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The Journal of Immunology, 2000, 165: 5831-5838.
Copyright © 2000 by The American Association of Immunologists

Monocytic Cells Synthesize, Adhere to, and Migrate on Laminin-8 ({alpha}4{beta}1{gamma}1)1

Claudio Pedraza*, Tarekegn Geberhiwot*, Sulev Ingerpuu{ddagger}, Daniel Assefa*, Zenebech Wondimu*, Jarkko Kortesmaa{dagger}, Karl Tryggvason{dagger}, Ismo Virtanen§ and Manuel Patarroyo2,*

* Microbiology and Tumor Biology Center, and {dagger} Division of Matrix Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden; {ddagger} Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia; and § Institute of Biomedicine, Department of Anatomy, University of Helsinki, Helsinki, Finland


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Laminins, a growing family of large heterotrimeric proteins with cell adhesive and signaling properties, are major components of vascular and other basement membranes. Expression, recognition, and use of laminin isoforms by leukocytes are poorly understood. In monoblastic THP-1 cells, transcripts for laminin {gamma}1-, {beta}1-, and {alpha}4-chains were detected by RT-PCR. Following immunoaffinity purification on a laminin {beta}1 Ab-Sepharose column, laminin {beta}1- (220 kDa), {gamma}1- (200 kDa), and {alpha}4- (180/200 kDa) chains were detected by Western blotting in THP-1 cells and in two other monoblastic cell lines, U-937 and Mono Mac 6. After cell permeabilization, a mAb to laminin {gamma}1-chain reacted with practically all blood monocytes by immunofluorescence flow cytometry, and laminin-8 ({alpha}4{beta}1{gamma}1) could be isolated also from these cells. Monoblastic JOSK-I cells adhered constitutively to immobilized recombinant laminin-8, less than to laminin-10/11 ({alpha}5{beta}1{gamma}1/{alpha}5{beta}2{gamma}1) but to a higher level than to laminin-1 ({alpha}1{beta}1{gamma}1). Compared with the other laminin isoforms, adhesion to laminin-8 was preferentially mediated by {alpha}6{beta}1 and {beta}2 integrins. Laminin-8 and, to a lower extent, laminin-1 promoted spontaneous and chemokine-induced migration of blood monocytes, whereas laminin-10/11 was inhibitory. Altogether, the results indicate that leukocytes, as other cell types, are able to synthesize complete laminin molecules. Expression, recognition, and use of laminin-8 by leukocytes suggest a major role of this laminin isoform in leukocyte physiology.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Monocytes are circulating mononuclear phagocytes derived from hemopoietic precursors present in bone marrow (1). During monopoiesis, progenitor cells progress to monoblasts, promonocytes, and, finally, monocytes, which migrate to the peripheral blood. In turn, blood monocytes extravasate and become macrophages, contributing to inflammation, immunity, and tissue repair (2). Resident macrophages are constitutively present in many tissues in the absence of inflammation, whereas monocytes are recruited to local sites following immune and/or inflammatory stimuli. Mononuclear phagocytes adhere to cells (lymphocytes, vascular endothelial cells, and other cell types) and to extracellular matrix proteins (fibronectin and laminins) by using integrins and other adhesion receptors. The adhesion participates in most monocyte/macrophage functions and activities, including Ag presentation, cytotoxicity, phagocytosis, migration (chemotaxis), tissue recruitment, and production of inflammatory mediators (3, 4, 5, 6).

