG control, similarly induced

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Fig. 5.Western blot analysis of laminin-8 from isolatedblood lymphocytes. (A) Laminin β1 antibody-immunopurified material from cell lysate. (B) Totalsecreted material from lymphocytes stimulated with200 nM TPA for 20 minutes. Mouse IgG (mIgG), rabbitIgG (rIgG), purified rabbit antibodies to laminin α4chain (anti-LNα4), mAb to laminin β1 chain (DG10)and mAb to laminin γ1 chain (22) were used. Arrowsindicate bands of 230, 220 and 180 kDa correspondingto laminin β1, γ1 and α4 chains, respectively. Reducingconditions, 6% acrylamide gel.

429Laminin-8 and lymphoid cells

a strong proliferative response in the lymphocytes (data notshown).

DISCUSSION

In the present study we demonstrate, for the first time, that T, Band NK cells synthesize laminin-8 and that blood lymphocytescontain and secrete this laminin isoform. We show expressionof laminin α, β and γ chains, and their assembly into acomplete laminin molecule in lymphoid cells. We alsodemonstrate promotion of lymphocyte migration by laminin-8, but not by laminin-10/11, activation-dependent adhesion ofblood lymphocytes to laminin-8 and -10/11, and costimulationof T cell proliferation by these laminin isoforms via α6β1integrin.

Jurkat, NAD-20 and NKL are relatively mature lymphoidcells of T, B and NK cell origin, respectively, and represent thethree major types of lymphocytes (Gillis and Watson, 1980;Patarroyo et al., 1988; Robertson et al., 1996). Expressionof mRNAs for laminin α4, β1 and γ1 chains in thelymphoid cells, as detected by RT-PCR, strongly suggestedbiosynthesis of laminin-8. Metabolic labelling followed byimmunoprecipitation with mAbs to laminin β1 and γ1 chainsrevealed two bands of 220 and 200 kDa, suggestingcomigration of the α chain with either the β or the γ chains. Incontrast to laminin β1 (220 kDa) and γ1 (200 kDa) chains,laminin α4 chain could not be detected by western blotting oftotal cell lysate, probably due to small amounts of the chainand/or masking of the polypeptide by other proteins. However,this chain (200/180 kDa) was readily detected following

immunoaffinity purification on the laminin β1 antibodycolumn. As in other laminin isoforms (Ekblom and Timpl,1996), the laminin α chain appeared to be rate-limiting inlaminin synthesis. The presence of laminin-8 wasunambiguously demonstrated by identification of physicallyassociated laminin α4, β1 and γ1 chains. In contrast to laminin-8 from lung and kidney (Miner et al., 1997), but similarly tolaminin-8 from other hematopoietic cells (Geberhiwot et al.,1999; Geberhiwot et al., 2000a; Pedraza et al., 2000), the α4chain was noncovalently associated to disulfide-bonded β1γ1chains in lymphoid cells. This was concluded from thedisparate electrophoretic mobility of, on the one hand, β1 andγ1 chains (slow) and, on the other hand, the α4 chain (fast),under nonreducing conditions.

Practically 100% of blood lymphocytes reacted with mAbsto laminin β1 and γ1 chains by immunofluorescence flowcytometry, suggesting that all lymphocytes, and not just asubpopulation, contained laminin. Since cell permeabilizationwas required for detection, laminin appears to have anintracellular localization. Similarly to the lymphoid cell lines,isolated blood lymphocytes contained laminin-8. Since RNAand protein synthesis is very low in these resting cells, theirlaminin-8 originated most likely from biosynthesis in precursorcells in thymus, bone marrow and/or other lymphoid tissues.Stimulation of blood lymphocytes with phorbol ester, anactivator of protein kinase C, followed by analysis of thesupernatant identified laminin-8 as a novel secretory productof lymphoid cells. This prompt secretion may be relevant tothe function of the laminin molecule.

The relationship between the laminin-like substance on thecell surface of mouse, rat and human NK cells reportedby another research group (Hiserodt et al., 1985a; Vujanovicet al., 1988; Schwarz et al., 1988) and the laminin-8 oflymphocytes is presently unknown. The antigen, detected byimmunofluorescence with a rabbit antiserum to mouse laminin-1, was not molecularly characterized. In preliminary studies,we have been unable to demonstrate staining of intact bloodNK cells (CD3−, CD56+) with mAbs to laminin β1 and γ1chains (unpublished data). The identity of the 200 and 400 kDaproteins from metabolically radiolabeled rat NK cellsimmunoprecipitated with rabbit antibodies to laminin-1reported by Morrone et al. (Morrone et al., 1989) is alsounknown. Although the 200 kDa band may correspond tolaminin α4, β1 and/or γ1 chains, it is unlikely that the 400 kDapolypeptide is laminin α1 since expression of this chain ismostly restricted to epithelial cells (Falk et al., 1999; Virtanenet al., 2000). Moreover, we have been unable to detect lamininα1 mRNA or full-length (300-400 kDa) laminin α chains inlymphoid cells (unpublished data). Similar to our results,Chang et al. (Chang et al., 1993) could not detect transcriptsfor laminin α1 or α2 chains in isolated mouse thymocytes byRT-PCR. It is noteworthy that the studies on NK cell lamininby the other groups were performed during the 1980s, whenonly one laminin α chain, namely α1, had been identified.

