Published online before print June 12, 2008
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5-laminin(s) in adhesion and migration of blood lymphocytes
* Departments of Odontology,
|| Physiology and Pharmacology, and
# Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden;
Institute of Biomedicine/Anatomy and
Haartman Institute, University of Helsinki, Finland;
Institut de Biologie et Chimie des Protéines, Université Lyon I, Lyon, France; and
¶ Department of Cell and Tissue Biology, University of California, San Francisco, California, USA
1 Correspondence: Department of Odontology, Karolinska Institutet at Huddinge, SE-141 04 Stockholm, Sweden. E-mail: manuel.patarroyo{at}ki.se
ABSTRACT
During extravasation and within lymph nodes (LNs), blood lymphocytes interact with laminins (Lms), major components of vascular basement membranes (BMs) and of reticular fibers (RFs), a fibrillar extracellular matrix. However, the identity and role of these laminin isoform(s) are poorly known. By using confocal microscopy examination of human LNs, we show that BMs of high endothelial venules (HEVs) express laminin 
3, 4, 5, β1, β2, and 
1 chains and that the same chains, in addition to 2, are found in RFs. In functional studies with laminin isoforms covering all Lm chains, 5-laminin (Lm-511) was the most adhesion- and migration-promoting isoform for human blood lymphocytes, followed by 3- (Lm-332) and 4- (Lm-411) laminins, and the lymphocytes used the 6β1 integrin as the primary receptor for the 5-laminin. Moreover, Lm-511 strongly costimulated T cell proliferation, and blood lymphocytes were able to secrete 4- and 5-laminins following stimulation. The LN cell number in laminin 4-deficient mice compared with wild-type did not differ significantly. This study demonstrates a predominant role for 5-laminin(s) in blood lymphocyte biology and identifies LN laminins and their integrin receptors in blood lymphocytes.
Key Words: leukocyte • extracellular matrix • basement membrane • chemotaxis
INTRODUCTION
Interaction of lymphocytes with extracellular matrix (ECM) components, such as collagens, fibronectin (FN), and laminins (Lms), regulates several lymphocyte activities in vitro, including adhesion, migration, differentiation, and proliferation/survival [1 ]. In lymph nodes (LNs) and other secondary lymphoid tissues, these ECM components are nonrandomly distributed and constitute barriers and scaffolds, such as vascular basement membranes (BMs) and reticular fibers (RFs) [2 ]. In addition to these structural functions, these large molecules may facilitate in situ-specific localization and/or migration of lymphocytes and other immune cells and hence, contribute to the generation of immune responses [3 , 4 ].
During trafficking, blood lymphocytes adhere to and migrate across high endothelial venules (HEVs) to enter LNs, tonsils, Peyer’s patches, and other secondary lymphoid tissues [5 , 6 ]. Whereas the initial steps of this extravasation process (rolling and firm adhesion) are well-characterized, the following transendothelial migration and the interaction with the HEV BM is poorly understood. Moreover, once within the lymphoid tissue, lymphocytes may also interact with the RFs [4 , 7 ].
Laminins are a growing family of large, heterotrimeric β glycoproteins, mainly found in BMs [8
]. Five , three β, and three laminin chains constitute, by combination, over 15 laminin isoforms, which are expressed in a cell- and tissue-specific manner [9
, 10
]. The chains of these molecules are recognized by at least seven different integrins (INTs) and strongly promote cell adhesion, migration, and differentiation [9
]. In secondary lymphoid tissues, laminins are present in the BMs of ordinary blood vessels and HEVs, as well as in RFs [2
, 11
12
13
]. The latter fibers, which also contain other BM components such as nidogen, perlecan, and type IV collagen, constitute the reticular network, a conduit system for transport of small, soluble molecules [11
12
13
14
].
Interactions of lymphocytes with laminin isoforms are poorly understood. Since identification of the prototype Lm-111 [laminin-1, laminin 1β11, Engelbreth-Holm-Swarm (EHS) laminin] in mice over 25 years ago, a large number of additional laminin isoforms with different tissue expression and biological properties have been found [8
9
10
]. Early studies describing laminin expression in lymphoid tissues and subsets of lymphocytes used polyclonal antibodies to Lm-111, which cross-react with most Lm isoforms or anti-Lm mAb of unknown or misassigned chain specificity [15
]. Moreover, early functional studies of lymphocytes and laminins examined the biological effect of Lm-111, which is neither synthesized by lymphoid cells nor expressed in lymphoid tissues [9
]. Alternatively, the studies used poorly characterized, commercial laminin preparations isolated from human placenta, which frequently consisted of large laminin fragments, a mixture of laminin isoforms, and/or contaminating proteins [16
].
More recently, mAbs of known laminin chain specificity and fully characterized, natural and recombinant (r) laminins have become available, as well as viable 2- and 4-laminin chain knockout (KO) mice [12
, 15
, 17
18
19
20
21
22
23
24
]. Studies with these tools have demonstrated a characteristic distribution of laminin isoforms in thymus and participation of 2-, 3-, and 5-laminins in adhesion, migration, proliferation, survival, and/or development of thymocytes and T cell malignancies [20
, 25
26
27
28
29
30
31
]. Moreover, adhesion and migration of neoplastic lymphocytes have been found to be promoted by 4- and 5-laminins [32
, 33
]. When compared with 1-laminin, which in adult life is highly restricted to a few epithelia, the latter laminins have a much wider tissue distribution and constitute the major vascular endothelial laminin isoforms [9
, 34
]. Although a few studies have described expression of laminin chains in LNs [12
, 13
, 35
, 36
], a systematic analysis of HEV laminins has not been reported nor has the effect of laminin isoforms covering all Lm chains, including Lm5, in the biology of normal blood lymphocytes. We recently described strong migration-promoting activities of 4-laminin (Lm-411, 4β11, laminin-8) on blood lymphocytes and neutrophils and impaired extravasation of neutrophils during peritonitis in Lm4 KO mice [24
, 37
].
As an attempt to determine the role of laminin isoforms in the extravasation and migration of blood lymphocytes, we have analyzed in the present study the expression of laminins in LNs, particularly in HEVs and RFs, by using a panel of mAb to all laminin chains, except Lm3, and confocal microscopy. With molecularly characterized, natural and recombinant laminins covering the five chains, we have determined the adhesion-, migration-, and costimulation-promoting activities of laminin isoforms on blood lymphocytes and the participation of laminin-binding integrins. Cell number of LNs from Lm4 KO mice and synthesis and secretion of 5-laminins by blood lymphocytes were also investigated.