Laminins are a growing family of large heterotrimeric proteins composed of {alpha}-, {beta}-, and {gamma}-chains (7, 8, 9). Five {alpha}-, three {beta}-, and three {gamma}-chains have been identified so far, assembling into at least 12 laminin isoforms (8, 9). Laminins are synthesized by numerous cell types of solid tissues, including vascular endothelial cells, and expression of the various laminin isoforms, particularly their {alpha}-chains, is cell and tissue specific (7, 8, 9). The prototype laminin-1 ({alpha}1{beta}1{gamma}1), originally isolated from a mouse tumor in 1979, has been well characterized in vitro, and its {alpha}-chain is recognized by several integrin receptors (7, 8, 9). However, laminin {alpha}1-chain, in contrast to the other laminin {alpha}-chains, has a most limited expression in vivo in newborn and adult tissues, and is mainly restricted to a subset of epithelia (10, 11). Laminin {alpha}-chains can be either full-length ({alpha}1, {alpha}2, {alpha}3B, and {alpha}5, ~400 kDa) or truncated ({alpha}3A and {alpha}4, ~200 kDa), and their various domains have 20–60% amino acid sequence identity (7, 8, 9, 12). Laminins are major components of basement membranes, but are also found in other tissue compartments. These molecules have structural, adhesive, and cell signaling functions, and are able to modulate cell behavior, including cell differentiation and migration, by interacting with cell surface (integrin) receptors (7, 8, 13). The biomedical relevance of laminin {alpha}-chains is illustrated by two human disorders, muscular dystrophy and epidermolysis bullosa (7, 8). These genetic diseases of muscle and skin are characterized by mutations in the genes for laminin {alpha}2- and {alpha}3-chains, respectively. Laminin-8 ({alpha}4{beta}1{gamma}1) and -10 ({alpha}5{beta}1{gamma}1) were described for the first time in 1997 (9) and constitute the major laminin isoforms of vascular endothelial cells (14, 15). During extravasation, blood leukocytes migrate through the vascular endothelium and its underlying basement membrane, where they encounter laminin-8 and -10.

Several studies concerning monocyte/macrophages and laminin have been reported. Laminin was found to affect the adhesion, phagocytosis, cytotoxicity, and migration of these cells (16, 17, 18, 19, 20, 21, 22), and stimulated mouse peritoneal macrophages were reported to express laminin at the cell surface as measured by indirect immunofluorescence (23). However, these studies were performed with laminin-1 and/or antisera to this laminin isoform. Because laminin-1 is not found in bone marrow, hemopoietic cells, or most blood vessels (10, 14, 15, 24), other laminin isoforms may be relevant for monocyte/macrophage physiology. We have recently demonstrated the presence of laminin-8 in blood platelets (25) and its synthesis by erythromegakaryocytic cells (26). To analyze expression, recognition, and use of laminin isoforms by monocytic cells, we have investigated in the present study synthesis and presence of laminin-8 in monoblastic THP-1, U-937, and Mono Mac 6 cell lines and blood monocytes. In addition, we have examined integrin-dependent adherence of monoblastic cells to recombinant laminin-8 and migration of blood monocytes on this laminin isoform. We report, for the first time, synthesis and expression of a complete laminin molecule by leukocytes, adhesion of the cells to laminin-8 via {alpha}6{beta}1 (CD49f/CD29) and {beta}2 (CD11/CD18) integrins, and promotion of monocyte migration by laminin-8.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells, Abs, and purified proteins

The human monoblastoid cell lines THP-1 (6), U-937 (6), Mono Mac 6 (27), and JOSK-I (28) were cultured in RPMI 1640 with 10% FBS (Life Technologies, Täby, Sweden) and antibiotics. The culture medium of Mono Mac 6 was supplemented with 1 mM sodium pyruvate, 1x nonessential amino acids, 1 mM oxaloacetic acid, and bovine insulin at 10 µg/ml. PBMC were obtained from healthy donors after Ficoll-Hypaque gradient centrifugation (Amersham Pharmacia AB, Uppsala, Sweden), and extensively washed to remove any contaminating platelets. Monocytes were isolated by adherence to tissue culture flasks (3, 6). By this procedure, more than 90% of the recovered cell population was positive for CD14, a monocyte marker.

Mouse mAbs DG10, LAM-89 (Sigma, St. Louis, MO), and 2G6 (Sera-Lab, Crawley Down, U.K) to human laminin {beta}1-chain, and 2E8, LN-41 (Takara Shuzo, Kyoto, Japan), and 22 (Transduction Laboratories, Lexington, KY) to human laminin {gamma}1, as confirmed by reactivity with recombinant human laminin {beta}1- and {gamma}1-chains (29), were used. Rabbit Abs to recombinant human laminin {alpha}4 I/II domains were produced and immunoaffinity purified as previously described (30). Their specificity was confirmed by reactivity against the recombinant protein (31) and by microsequencing of the immunoreactive band from platelets (25). Moreover, these Abs did not stain muscle tissue sections or cell extracts from laminin {alpha}4-chain knockout mice (our unpublished data). The rabbit Abs were not used for immunoprecipitation or immunofluorescence, because they recognize denaturated Ag. Blocking mAbs to integrin chains included H12 ({alpha}L or CD11a, kindly provided by Prof. Hans Wigzell, Karolinska Institutet), 13 ({beta}1 or CD29, generous gift from Prof. Kenneth Yamada, National Institutes of Health, Bethesda, MD), GoH3 ({alpha}6 or CD49f, Immunotech, Marseille, France), and IB4 (32) ({beta}2 or CD18, kindly provided by Dr. Claes Lundberg, Amersham Pharmacia AB). mAbs TUK4 to CD14 (monocyte marker) and MPO-7 to myeloperoxidase (myelomonocytic intracellular Ag) were purchased from Dakopatts (Copenhagen, Denmark), as well as rabbit IgG control. Purified mouse IgG (Coulter, Hialeah, HI) was used as negative control.