Vascular laminin-8 may contribute to extravasation of bloodlymphocytes, whereas endogenous laminin-8 may mediatelymphocyte migration and chemotaxis in extravascular loci.Compared to HSA, exogenous laminin-8 and, to a lower extent,laminin-1, reproducibly enhanced lymphocyte migration inpresence of SDF-1, a potent chemoattractant for mononuclearleukocytes, which binds to the chemokine receptor CXCR4. In

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Fig. 6.Laminin-8, but not laminin-10/11, promotes lymphocytemigration. Lymphocyte migration assays were performed by using 5µm pore insert filters precoated with human serum albumin (HSA),laminin-1 (LN-1), laminin-8 (LN-8) or laminin-10/11 (LN-10/11).The number of transmigrated cells was determined microscopically,and the percentage of lymphocytes in the cell population wasestablished by flow cytometry. Starting with 5×105 bloodmononuclear cells (55% lymphocytes) in the upper chamber,migration to the lower chamber was measured after incubating thecells for 3 hours at 37°C. SDF-1, stromal cell derived factor 1. Valuesare means ± s.d. of four experiments (*P<0.05).

430

contrast, laminin-10/11 tended to inhibit cell migration. Asonly a fraction (15%) of the lymphocytes transmigrated, aparticular lymphocyte subpopulation appears to be motile. Thepromoting and inhibitory effects of laminin-8 and laminin-10/11, respectively, on leukocyte migration are more evidentwhen blood monocytes are used (Pedraza et al., 2000). Theopposite effects of these laminin isoforms on lymphocytemigration suggest that laminin-8 promotes migration oflymphoid cells in tissues, whereas laminin-10/11 determinestheir tissue localization (arrest). Interestingly, considerablelymphocyte 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 locomotionof T lymphocytes in a collagen matrix is abolished by a mAbto α6 integrin (Friedl et al., 1995). Since laminin was notpresent in the matrix and α6 integrins do not bind collagen,endogenous laminin-8 could mediate the lymphocytemigration. Keratinocyte migration is known to depend onendogenously secreted laminin-5 (α3β3γ2) (Zhang andKramer, 1996).

In other studies, laminin-1 promoted in vitro migration ofspleen lymphocytes and NK cells (Li and Cheung, 1992;

JOURNAL OF CELL SCIENCE 114 (2)

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Fig. 7.Adherence of isolated blood CD4 T cells to laminin-8 andlaminin-10/11 and identification of the integrin receptor.(A) Adherence of the T cells to plastic surfaces coated with humanserum albumin (HSA), mouse laminin-1 (LN-1), recombinantlaminin-8 (LN-8) and placenta laminin-10/11 (LN-10/11) in absenceor presence of phorbol ester (200 nM TPA). (B) Effect of monoclonalantibodies to α6β1 integrin on adherence of T cells to laminin-8 or-10/11 in the presence of phorbol ester. IgG, control IgG; 4B4, mAbto integrin β1 chain; GoH3, mAb to integrin α6 chain. Plasticsurfaces were coated with laminins at 20 µg/ml. Adhesion blockingantibodies were used at 20 µg/ml. Adherence was measured afterincubating the cells (2×105 cells/well) in the coated plates for 1 hourat 37°C. Results are shown as optical density (A) and% adherence(B). Values are means ± s.d. for four experiments (*P<0.05;** P<0.01; ***P<0.001).

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Fig. 8.Costimulation of T cell proliferation by laminin-8 andlaminin-10/11 and participation of α6β1 integrin. (A) Proliferation ofisolated CD4 T cells on immobilized mAb CD3 alone, laminin aloneor in combination. CD3, mAb UCHT1 to CD3; HSA, human serumalbumin; LN-1, mouse laminin-1; LN-8, recombinant laminin-8; LN-10/11, placenta laminin-10/11; Medium, plain medium. (B) Effect ofmonoclonal antibodies to α6β1 integrin on T cell proliferationinduced by mAb CD3 plus laminin. CD3, mAb UCHT1 to CD3; LN-1, mouse laminin-1; LN-8, recombinant laminin-8; LN-10/11,placenta laminin-10/11; IgG, control IgG; 4B4, mAb to integrin β1chain; GoH3, mAb to laminin α6 chain. Plastic surfaces were coatedwith mAb UCHT1 to CD3 at 0.5 µg/ml and/or laminins at 2.5 µg/ml.Cell proliferation was measured after culturing cells for 4 days.Adhesion blocking antibodies were used at 20 µg/ml. Values aremeans ± s.d. of four experiments in A and B (*P<0.05; **P<0.01).