MATERIALS AND METHODS
Cells and purified laminins
PBMCs were obtained from healthy donors after Ficoll-Hypaque gradient centrifugation (Amersham Pharmacia AB, Uppsala, Sweden) with approval of the local ethical committee and informed consent of the participating subjects. Lymphocytes were subsequently isolated by removing tissue-culture flask-adherent cells or by gradients of 60%, 47.5%, and 34% Percoll (Amersham Pharmacia AB) in complete medium (10% FCS in RPMI) with modifications of a method described previously [38
]. CD4+ T cells were obtained from PBMCs by positive selection using magnetic beads coated with anti-CD4 antibody according to instructions from the manufacturer (Dynal AS, Oslo, Norway).
Mouse Lm-111 (mLm-111, 1β11, laminin-1), isolated from the EHS tumor, and human merosin (Lm-211, 2β11, laminin-2) from placenta were obtained from Chemicon International (Temecula, CA, USA). Human Lm-332 (hLm-332; 3β32, laminin-5) was immunopurified from the conditioned medium of SCC25 cells [21
]. rhLm-211 (2β11, laminin-2), rhLm-411 (4β11, laminin-8), and rhLm-511 (5β11, laminin-10) were produced in a mammalian expression system and purified and characterized as described previously [17
, 18
, 22
]. Another 5-laminin preparation, Lm-511, was purchased from Sigma-Aldrich (St. Louis, MO, USA) as human laminin. This preparation, isolated from placenta by mild pepsin digestion and purified by immunoaffinity chromatography, was molecularly characterized and used only when reactive with mAb 4C7 to Lm5 chain globular domain [16
]. Human serum albumin (HSA) and plasma FN (pFN) were also obtained from Sigma-Aldrich.
Immunofluorescence and confocal laser-scanning microscopy (CLSM)
For immunofluorescence studies, four normal LN specimens were retrieved from the files of the Institute of Biomedicine/Anatomy and cut at 5 µm. The following mAbs, all mouse IgG, against laminin chains were used in frozen sections: 161EB7 (Lm1), 5H2 (Lm2), BM-2 (Lm3), 168FC10 (Lm4), 4C7 (Lm5), 114DG10 (Lmβ1), S5F11 (Lmβ2), 6F12 (Lmβ3), 113BC7 (Lm1), and D4B5 (Lm2 chain). These antibodies were detected with Alexa Fluor 594 goat anti-mouse IgG antibodies (Molecular Probes, Eugene, OR, USA). mAbs M3F7 (collagen type IV 1/2) and A9 (nidogen-1) were also tested. The source of the mAbs was described recently [39
]. For double-immunolabeling, mAb 3G4 against type III collagen (mouse IgM, Life Technologies, Gaithersburg, MD, USA) or mAb MECA-79 against sulfation-dependent, HEV-associated carbohydrate (rat IgM, BD Biosciences, Stockholm, Sweden) were used, together with Alexa Fluor 488 goat anti-mouse IgM (µ chain) antibody or Alexa Fluor 488 goat anti-rat IgM (µ chain), respectively. Tetramethylrhodamine-isothiocyanate-coupled Ulex Europaeus I-lectin (Vector Laboratories, Burlingame, CA, USA) was also used in double-immunostainings to identify blood vessel endothelia. Double-immunolabeling and CLSM were carried out as described recently [39
].
Immunofluorescence flow cytometry
Lymphocytes, gated from isolated PBMCs by forward- and side-scatter, were analyzed for cell surface (nonpermeabilized) and intracellular (permeabilized) expression of Lm chains as well as for cell surface expression of integrins and differentiation markers by immunofluorescence flow cytometry in a FACScan flow cytometer (Beckton Dickinson, Mountain View, CA, USA), as described previously [40
]. For integrin expression, mAb 6S6 (INTβ1; Chemicon), FB12 (INT1; Chemicon), P1E6 (INT2; Chemicon), P1B5 (INT3; Chemicon), BQ16 (INT6; Ancell Corp., Bayport, MN, USA), 9.1 (INT7) [41
], 69.6.5 (INTV; Beckman Coulter, Bromma, Sweden), IB4 (INTβ2) [24
] (generous gift from Prof. Samuel Wright, Merck Research Labs, Rahway, NJ, USA), H12 (INTL; kindly provided by Hans Wigzell, Karolinska Institutet, Stockholm, Sweden), 2LPM (INTM; Dakopatts, Copenhagen, Denmark), 3.9 (INTX; Harlan SeraLab, Loughborough, UK), SZ.21 (INTβ3; Beckman Coulter), SZ.22 (INTIIb; Beckman Coulter), LM609 (INTVβ3; Chemicon), ASC-9 (INTβ4; Chemicon), and P1F6 (INTVβ5; Chemicon) were used. For Lm chain expression, mAb 15H5 (Lm5) [42
] (kindly provided by Prof. Kiyotoshi Sekiguchi, Osaka University, Japan), LN-26 (Lmβ1; Takara Shuzo, Kyoto, Japan) [15
], C4 (Lmβ2; Neomarkers, Fremont CA, USA), and LN-82 (Lm1; Takara Shuzo) [15
] were used. mIgG and mAb 9.6 to CD2, a lymphocyte marker, were also included as negative and positive controls, respectively. Over 90% of the gated cells (lymphocytes) expressed CD2. INT, Lm, and CD2 antibodies, all mouse IgG, were detected with FITC-conjugated F(ab')2 fragments of rabbit anti-mouse Igs (Dakopatts), whereas lymphocyte populations were identified by using PerCP-labeled anti-CD4 (Th cells), APC-labeled anti-CD8 (cytotoxic T cells), and PE-labeled anti-CD19 (B cells) antibodies (BD Biosciences, San Diego, CA, USA). Integrin expression in different lymphocyte populations was investigated by multicolor immunofluorescence.