Mouse laminin-1 ({alpha}1{beta}1{gamma}1, isolated from Engelbreth-Holm-Swarm tumor) was obtained from Life Technologies, as well as human placenta laminin, which was purified with mAb 4C7 to laminin {alpha}5-chain and recently classified as laminin-10/11 ({alpha}5{beta}1{gamma}1/{alpha}5{beta}2{gamma}1) (33). Full-length recombinant laminin-8 ({alpha}4{beta}1{gamma}1) was produced in a mammalian expression system and purified by affinity chromatography as described (31). The recombinant molecule was a Y-shaped heterotrimer, as expected for the native protein.

RNA extraction and PCR

Total RNA was purified from cultured cells by using RNazol B (AMS Biotechnology, Täby, Sweden). Purity and quantification of RNA were assessed by its absorbance (Gene Quant, Pharmacia). cDNA synthesis and PCR were conducted using the Advantage RT-for-PCR kit (Clontech Laboratories, Palo Alto, CA). Briefly, after oligo(dT)18 priming, mRNA was reverse transcribed into cDNA by incubation for 60 min at 42°C with 200 U of Moloney murine leukemia virus reverse transcriptase in 20 µl of reaction buffer containing 20 U of recombinant RNase inhibitor and 10 mM of each dNTP. The reactions were terminated by heating the samples at 94°C for 5 min, and the mixture was diluted to 100 µl with diethyl pyrocarbonate-treated water. Single-stranded cDNAs in 20 µl of reaction buffer containing 1.5 mM MgCl2, 10 µM of each primer and dNTP mix (10 mM each) were amplified by 35 cycles of PCR using 1.25 U of Ampli Taq DNA polymerase (Perkin-Elmer/Roche Molecular Systems, Branchburg, NJ). The conditions for PCR were 94°C, 5 min; 60°C, 1 min; 72°C, 1 min. For PCR of human laminin {alpha}4 cDNA, paired primers 5'-TGCCTACTTTACCAGGGTGG-3' (sense strand) and 5'-AAACATGTAAACCAAGCGGC-3' (antisense strand) (nucleotides 4287–4767) (26) were used to direct synthesis of a 481-bp product. For human laminin {beta}1 cDNA, paired primers 5'-TTGGACCAAGATGTCCTGAG-3' (sense strand) and 5'-CAATATATTCTGCCTCCCCG-3' (antisense strand) (nucleotides 955-1633) (26) were used and a 679-bp product was expected. For human laminin {gamma}1 cDNA, paired primers 5'-GCAAGACTGAACAGCAGACC-3' (sense strand) and 5'-TCCTATCAAGATCGCTGACC-3' (antisense strand) (nucleotides 4241–4927) (26) were used and a 687-bp product was expected.

Nested PCR was performed for amplification of the {alpha}4-chain with 5 µg of total RNA for cDNA synthesis. PCR 1 included the {alpha}4 primers and 2 mM MgCl2, and proceeded for 45 cycles. For PCR 2, 2 µl of the initial RT-PCR were used as template and the {alpha}4 antisense strand primer was combined with the {alpha}4 primer 5'-CATGGGATCCTGTTGCTCT-3' (sense strand) (nucleotides 4481–4767) (34), with expected product of 287 bp. The PCR conditions were the same as for amplification of {beta}1- and {gamma}1-chains. PCR products were analyzed on 1.5% (w/v) agarose gels. Human placenta total RNA and human GAPDH primers provided with the kit were used as positive control.