431Laminin-8 and lymphoid cells

Somersalo and Saksela, 1991), and bulk placenta lamininfacilitated chemotaxis of malignant plasma cells (Shibayamaet al., 1995). Compared to type 2 T helper (Th2) cells, Th1cells preferentially migrated on laminin-1 by using α6β1integrin (Colontonio et al., 1999), whereas replacement of theα6A integrin splice variant by α6B in lymph node T cellsreduced migration of the cells on laminin-1, but not onfibronectin (Gimond et al., 1998). Notably, vascular α6 integrinmediated homing of pro-T cells to the thymus (Dunon andImhof, 1993). Modulation in vivo of lymphocyte traffic,particularly inhibition of lymphocyte accumulation inperipheral lymph nodes, by anti-laminin-1 antibodies (Kupiec-Weglinski and de Sousa, 1991) indicated the role of lamininsin lymphocyte migration.

In spite of several immunohistochemical studies (Berrih etal., 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 lamininisoforms in lymphoid tissue is poorly understood. Vessels,including high endothelial venules (HEVs), areimmunoreactive with antibodies to laminin-1 and laminin β1and γ1 chains (van den Berg, 1993; Jaspars et al., 1995;Castanos-Velez et al., 1995; Jaspars et al., 1996). Since inlymphoid tissues laminin α1 chain is not detected and lamininα2 chain expression appears to be rather limited (Chang et al.,1993; Virtanen et al., 1996; Falk et al., 1999), other laminin αchains could be present. Indeed, laminin α5 chain, as detectedby mAb 4C7, is found in the subepithelial basement membraneof the capsule and in the basal laminae of stromal andparenchymal vessels in thymus (Virtanen et al., 1996). Thislaminin chain is also found in basement membranes ofcapillaries and HEVs and, most likely, in the reticular fibers oflymphoid tissue (Sorokin et al., 1997). These laminin-containing fibers, which radiate from the basement membraneof HEVs and are more abundant in the interfollicular area,could form a scaffold for lymphocyte localization and/or apathway for lymphocyte migration within the lymphoid tissue(Kramer et al., 1988). In addition, a granular staining oflymphoid follicles, probably corresponding to folliculardendritic cells, was revealed with antibodies to laminin-1(Jaspars et al., 1995). Antibodies to laminin-5 (α3β3γ2) alsoreacted 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 antigencan be also found on the luminal surface of HEVs and lungcapillaries (Girard and Springer, 1995; Hilario et al., 1996),suggesting a role of vascular laminins in early leukocyte-endothelium interactions. Immunolocalization of laminin α4chain in lymphoid tissue is presently unknown. Since laminin-8 is a major endothelial laminin (Frieser et al., 1997), it couldbe expressed by HEVs. Moreover, upregulation of laminin α4mRNA in vascular endothelial cells by IL-1 (Frieser et al.,1997) suggests participation of laminin-8 in inflammatoryresponses. Considering the cell specific expression, tissuedistribution and biological effects of laminin-8 and -10/11,these laminin isoforms appear to be most relevant inlymphocyte physiology.

Adhesion of lymphoid cells to laminin-8 and -10/11 has notbeen previously reported or recognized. Laminin-8 was not

available for adhesion studies, and presence of laminin-10/11in the commercial placenta laminin preparation was onlyrecently established (Ferletta and Ekblom, 1999). Adherenceof blood CD4 T cells to laminin-8 and -10/11 followingstimulation with phorbol ester indicated that the adhesion wasactivation-dependent and suggested that these laminin isoformscould be relevant to adhesion-dependent lymphocyte function/activities. The low levels of adhesion of blood lymphocytes tomouse laminin-1 described by us and by others (Weeks et al.,1994) may be due to its α1 chain, which is absent inhematopoietic and lymphoid tissues and in most bloodvessels (Chang et al., 1993; Falk et al., 1999; Gu et al., 1999;Virtanen et al., 2000). Alternatively, glycosylation or othermodifications of this mouse tumor laminin may be responsible.The receptor for laminin-8 and -10/11 in the bloodlymphocytes was identified as α6β1 integrin. Platelets andmonocytic cells also use this integrin to adhere to laminin-8(Geberhiwot et al., 1999; Pedraza et al., 2000). Thymocytesand blood lymphocytes express α6β1 integrin (Shimizu et al.,1990a; Chang et al., 1993; Chang et al., 1995), and phorbolesters are known to enhance avidity of leukocyte integrins fortheir ligands (Patarroyo et al., 1985; Shimizu et al., 1990).