Cell adhesion, migration, and proliferation assays
Lymphocyte adhesion to laminin isoforms was performed as described previously [37
] with some modifications. Briefly, lymphocytes were isolated from PBMCs by Percoll density gradients and thereafter, resuspended in RPMI 1640 (2x106 cells/ml), and 96-well flat-bottom polystyrene plates (BD Biosciences, Stockholm, Sweden) were coated with 50 µl/well PBS or 20 µg/ml HSA, Lm-111, Lm-411, or Lm-511 in PBS for 3 h at 37°C. After rinsing with PBS, free sites were blocked with 2% polyvinylpyrrolidone (PVP; molecular weight 360 kDa; Sigma-Aldrich) for 1 h at room temperature, and after further washing, 2 x 105 cells/well (100 µl) were incubated in the presence of 200 nM tetradecanoyl phorbol acetate (TPA; also known as PMA, Sigma-Aldrich) at 37°C for 1 h. Following five washes, adherent cells were fixed and stained overnight. The plate was then washed, and adherent cells were quantified in a microplate reader. Transmigration of lymphocytes through protein-coated filters was measured microscopically and by flow cytometry as described before [37
]. Briefly, human PBMCs (5x105 containing 
70% lymphocytes) were added to the top chamber of 3 µm pore polycarbonate Transwell culture inserts (Costar, Cambridge, MA, USA) and incubated at 37°C for 3 h in the presence (chemokine-stimulated migration) of 500 ng/ml stromal cell-derived factor 1 (SDF-1; R&D Systems, Abingdon, UK). Thereafter, the number of cells in the lower chamber was measured microscopically, and the percentage of lymphocytes among the transmigrated was established by flow cytometry, according to forward- and side-scatter. Lymphocyte populations CD4+ (Th) cells, CD8+ (cytotoxic T) cells, and CD19+ (B) cells, before and after transmigration, were identified by immunofluorescence flow cytometry. The filters had been coated initially with proteins and blocked with PVP, as in the cell adhesion assay. To identify integrin receptors participating in cell adhesion and migration assays, isolated PBMCs were resuspended at 5 x 106 cells/ml in RPMI 1640 and incubated separately with 20 µg/ml mIgG (negative control) or function-blocking mAb IB4 (INTβ2), 60.1 (INTM) [24
] (kindly provided by Prof. Patrick Beatty, University of Utah, Salk Lake City, UT, USA), 6S6 (INTβ1; Chemicon), 13 (INTβ1; BD Biosciences, Stockholm), P1B5 (INT3; Chemicon), or GoH3 (INT6; BD Biosciences, Stockholm) at room temperature for 20 min. Cell migration in the presence of control IgG was defined as 100% migration. All mAbs, gift or commercial ones against INTs used in the functional assays, were protein A- or protein G-affinity chromatography-purified IgG. Proliferation of isolated CD4+ T cells (99% purity) was measured as 3H-thymidine incorporation after 4 days stimulation, as described previously [37
]. Immobilized mAb UCHT1 to CD3 (BD Biosciences, Stockholm) was tested in the absence or presence of HSA or various laminin isoforms. For statistical analysis, Student’s t-test, mean, and SD values were calculated, and the level of significance (*, P<0.05; **, P<0.01; ***, P<0.001) was determined by comparing laminin isoforms with HSA or mAb with mIgG.
Flow cytometric determination of LN cell number in Lm4-deficient mice
Lm4 KO mice were generated by gene targeting in embryonic stem cells, as described previously [23
]. Wild-type (WT; +/+) and KO (–/–) adult male mice were used to determine the cell number in peripheral LN. Animals were killed by cervical dislocation, and cells were isolated from inguinal LNs by using a mesh iron screen and a 70-µm cell strainer. Total isolated cells resuspended in 0.3 ml PBS were quantified by flow cytometry using a FACSort (BD Biosciences, San Jose, CA, USA) and identified according to forward- and side-scatter characteristics. All experiments were approved by the regional ethical committee for animal experimentation.
RT-PCR
RT-PCR was performed as described previously [37
] with a few modifications. Total RNA was extracted from blood lymphocytes by using the Ribopure TM-WBC kit (Ambion Europe Ltd., Huntingdon, UK). RT and PCR conditions and primers for Lm5, Lmβ1, Lmβ2, and Lm1 have been described [37
, 40
].
Gel electrophoresis, immunoblotting, ECL, and purification of laminin from cell lysate by immunoaffinity chromatography and induction of lymphocyte secretion
Cell lysate from 1 x 109 isolated blood lymphocytes was obtained as described previously [37
], and laminin was purified from it by immunoaffinity chromatography with mAb 4C7 (Lm5; kindly provided by Eva Engvall, Burham Institute, La Jolla, CA, USA), which had been coupled to cyanogen bromide-activated Sepharose 4B (Amersham Pharmacia AB). Following extensive washing, the protein bound to the column was eluted using high pH, and the samples were collected in neutralizing buffer, as reported previously [24
, 37
]. To study laminin secretion, isolated lymphocytes were resuspended at 5 x 107 cells/ml in plain RPMI 1640. After preincubation for 15 min, the cells were stimulated with 200 nM TPA for 20 min at 37°C. Following addition of protease inhibitors and centrifugation of the cells, supernatant was collected and analyzed for secreted laminin. After concentration, the purified and secreted materials were separated by SDS-PAGE, and blotted filters were blocked with 0.1% Tween 20/5% dry, skimmed milk in PBS. mAb 6C3 (Lm4) [24
], 15H5 (Lm5), DG10 (Lmβ1), C4 (Lmβ2), and 22 (Lm1; BD Biosciences, Stockholm) were used as immunoblotting mAbs. mIgG (BD Biosciences, Stockholm) was included as a negative control. Peroxidase-conjugated anti-mIg (Dakopatts) was added as secondary antibody, and ECL (Amersham Pharmacia AB) was used as a developer.
RESULTS
Expression of laminins in HEVs and RFs
We studied first the localization of Lm chains in normal human LNs by CLSM in HEVs (Fig. 1
). Lm1 chain immunoreactivity (B) was broad in the basal aspect of HEVs, visualized with mAb MECA-79 (A), as demonstrated by a variable yellow-red color in the merged figure (C). Immunoreactivity for Lm1 chain was also found in BM of ordinary blood vessels (B) and in fibril-like structures outside of the vessels. Merged figures for HEV and Lm1 (D) and Lm2 (E) chains, respectively, showed that these components were not found in BM of HEVs or other vessels but that the Lm2 chain was present in stromal fibers. In contrast, immunoreactivity for Lm3 (F), Lm4 (G), Lm5 (H), Lmβ1 (I), and Lmβ2 (J) chains located variably to the BM region of practically all HEVs and other blood vessels and to stromal fibers. Instead, immunoreactivities for Lmβ3 (K) and Lm2 (L) chains were confined solely to capillaries and fibers in the germinal center area. Under the present experimental conditions, neither apical staining nor double-layered BM could be observed in HEVs with the Lm antibodies. Immunoreactivity of Lm chains in ordinary blood vessels could be confirmed by double-immunoreaction with Ulex europaeus I lectin-conjugate (data not shown).