Metabolic labeling of THP-1 cells with [35S]methionine and [35S]cysteine and immunoprecipitation

Following incubation at 37°C for 30 min in methionine- and cysteine-free medium, 30 x 106 cells were labeled in 10 ml of methionine- and cysteine-free RPMI 1640 medium with 10% dialyzed FBS containing 0.20 mCi/ml Trans 35S-label (ICN Radiochemical, Costa Mesa, CA) for 4 h at 37°C. After washing three times with cold PBS, the cells were lysed by adding 1 ml of lysis buffer (1% Triton X-100 in PBS with 1 µg/ml of aprotinin, 2 µM leupeptin, 2 µM pepstatin, 1 mM PMSF, and 2 mM EDTA as protease inhibitors). The soluble fraction (cell lysate) was precleared with protein G-Sepharose beads (Amersham Pharmacia AB), and immunoprecipitation was performed by adding to 200 µl of cell lysate 5 µg of mAb followed by addition of 50 µl of protein G-Sepharose beads previously coated with 5 µl of rabbit anti-mouse Ig (Dakopatts).

Gel electrophoresis, immunoblotting, enhanced chemiluminescence, and purification of laminin by immunoaffinity chromatography

Protein samples were analyzed by SDS-PAGE. In Western blots, filters were blocked with 0.1% Tween 20/5% dry milk in PBS. Peroxidase-linked anti-mouse or anti-rabbit Ig (Dakopatts) was used as secondary Ab, and ECL (Amersham, U.K.) was used as developer. Laminin was purified from cell lysates by immunoaffinity chromatography as previously described (25). Briefly, Sepharose CL-4B preadsorbed cell lysate was applied to laminin {beta}1 Ab column (mAb DG10 coupled to CNBr-activated Sepharose 4B), which had been equilibrated with lysis buffer, and cycled twice. Following extensive washing, the proteins bound to the column were eluted by using high pH, and the samples were collected in neutralizing buffer. After concentration, the purified material was analyzed by SDS-PAGE and Western blotting.

Immunofluorescence flow cytometry

Isolated blood mononuclear cells were analyzed for cell surface (nonpermeabilized) and intracellular (permeabilized) Ag expression by immunofluorescence. Cell permeabilization was performed by fixation and permeabilization with IntraStain kit (Dakopatts). Indirect immunofluorescence was performed by incubating cells, after blocking with 2 mg/ml of heat aggregated human IgG (Sigma) for 30 min at 4°C, with saturating amounts (1 µg IgG to 1 x 106 cells) of mAbs, followed by fluorescein-conjugated F(ab')2 of rabbit anti-mouse Ig (Dakopatts) at 1:20 dilution. After staining, monocytes were gated (according to forward and side scatter) and analyzed in a FACScan flow cytometer (Becton Dickinson, Mountain View, CA). Monocyte identity and permeabilization were confirmed by reactivity with mAbs to CD14 and myeloperoxidase, respectively.

Cell adhesion assay

In studies of cell adhesion to immobilized proteins, cells were washed twice with PBS and resuspended in RPMI (106/ml) with 0.5% human serum albumin (HSA)3 (Sigma). Then, 96-well plates (MaxiSorp; Nunc, Roskilde, Denmark) were coated overnight with HSA, mouse Engelbreth-Holm-Swarm laminin-1, recombinant laminin-8, or human placenta laminin-10/11 at 20 µg/ml. After blocking with BSA, 105 cells were added per well and incubated for 1 h at 37°C. Following five washes with plain medium, adherent cells were fixed by adding 50 µl of fixative solution (paraformaldehyde 40 g/liter, NaH2PO4·H2O, 16.97 g/liter; NaOH, 3.86 g/liter; and D-glucose, 5.4 g/liter; prepared at 65°C, pH 7.4) for 15 min and thereafter 50 µl of filtered toluidine blue dye (Sigma, 0.5% w/v in PBS) was added overnight at room temperature. The plate was then washed with copious amounts of distilled water. Adherent cells were quantified in a microplate reader (Multiskan Bichromatic, Labsystems, Helsinki, Finland) at 690 nm by releasing the blue dye with 100 µl of 2% SDS (Bio-Rad Laboratories, Richmond, CA). Effect of blocking mAbs to integrins on the laminin-specific cell adhesion was performed by pretreating the cell suspension with Abs (20 µg/ml) for 15 min before adding the cells to the laminin-coated wells. Cell adhesion in presence of control IgG was defined as 100% adherence. In statistical analysis (Student’s t test), mean and SD were calculated as well as level of significance (*, p < 0.05; **, p < 0.01; ***, p < 0.001), comparing the blocking Abs to the IgG control.