In related studies by other groups, mouse and humanthymocytes and human blood lymphocytes were found toadhere to bulk laminin isolated from human placenta, but atlow levels or not at all to mouse laminin-1 (Cardarelli andPierschbacher, 1986; Shimizu et al., 1990a; Chang et al., 1993;Weeks et al., 1994; Chang et al., 1995). Adhesion ofthymocytes to the bulk laminin, which probably containedlaminin-10/11, was mediated by α6β1 integrin, but α3β1integrin also contributed (Chang et al., 1995), and the cellsadhered to a similar extent to a merosin preparation (laminin-2/4, α2β1γ1/α2β2γ1) (Chang et al., 1995). Stimulated NK cellsalso adhered to laminin (unspecified isoform) via α6β1 integrin(Gismondi et al., 1992). Notably, lymphoid cells of B cellorigin were also able to adhere to bulk placenta laminin (Segatet al., 1994), and blood leukocytes, including lymphocytes,were found to tether and arrest on laminin under physiologicalshear flow via α6β1 integrin (Kitayama et al., 2000).

Antigen-specific activation by T cells requires not onlyCD3/TCR engagement by antigen/major histocompatibilitycomplex (Ag/MHC) but additional costimuli (Janeway et al.,1999). In the present study, laminin-8 or mAb to CD3 on theirown failed to drive the CD4 T cells to proliferate, but togetherhad a synergistic effect. Laminin-10/11 was also comitogenic,whereas laminin-1 was inactive. The costimulatory signal wastransduced via α6β1 integrin, the same integrin that mediatedcell adhesion. Our data confirm the inability of the mouse EHSlaminin-1 to costimulate proliferation of human lymphoid cellsdescribed by other groups (Matsuyama et al., 1989; Shimizuet al., 1990b). However, the laminin-1 has been reported tocostimulate mouse lymph node T cell proliferation (Gimond etal., 1998), and to inhibit, to some extent, growth of mousespleen lymphocytes stimulated by Concanavalin A (Li andCheung, 1992). Bulk placenta laminin (presumably containinglaminin-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). Bothlaminin-2/4 and laminin-5 supported survival of earlythymocytes (Iwao et al., 2000; Kim et al., 2000). Inasmuch as

432

T cells normally recognize antigen present on cells (with MHCmolecules) rather than on extracellular matrices, possibilitiesmust be considered that laminin-8 and other laminin isoformsbecome important for T cell activation when presented on thesurface of other cells, such as antigen-presenting cells. Insupport of this hypothesis, laminin epitopes can be detected onthe surface of cells of the monocytic lineage, includingactivated 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 otherlymphocyte function/activities. Laminin-1 and -10/11 triggeredCa2+ signalling in Jurkat cells via α6β1 integrin (Weismann etal., 1997), and laminin (unspecified isoform) induced TNFαsecretion by interacting CD4 T cells and macrophages, inthe absence of antigenic stimulus (Hershkoviz et al., 1993).Laminin-1 inhibited the lysis of target cells by NK cells buthad no effect on the cytotoxicity mediated by T lymphocytes(Hiserodt et al., 1985b), and laminin receptor expression of thetarget cells correlated with their NK sensitivity (Laybourn etal., 1989). Antibodies to laminin-1 inhibited cytotoxicitymediated by NK cells, but not by cytotoxic T lymphocytes(Hiserodt et al., 1985a), and α6β1 integrin was found toparticipate in natural killing (Lowdell et al., 1995). Based onthese studies and our data, it is tempting to speculate thatlaminin-8 participates in NK recognition.

Synthesis, secretion, recognition and use of laminin-8 bylymphoid cells indicate the relevance of this laminin isoformin lymphocyte function/activities. In addition to stimulatingcell migration and proliferation, recognition of exogenousand/or endogenous laminin-8 may contribute to lymphocytesurvival, maturation, cytotoxicity and tissue localization. Allthese processes participate in the development of immune/inflammatory responses and in the pathophysiology oflymphoid malignancies.

This project was supported by Cancerfonden and the KarolinskaInstitute. The authors gratefully acknowledge Drs E. Engvall, D.Gigliotti and A. Iivanainen for providing antibodies, and Drs GeorgeKlein, Michael J. Robertson and Mikael Jondal for providing celllines. We also thank Dr Timo Pikkarainen for comments on themanuscript and Dr B. Chambers for revising the English text.

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