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Figure 1. Expression of Lm chains in LN HEVs. HEVs (green), defined by MECA-79-positive reactivity, and Lm chains (red), examined with the corresponding mAb, were analyzed in LNs by confocal microscopy. Following merging, colocalization was revealed by yellow staining. (A) mAb MECA-79 alone; (B) mAb 113BC7 to Lm1 alone; (C) merge of A plus B; and (D–L) Merge of MECA-79 with mAbs to different Lm chains: (D) mAb 161EB7 to Lm1; (E) mAb 5H2 to Lm2; (F) mAb BM-2 to Lm3; (G) mAb 168FC10 to Lm4; (H) mAb 4C7 to Lm5; (I) mAb 114DG10 to Lmβ1; (J) mAb S5F11 to Lmβ2; (K) mAb 6F12 to Lmβ3; and (L) mAb D4B5 to Lm2. HEVs expressed Lm3, -4, -5, -β1, -β2, and -1 chains and lacked reactivity with antibodies to Lm1, -2, -β3, and -2 chains. In contrast to HEVs, germinal center vessels expressed Lm2 staining (L). Original scale bar (100 µm) in L applies to all pictures.
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1 chain showed a rather similar localization (B), and the merged figure (C) showed an extensive colocalization for these proteins in the fiber-like structures. Immunoreactitivity of the Lm1 chain was not found in the LNs (D), and immunoreactivities for Lm2 (E), Lm3 (F), Lm4 (G), Lm5 (H), Lmβ1 (I), and Lmβ2 (J) chains showed an extensive colocalization with that of type III collagen. Immunoreactivities for Lmβ3 (K) and Lm2 (L) chains were found in colocalization with type III collagen fibers only in the germinal center area. Laminin expression in HEVs and RFs of tonsils was similar to the one of LNs (data not shown). Collagen type IV 1/2 and nidogen-1 were located in all BMs, including those of HEVs, and were also present in RFs (data not shown).
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Figure 2. Expression of Lm chains in LN RFs. RFs (green), defined by type III collagen-positive immunoreactivity, and Lm chains (red), examined with the corresponding mAb, were analyzed in LNs by confocal microscopy. Following merging, colocalization was revealed by yellow staining. (A) mAb 3G4 against type III collagen alone; (B) mAb 113BC7 to Lm1 alone; (C) Merge of A plus B; and (D–L) Merge of mAb 3G4 with mAbs to different Lm chains: (D) mAb 161EB7 to Lm1; (E) mAb 5H2 to Lm2; (F) mAb BM-2 to Lm3; (G) mAb 168FC10 to Lm4; (H) mAb 4C7 to Lm5; (I) mAb 114DG10 to Lmβ1; (J) mAb S5F11 to Lmβ2; (K) mAb 6F12 to Lmβ3; and (L) mAb D4B5 to Lm2. RFs expressed Lm2, -3, -4, -5, -β1, -β2, and -1 chains and lacked reactivity with antibodies to Lm1, -β3, and -2 chains. Staining of germinal center vessels with mAb to Lmβ3 and -2 chains was also observed in K and L, respectively. Original scale bar (100 µm) in L applies to all pictures.
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6, -β1, -L, -M, and -β2 chains. A low expression of INT1, -3, and -Vβ3 was also noted. INT2, -7, -IIb, -β4, and -Vβ5 were minimally expressed or not detected. By using multicolor immunofluorescence flow cytometry, expression of the "classical" laminin-binding integrins 6β1 and 3β1 by the blood lymphocytes was further investigated. INT6 was found to be expressed by the majority of CD4+ (Th) cells (77.7%) and CD8+ (T cytotoxic) cells (78.0%) but only by a few CD19+ (B) cells (6.3%). INTβ1 was detected in almost all CD4+ cells (87.1%) and CD8+ cells (95.5%) but only on half of the CD19+ cells (49.6%), whereas a low percentage of CD4+ cells, CD8+ cells, and CD19+ cells expressed INT3 (16.1%, 15.1%, and 10.8%, respectively; mean value of percentage of positive cells from three donors).
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Figure 3. Expression of laminin-binding integrins in blood lymphocytes. Flow cytometry analysis of integrin expression on lymphocytes: mIgG (open peak) and mAbs (shaded peak) 6S6 (INTβ1), FB12 (INT1), P1E6 (INT2), P1B5 (INT3), BQ16 (INT6), 9.1 (INT7), 69.6.5 (INTV), IB4 (INTβ2), H12 (INTL), 2LPM (INTM), 3.9 (INTX), SZ.21 (INTβ3), SZ.22 (INTIIb), LM609 (INTVβ3), ASC-9 (INTβ4), and P1F6 (INTVβ5). The vertical axis shows the cell number, and the horizontal axis shows the log fluorescence intensity [fluorescence 1-height (FL1-H)]. Mean values of mean fluorescence intensity and percentage of positive cells (%) of three experiments were as follows: mIgG: 4.3 (0%), INTβ1: 60.4 (86.9%), INT1: 8.6 (28.0%), INT2: 5.4 (3.9%), INT3: 9.0 (29.0%), INT6: 29.9 (72.6%), INT7: 4.8 (1.7%); INTV: 6.0 (4.9%), INTβ2: 302.0 (92.9%), INTL: 214.4 (92.7%), INTM: 35.7 (34.3%), INTX: 5.9 (7.7%), INTβ3: 5.6 (3.4%), INTIIb: 5.3 (1.6%), INTVβ3: 6.6 (12.4%), INTβ4: 5.0 (1.1%), and INTVβ5: 5.1 (0.5%).