Cell migration assay

Transmigration of monocytes through protein-coated filters was measured microscopically and by flow cytometry. Briefly, human blood mononuclear cells (5 x 105, containing 45% monocytes) in 100 µl RPMI 1640 medium were added to the top chamber of a 6.5-mm diameter, 5-µm pore polycarbonate Transwell culture insert (Costar, Cambridge, MA) and incubated at 37°C for 3 h in absence (spontaneous migration) or presence (chemokine-stimulated migration) of 500 ng/ml stromal cell-derived factor 1 (SDF-1{alpha}; R&D Systems, Abingdon, U.K.) in the lower chamber. In preliminary experiments, these chemokine dose was found to be optimal (data not shown). The filters had been previously coated overnight with 20 µg/ml of various laminin isoforms or HSA, and blocked with 0.5% HSA for 1 h. To effectively remove all transmigrated cells from the lower chamber, a final concentration of 10 mM EDTA was added to each well and cells were resuspended vigorously. Thereafter, the resuspended cells were fixed with 1% formaldehyde and their cell number was determined microscopically by counting five fields with a 40x objective. The percentage of monocytes in the cell population was determined in a FACScan (Becton Dickinson, San Jose, CA) by gating on their forward and side scatter. In statistical analysis (Student’s t test), mean and SD were calculated as well as level of significance (*, p < 0.05; **, p < 0.01; ***, p < 0.001), comparing the various laminin isoforms to HSA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of laminin {alpha}4, {beta}1, and {gamma}1 mRNAs in monoblastic THP-1 cells

To investigate whether mRNAs encoding for the chains of laminin-8 are expressed in monocytic cells, RT-PCR was conducted using pairs of primers that were designed based on the reported cDNAs of human laminin {alpha}4-, {beta}1-, and {gamma}1-chains. After 35 cycles of PCR with reversed transcribed THP-1 mRNA, amplified products with the expected size were detected with primers for {gamma}1 (687 bp, strong) and {beta}1 (679 bp, weak), but not for {alpha}4 (Fig. 1GoA). In contrast to the {gamma}1 product, the {beta}1 product was not always found. Similar results were obtained with Mono Mac 6, whereas only the {gamma}1 transcript was detected in U-937 cells and in blood mononuclear cells, which contains both monocytes and lymphocytes (data not shown). To detect laminin {alpha}4 mRNA in THP-1 cells, the products amplified with the {alpha}4 primers were further amplified with an additional {alpha}4 primer in a nested PCR. Under these conditions, a product with the expected size (287 bp) was obtained from both THP-1 and human placenta (Fig. 1GoB).



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  		FIGURE 1. RT-PCR of laminin-8 chains in monoblastic THP-1 cells. A, Amplification of laminin {beta}1 and {gamma}1 cDNA fragments by RT-PCR of total RNA from THP-1 cells. B, Amplification of laminin {alpha}4 by nested PCR of total RNA from placenta and THP-1 cells.

 
Synthesis and expression of laminin {gamma}1 and {beta}1 polypeptides and overproduction of the {gamma}1-chain in THP-1 cells

To analyze synthesis of the laminin chains, the lysate of metabolically radiolabeled THP-1 cells was immunoprecipitated with mAbs to either {beta}1- or {gamma}1-chains (Fig. 2Go). Immunoprecipitation with Abs to laminin {gamma}1 revealed a single band of 200 kDa, whereas no radiolabeled polypeptides were immunoprecipitated with mAbs to laminin {beta}1-chain. A strongly labeled polypeptide of 180 kDa and a minor one of 160 kDa were immunoprecipitated with mAb H12 to CD11a, which was used as a positive control.



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  		FIGURE 2. Immunoprecipitation from [35S]methionine/cysteine-labeled THP-1 cells with mAbs to laminin {beta}1- and {gamma}1-chains. Name of the Abs and their specificity (in parenthesis) are shown. mIgG, Mouse IgG. Arrows indicate bands of 180 kDa (left) and 200 kDa (right), corresponding to CD11a and laminin {gamma}1-chain, respectively. Immunoprecipitated materials were separated by SDS gel electrophoresis under reducing conditions (5% acrylamide gel).