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6β1 integrin1 chains but differ in their chain. Natural mLm-111 (EHS-laminin), human pFN, and HSA were also used. The constitutive cell adhesion to all proteins was minimal, if any (data not shown). However, following stimulation of the cells with phorbol ester for 1 h, the laminin isoforms were more adhesive than HSA, and Lm-511 was more active than Lm-411, followed by Lm-111 (P<0.01; Fig. 4A
). Correlation of OD values of the adhesion assay with the cell number indicated that nearly 23% of the blood lymphocytes adhered on Lm-511. Under similar experimental conditions, IL-2 (at 1, 5, and 25x103 units/ml) and SDF-1 [at 0.5, 2.5, and 8 µg/ml (1 µM)] were unable to induce lymphocyte adhesion (data not shown). Lm-211 and Lm-332 were less adhesive than Lm-111 and hence, poorly active (data not shown). When compared with 1 h, lymphocyte adhesion on Lm-511 slightly increased at 3 h but drastically decreased at 12 h (data not shown). To identify the adhesive receptor, blocking mAbs to INTβ2 (IB4), INTβ1 (6S6), and INT6 (GoH3) were tested on lymphocyte adhesion to Lm-111, Lm-411, Lm-511, and pFN. Statistically significant inhibition (P<0.01; Fig. 4B
) was obtained with mAb GoH3 and 6S6 on Lm-411 and Lm-511 only, indicating a role of 6β1 INT as a receptor for both vascular laminin isoforms. Although mAb IB4 to INTβ2 was partially inhibitory on all substrata, including HSA, its effect alone did not reach statistical significance. Of INTs analyzed in Figure 3
, cell-surface expression of Mβ2 was almost doubled by the TPA treatment, whereas expression of all other INTs was slightly reduced or not affected (data not shown).
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Figure 4. Adhesion of isolated blood lymphocytes to Lm-511 and to other laminin isoforms and participation of integrin. (A) Adhesion of isolated blood lymphocytes to plastic surfaces coated with HSA, Lm-111, Lm-411, rLm-511, and pFN in the presence of phorbol ester (200 nM TPA). Adhesion was measured after incubating the cells (200x103 cells/well) in the coated plates for 1 h at 37°C and shown as OD. Plastic surfaces were coated with proteins at 20 µg/ml and blocked with 2% PVP. (B) Effect of mAbs to integrins on lymphocyte adhesion to plastic surfaces coated with HSA, Lm-111, Lm-411, rLm-511, and pFN. mIgG (negative control), mAb IB4 (INTβ2), mAb 6S6 (INTβ1), and mAb GoH3 (INT6). Adhesion-blocking mAbs were used at 20 µg/ml. Mean and SD of four to six experiments are shown. P values (*, P<0.05; **, P<0.01) compare (A) laminin isoforms with HSA and (B) mAbs with mIgG on each laminin isoform.
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6β1 integrin as a primary laminin receptor as a chemoattractant (Fig. 5A
; P<0.01 and P<0.001, respectively). The level of migration on rLm-511 was by far higher than that on Lm-111, rLm-411, and pFN, and the difference with rLm-411, the other major endothelial laminin isoforms [34
], was highly significant (P<0.001). In a second group of experiments, placenta Lm-511 was tested and compared with Lm-111, rLm-211, Lm-332, and rLm-411. This natural Lm-511 was also found to strongly promote migration of blood lymphocytes (Fig. 5B
; P<0.001) and to a higher level than the other laminin isoforms. Natural and rLm-511 promoted migration of 80–100 x 103 lymphocytes, which correspond to 23–24% of the initial lymphocyte population (i.e., 70% of 500x103 PBMCs). Natural Lm-211 (merosin) behaved similarly to rLm-211 (data not shown). In decreasing order, the migration-promoting activity of the various laminin isoforms could be summarized as follows: Lm-511 > Lm-332 > Lm-411 > Lm-111 > Lm-221, indicating that Lm-511 predominantly promotes migration of blood lymphocytes compared with other laminin isoforms. Interestingly, Lm-511 was also more migration-promoting than pFN. Analysis of lymphocyte populations by immunofluorescence flow cytometry before and after transmigration demonstrated that CD4+ (Th) cells and CD8+ (cytotoxic T) cells but not CD19+ (B) cells readily migrated on Lm-511, as indicated by a shift in the percentage of CD4+ cells, CD8+ cells, and CD19+ cells from 34.6%, 23.9%, and 12.9% to 49.6%, 23.2%, and 1.8%, respectively (mean value of three different donors). Whereas 12.4% of CD4+ cells (15,168 out of 122,688) and 8.4% of CD8+ cells (7093 out of 84,575) migrated on Lm-511, only 1.3% of CD19+ cells (537 out of 45,785) migrated on this laminin isoform. The effect of function-blocking mAbs to integrins indicated that migration of blood lymphocytes on Lm-511 was largely mediated by 6β1 integrin, as antibodies to INT6 and INTβ1 chains exerted a major and highly statistically significant inhibition (Fig. 5C)
. Antibodies to INT3 alone were inactive, but together with INT6 antibodies, they exerted 10% more inhibition than INT6 antibodies alone, suggesting a small contribution of 3β1 integrin (data not shown). On Lm-411, mAb to INT6 were also inhibitory. In contrast, mAb to β2 INT exerted partial inhibition on both laminin isoforms, but the effect was not statistically significant. On Lm-332, mAbs to INT3 and INTβ1 inhibited significantly, as well as mAbs to INTM and INTβ2, indicating participation of 3β1 and Mβ2 integrins in the cell migration (Fig. 5D)
. On the other hand, antibodies to INT6 were modestly inhibitory, and their effect did not reach statistical significance (Fig. 5D)
.
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Figure 5. Lm-511 strongly promotes migration of blood lymphocytes via 6β1 integrin and costimulates T cell proliferation. (A–D) Cell migration assay; (E) T cell proliferation assay. Lymphocyte transmigration through 3 µm pore filters precoated with proteins was measured after 3 h incubating. (A) HSA, Lm-111, Lm-411, rLm-511, and pFN. (B) HSA, Lm-111, Lm-211, Lm-332, Lm-411, and Lm-511 (placenta). (C) Effect of blocking mAbs to integrins on lymphocyte transmigration through filters precoated with Lm-411 and rLm-511. mIgG (negative control), mAb IB4 (INTβ2), mAb 6S6 (INTβ1), and mAb GoH3 (INT6). (D) Effect of blocking mAbs to integrins on lymphocyte transmigration through filters precoated with Lm-332. mIgG, mAb P1B5 (INT3), mAb GoH3 (INT6), mAb 13 (INTβ1), mAb IB4 (INTβ2), and mAb 60.1 (INTM). (E) Proliferation of isolated CD4 T cells on immobilized mAb CD3 alone, laminin alone, or in combinations: CD3 (mAb UCHT1), HSA, Lm-111, Lm-411, rLm-511, Lm-511 (placenta). Cell proliferation was measured by H3-thymidine incorporation after culturing cells for 4 days. In all figures, mean and SD of at least four experiments are shown. P values (*, P<0.05; **, P<0.01, ***, P<0.001) compare laminins with HSA (A, B, and E) or mAb with mIgG, defined as 100% in all experiments (C and D).
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LN cell number in Lm4-deficient mice compared with WT does not differ significantly
As an initial approach to investigate the role of laminins in lymphocyte biology in vivo, the total cell number of inguinal LNs was compared in Lm4-deficient and WT mice. This parameter appears to be influenced by entrance/exit and proliferation/survival of lymphocytes. Mean value and SD of the total cell number were 405 ±115 (x103) and 464 ±186 (x103) for Lm4-deficient and WT mice, respectively. This difference was not statistically significant (P=0.67). Six animals were tested in each group. Histological examination of Peyer’s patches, a lymphoid tissue that is highly exposed to antigenic stimulation, demonstrated that Lm4-deficient mice retained the ability to develop germinal centers (data not shown).
Blood lymphocytes contain and, following stimulation, secrete Lm- 511 and Lm-521
To investigate expression of 5-laminins by blood lymphocytes, the presence of mRNAs encoding for Lm-511 and Lm-521 chains was first studied by RT-PCR, using pairs of primers based on the reported cDNA sequences of hLm5, -β1, -β2, and -1 chains. Amplified products with the expected size were detected with primers for 5 (655), β2 (632), and 1 (687) after 30 cycles of PCR with reverse-transcribed transcripts from blood lymphocytes (Fig. 6A
). Results for β1 transcripts were inconclusive (data not shown).
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Figure 6. Blood lymphocytes contain Lm-511 and Lm-521 and secrete 4- and 5-laminins. (A) RT-PCR of Lm-521 components in isolated lymphocytes. Amplification of Lm5, -β2, and -1 cDNA fragments by RT-PCR of total RNA. GAPDH and buffer were used as positive and negative controls, respectively. (B) Detection of Lm5, -β1, -β2, and -1 chains in blood lymphocytes by immunofluorescence flow cytometry. Reactivity of mAb with nonpermeabilized and permeabilized cells is shown. mIgG (shaded peak) and mAbs (open peak): 9.6 (CD2), 15H5 (Lm5), LN-26 (Lmβ1), C4 (Lmβ2), and LN-82 (Lm1). CD2 was used as a lymphocyte marker. (C) Western blot analysis of Lm5 mAb (4C7) immunoaffinity-purified protein from isolated blood lymphocyte lysate (upper panel) and of total secreted material from isolated blood lymphocytes stimulated with 200 nM TPA for 20 min (lower panel). Name of the antibodies and their specificity (in parentheses) are shown. Arrows indicate bands of 350, 230, 220, and 190 kDa, corresponding to Lm5, -β1, -1, and -β2, respectively (upper panel), and bands of 300/280, 230, 220, 190, and 180, corresponding to Lm5, -β1, -1, -β2, and -4 chains, respectively (lower panel). *, A band of approximately 200 kDa reactive with mAb 15H5 (Lm5), which was observed occasionally. Proteins were separated by SDS gel electrophoresis under reducing conditions (5% acrylamide).
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5-laminins in blood lymphocytes, immunofluorescence flow cytometry was performed. Lymphocytes, gated by forward- and side-scatter from PBMCs, displayed reactivity with mAb to CD2, a marker of most lymphocytes. mAb 15H5 (Lm5), LN-26 (Lmβ1), C4 (Lmβ2), and LN-82 (Lm1) did not react, or only minimally, with intact lymphocytes, whereas practically all cells were stained with the antibodies following cell permeabilization (Fig. 6B)
.
To further investigate expression of 5-laminins, the cell lysate of isolated blood lymphocytes was immunopurified on a Lm5 (4C7) antibody column, and the purified material was then analyzed by Western blotting. This technique allows identification of Lm chains and demonstrates their physical association. Under reducing conditions, mAb DG10 (Lmβ1), C4 (Lmβ2), 22 (Lm1), and 15H5 (Lm5) recognized polypeptides of 230, 190, 220, and 350 kDa, respectively (Fig. 6C
, upper panel). The intensity of Lmβ1 and -β2 bands suggested that Lm-521 was more abundant than Lm-511. In the total supernatant of blood lymphocytes stimulated with phorbol ester for 20 min, Lmβ1, -β2, and -1 were also detected, in addition to the Lm4 chain (Fig. 6C
, lower panel), and Lm5 appeared as smaller 300/280 kDa bands (Fig. 6C
, lower panel). Occasionally, an additional band (*) of approximately 200 kDa, reactive with mAb 15H5 (Lm5), was observed in the secreted material, suggesting further proteolytic cleavage.
DISCUSSION
In the present study, laminins of HEVs and RFs of lymphoid tissue and their integrin receptors in blood lymphocytes are identified, and the effect of laminin isoforms covering all chains on the lymphocytes is described. Moreover, a strong and predominant, migration-promoting effect of 5-laminins on blood lymphocytes, particularly T cells, is reported for the first time, as well as synthesis and secretion of 5-laminins by lymphocytes.
In spite of its apparent relevance to lymphocyte recirculation, the role and composition of HEV BMs are poorly understood. Early morphological studies reported a thick, dense, and strongly negatively charged BM, distinct from that of flat endothelium [44
45
46
]. Our confocal microscopy studies suggested the expression of Lm-311/321, Lm-411/421, and Lm-511/521 by the HEV BM. Neither Lm-332 nor 1- or 2-laminins were detected in these vessels. As all HEVs expressed the same Lm chains, each HEV BM should contain simultaneously at least three different laminin isoforms. The HEV BMs may provide an adhesive and migration-promoting substrate for the lymphocytes, as demonstrated for laminins in the present study. Moreover, the laminins may also be able to bind and present chemokines and other cytokines, as reported for other BM proteins [5
]. In contrast to Lm4 and -5, Lm3 is rarely found in blood vessels, as only vasculatures of lymphoid and a few other tissues express this Lm chain [28
, 34
] (to be published). Among the lymphoid vessels, selective staining of the capillaries in the lymphoid follicles by antibodies to β3 and 2 Lm chains indicated the presence of Lm-332 in this vasculature but not in HEVs. Others [35
] have reported similar results. Expression of Lm5 by LN blood vessels, including HEV, has been described in mice [47
], and broad staining of human LN vasculature, without specifying HEVs, with antibodies to Lm3, -4, and -5 chains has been reported recently [12
].
As major components of the reticular network, RFs connects the subcapsular sinus to HEVs in lymphoid tissues, allowing the transport of small molecules from the afferent lymph in a conduit system [13
, 14
, 36
]. With a core of collagen types I and III and surrounded by BM components, these fibers are largely, but not fully, ensheathed by fibroblastic reticular cells [11
, 13
, 36
]. Nearly 10% of the surface of these interfollicular fibers appears to be accessible to migratory cells, allowing physical interaction with lymphocytes, dendritic cells, and macrophages [7
]. In the present study, the confocal microscopy suggested expression of Lm-211/221, Lm-311/321, Lm-411/421, and Lm-511/521, but not 1-laminins, by the RFs. Lack of reactivity with mAb to β3 and 2 Lm chains indicated that RFs in LNs, in contrast to thymus [48
], do not express Lm-332. Although laminins appear to be more accessible to lymphocytes in HEV BMs than in RFs, laminins and other outer components of the RFs may still function, not only as cell-adhesive substrates but also as migration-promoting proteins for lymphocytes and other migrating immune cells. Others [12
, 13
, 36
] have also reported expression of Lm2, -3, -4, and -5 but not -1 chains by LN RFs.
Among laminin-binding integrins expressed by blood lymphocytes, 6β1 was the most abundant, followed by 1β1, 3β1, and Vβ3 at much lower levels. Analysis of lymphocyte populations indicated that 6β1 integrin was expressed by most T cells, including CD4+ cells and CD8+ cells, and that most B cells lacked this integrin, and integrin 3β1 was expressed by a minority of cells in each lymphocyte population. Integrin Mβ2, which has been proposed to be a laminin receptor [24
], was detected in half of the lymphocyte population. NK cells, CD8+ T cells, and B cells are known to express this integrin [49
]. In functional assays of the present study, laminins were adhesive for blood lymphocytes after stimulation of the cells with TPA. 5-laminin was the most active isoform, and vascular 4- and 5-laminins used the 6β1 integrin as a primary receptor with some contribution of (M)β2 integrin. Through protein kinase C activation and protein phosphorylation, TPA may induce integrin activation by cytoskeleton-mediated clustering of cell membrane integrins and/or conformational changes of these adhesive receptors. Mβ2 integrin may be responsible for the relatively high, "nonspecific" lymphocyte adhesion, as reported for neutrophils [24
]. The extent of adhesion of stimulated lymphocytes to HSA, heat-denatured HSA, and OVA was similar (data not shown). When compared with our early study that focused on the interaction of lymphocytes with Lm-411 [37
], the present adhesion results with stimulated cells were largely similar, but the adhesion to Lm-411, although higher than on HSA and lower than on Lm-511, did not reach statistical significance. Moreover, specific adhesion of the cells to laminins was undetectable in the absence of stimulus. These differences may be explained by the use of isolated blood CD4 T cells in the early study, in contrast to total blood lymphocytes (Percoll-isolated) in the present study. The later preparation contains, in addition to CD4+ T cells, NK cells, CD8+ T cells, and B cells, which in contrast to CD4+ T cells, express considerable amounts of Mβ2 integrin [49
]. Participation of this integrin apparently decreased the adhesive ratio between laminins and HSA.
A predominant, cell-adhesive property of 5-laminin, when compared with other isoforms, has also been reported for hematopoietic cell lines, encephalitogenic T cell lines, malignant lymphoid cells, a subpopulation of thymocytes, and bone marrow progenitor cells and was similarly mediated via 6β1 integrin [27
, 33
, 50
51
52
]. Correspondingly, migration of bone marrow progenitor cells and malignant lymphoid cells was higher on 5-laminin than on other laminin isoforms via the same integrin receptor [32
, 50
]. In our present study, the migration-promoting activity of 5-laminin on blood lymphocytes was superior to that of 1-, 2-, 3-, and 4-laminins, as well as to that of FN, probably the most extensively used ECM protein in lymphocyte studies. Interestingly, analysis of lymphocyte populations demonstrated that the CD4+ cells and the CD8+ cells, but not the B cells, readily migrated on Lm-511, suggesting a migration-promoting activity of this laminin isoform preferentially on T cells. Studies in progress investigate the effect of various laminin isoforms in several lymphocyte populations and the contribution of integrins. In our early Lm-411/lymphocyte study [37
], a commercial placenta laminin preparation, referred to as LN-10/11 (Lm-511/521), was used for comparison and found not to promote lymphocyte migration. We did not molecularly characterize the batches of this preparation. Recent studies from our group have demonstrated major molecular and functional heterogeneity among different batches of the same commercial laminin product [16
]. In the present study, only those batches reactive with a mAb 4C7 to Lm5 globular domain [16
], which is recognized by 3β1 and 6β1 integrins [53
], were used as Lm-511. These molecularly characterized preparations behaved similarly to rLm-511 in the cell migration assays.
The statistically significant, inhibitory effect of mAb to INT6 on the adhesion and migration of the lymphocytes on Lm-411 and Lm-511 and a similar effect of mAb to INTβ1 identified 6β1 integrin as a primary receptor for these vascular laminins. Interestingly, expression of this integrin on blood CD4+ (Th) cells, CD8+ (cytotoxic T) cells, and CD19+ (B) cells roughly correlated with the ability of these lymphocyte populations to migrate on Lm-511. Moreover, the additive effect of mAb to INTβ2 suggested an additional contribution of the leukocyte integrins. Treatment of rats with rabbit antibodies to a rat laminin has been found to decrease the accumulation of adoptively transferred lymphocytes in peripheral LNs [54
]. The role of laminins in lymphocyte migration in vivo is similarly supported by studies with mice lacking 6A INT, the laminin-binding integrin splice variant expressed by the immune cells [55
]. In these INT6A KO mice, lymphocytes displayed decreased migration on a 1-laminin substrate and a reduction in the number of T cells isolated from peripheral LNs. However, lymphocyte homing to the LNs, which involves several receptor-ligand interactions, was not affected in these animals. As a classical laminin receptor, 6β1 integrin binds laminins covering all chains but with some preference for 5-laminin [53
].
Antigen-specific activation of T cells is known to require not only CD3/TCR engagement by antigen/MHC but also additional costimuli. In a previous study, we showed a synergistic effect of Lm-411 and a placenta Lm-511 preparation together with mAb to CD3 to drive CD4+ T cells to proliferate, in line with previous reports [1
, 37
]. In the present study, we reproduced these findings and observed, in addition, a stronger, synergistic effect of Lm-511 when compared with Lm-411. The higher costimulatory effect of placenta Lm-511, when compared with rLm-511, may be a result of in vivo processing of the natural form. Inasmuch as T cells normally recognize antigen present on cells (with MHC molecules) rather than on ECMs, it is unlikely that laminins may exert such an effect as ECM components. Instead, laminins may be costimulatory to T cells when exposed on the surface of monocyte/macrophages and other APCs. In accordance, we recently found the presence of 4- and 5-laminins in monocytes, and some of these moieties were localized on the cell surface [43
] (to be published). In contrast to other laminins, Lm-332, which is expressed by the thymus, has been described not to be comitogenic to thymocytes [31
]. On the other hand, this isoform was reported to promote thymocyte migration via 3β1 integrin and to be poorly adhesive for blood lymphocytes, which expressed low amounts of the latter integrin [28
, 30
]. In the present study, Lm-332 was similarly poorly adhesive for blood lymphocytes but significantly promoted migration of these cells via 3β1 and Mβ2 integrins. Recently, we reported strong migration-promoting activity of Lm-332 and Lm-511 in glioma cells via 3β1 integrin [56
]. It should be noted that we use Lm-332 as a source of 3-laminin, as Lm-311/321 are not available. Integrins 3β1, 6β1, and 6β4 are known to bind the Lm globular tandem of the Lm3 chain [9
], which may also be important for the interaction of extravasated lymphocytes with epithelial BMs [28
].
When compared for adhesion, migration, and costimulation, rLm-511 was more active than rLm-411. As both laminin isoforms share β1 and 1 chains, their different biological effects should be ascribed to their distinct chain. Lm 5, the last chain to be identified, is the most widely distributed chain and is highly expressed by epithelia and endothelia [57
]. It has an apparent molecular weight of 350 kDa, in contrast with the shorter 200 kDa Lm4 chain, and it is recognized by 3β1, 6β1, and 6β4 integrins [53
]. Interestingly, its vascular expression appears to be developmentally regulated, as it is widely expressed in blood vessels in adult life [34
, 47
]. Lm5 genetic deletion leads to death late in embryogenesis, probably as a result of defects in the placental vasculature and other developmental abnormalities [58
]. In contrast, Lm4 KO mice survive, in spite of transient hemorraghes at birth, which reflect impaired microvessel maturation [23
]. Although we have previously reported defective recruitment of neutrophils in a peritonitis model in Lm4 KO mice [24
], we were unable to detect significant alterations in the LN cell number of these animals in the present study. Considering that lymphocyte trafficking (homing and egress), proliferation, and survival in the lymphoid tissue may all affect the LN cell number, this result does not support a pivotal role for 4-laminins in these lymphocyte activities. It may, instead, suggest a major role of 5-laminins.
A recent study showing colocalization of extravasating mononuclear leukocytes with Lm4- but not Lm5-expressing brain blood vessels in experimental autoimmune encephalitis in mice has led to the assumption that the 4-laminin promotes cell extravasation, whereas 5-laminin is nonpermissive and even inhibitory [52
]. A more recent study of normal and pathological (multiple sclerosis) human brain has not confirmed this exclusive expression of 4- or 5-laminins by brain vessels but demonstrated instead simultaneous expression of the two isoforms by the vasculature and accumulation of leukocytes around vessels expressing Lm5 [59
]. Whereas the role of vascular laminin isoforms may differ in inflammatory and homeostatic lymphocyte extravasation, our results do not support the concept of 5-laminins as a nonpermissive or inhibitory substrate for blood lymphocyte migration. On the contrary, the functional studies, the laminin isoform recognition by 6β1 integrin, and the isoform expression by HEVs strongly suggest a predominant role of 5-laminins in lymphocyte extravasation.
We have described previously synthesis and secretion of 4-laminin, as Lm-411, by lymphoid cells [37
]. Now we report, for the first time in leukocytes, the presence of 5-laminin, mainly as Lm-521 with some Lm-511, in blood lymphocytes and secretion of 5-laminin following stimulation of the cells. As recently reported by us for platelets [40
], the Lm5 chain associates more to the Lmβ2 chain than to the Lmβ1 chain. The weak Lm bands obtained from the blood lymphocytes by RT-PCR, which may reflect a low amount of transcripts present in these resting cells, suggest synthesis of 5-laminins. Immunofluorescence flow cytometry indicated that practically all blood lymphocytes expressed 5-laminins but at relatively low amounts and mostly intracellularly. 4- and/or 5-laminins may correspond to the reactivity of polyclonal anti-Lm-111 antibodies with mouse, rat, and human NK cells demonstrated in early studies [37
]. The function of the endogenous laminins of lymphocytes is presently unknown. Interestingly, 4- and 5-laminins were secreted following stimulation of the cells, and the larger Lm5 chain appeared to suffer proteolytic cleavage. In the absence of BM and RF laminins, laminin secreted and deposited by activated lymphocytes may be used for cell migration. On the other hand, laminins expressed on the lymphocyte cell surface may allow interaction of activated lymphocytes with other cells. Studies in progress address these issues.
Altogether, the present results suggest that during lymphocyte extravasation into secondary lymphoid tissues, 5-laminins and to a lower extent, 3- and 4-laminins found in HEV BM contribute to lymphocyte migration through the vessel wall. In the RFs, these laminins may constitute pathways for the migration of lymphocytes and other cell types within the lymphoid tissue. This principle may also apply to HEV-like vessels found in chronically inflamed, nonlymphoid tissues of patients with rheumatoid arthritis and other inflammatory diseases. On the other hand, HEV and RF laminins may also contribute to the extravasation of cancer cells and the migration of these cells throughout the lymphoid tissue, respectively, promoting hematogenous and lymphatic dissemination of cancer. In the absence of exogenous laminins, stimulated lymphocytes may secrete, use, and provide 5-laminins. When compared with other laminin isoforms and ECM proteins, 5-laminin appears to exert the strongest migration-promoting activity for blood lymphocytes, particularly T cells.
ACKNOWLEDGEMENTS
This research work was supported by the Swedish Cancer Society and Karolinska Institutet (M. P.) and Erifyisvaltioapu research grants from the Helsinki University Hospital (I. V.). The authors thank Drs. Eva Engvall, Kiyotoshi Sekiguchi, Samuel Wright, Patrick Beatty, Sergei Smirnov, and Peter Yurchenco for providing mAb and recombinant proteins, Dr. Björn Rozell for histological examination of mouse lymphoid tissues, and Ms. Ingegerd Andurén and Mr. Hernán Concha for excellent technical assistance.
Received January 18, 2008; revised May 5, 2008; accepted May 5, 2008.
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