 
In further studies, Western blot analysis of THP-1 cell lysate was performed. Under reducing conditions, mAb 22 to laminin {gamma}1-chain and mAb DG10 to laminin {beta}1-chain recognized polypeptides with the expected size of 200 (strong) and 220 (weak), respectively (data not shown). Because rabbit Abs to laminin {alpha}4-chain reacted with many polypeptides in the total cell lysate, the results concerning presence of this chain were inconclusive. Under nonreducing conditions, mAb 2E8 to {gamma}1 strongly labeled a component of 200 kDa and, very faintly, a band with a much slower electrophoretic mobility (~420 kDa), which was also labeled by mAb DG10 to {beta}1 (data not shown). This component had a migration slightly faster than that of fibronectin (450 kDa), and corresponds to a disulfide-bonded {beta}1/{gamma}1 heterodimer. The laminin {beta}1- and {gamma}1-chains were also detected by Western blotting in the total cell lysate of THP-1 cells, which had been grown in serum-free conditions for several months (data not shown), confirming the endogenous synthesis of the chains. Thus, THP-1 cells produced much more {gamma}1-chain than {beta}1-chain, the majority of {gamma}1-chain was present apparently as monomer, whereas most {beta}1-chain was associated to {gamma}1-chain.

Laminin {alpha}4-, {beta}1-, and {gamma}1-chains are physically associated as laminin-8 in monoblastic THP-1, U-937, and Mono Mac 6 cells

To identify the {alpha}-chain and to demonstrate its physical association to {beta}1- and {gamma}1-chains, laminin was isolated from THP-1 lysates by immunoaffinity chromatography on a laminin {beta}1 (DG10) Ab column. The isolated material was then analyzed by Western blotting (Fig. 3Go). Under reducing conditions, mAbs to {beta}1- and {gamma}1-chains strongly reacted with 220- and 200-kDa polypeptides, respectively. Affinity purified rabbit Abs to laminin {alpha}4-chain were strongly reactive with polypeptides of 180 and, to a lesser extent, 200 and 130 kDa. Almost identical results were obtained with the material isolated from U-937 and Mono Mac 6, two other monoblastic cell lines (Fig. 3Go).



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  		FIGURE 3. Western blot analysis of {beta}1 Ab-immunoaffinity-purified laminin from monoblastic THP-1, U-937, and Mono Mac 6 cell lines. Mouse IgG (mIgG), rabbit IgG (rIgG), purified rabbit Abs to laminin {alpha}4-chain (anti-LN{alpha}4), mAb to laminin {beta}1-chain (DG10), and mAb to laminin {gamma}1-chain (22) were used. Arrows to the right indicate bands of 220, 200, and 180 kDa corresponding to laminin {beta}1-, {gamma}1-, and {alpha}4-chains, respectively. Five percent acrylamide gels under reducing conditions.

 
Blood monocytes contain laminin-8

THP-1, U-937, and Mono Mac 6 are monoblastic cell lines of malignant origin and represent monocyte precursors present in bone marrow (6, 27). Monocytes are more differentiated cells and constitute 20–30% of blood mononuclear leukocytes (1, 2, 6, 35). As a first approach to determine presence of laminin in monocytes, immunofluorescence flow cytometry of purified blood mononuclear cells was used (Fig. 4Go). Monocytes were gated by forward and side scatter and their identity confirmed by reactivity with mAb TUK4 to CD14, a monocyte marker. mAb LN-41 to {gamma}1, the most widely expressed laminin chain, bound minimally to intact monocyte cell surfaces, whereas practically all monocytes examined following permeabilization were consistently reactive. In two of seven experiments, cell surface expression of the laminin epitope was also observed (data not shown). Myeloperoxidase, an intracellular marker of myelomonocytic cells, was detected only in permeabilized cells, as expected. To determine whether monocytes also contain laminin {beta}1- and {alpha}4-chains to form laminin-8, the cell lysate of monocytes isolated by plastic adherence was immunopurified on the laminin {beta}1 Ab column, and the isolated material was analyzed by Western blotting (Fig. 5Go). Similar to the observations with monoblastic cell lines, mAb DG10 to laminin {beta}1-chain and mAb 22 to laminin {gamma}1-chain recognized polypeptides of 230 and 220 kDa, respectively, whereas rabbit Abs to {alpha}4 reacted with bands of 180 and 130 kDa, under reducing conditions.



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  		FIGURE 4. Detection of laminin {gamma}1-chain in blood monocytes by immunofluorescence flow cytometry. Reactivity of mAbs with nonpermeabilized and permeabilized cells is shown. Mouse IgG control (dotted line) and mAbs (shaded peak) TUK4 (CD14), MPO-7 (myeloperoxidase), and LN-41 (laminin {gamma}1-chain). From isolated blood mononuclear cells, monocytes were gated according to side and forward scatter. Their identity and permeability were confirmed by reactivity with mAbs to CD14 and myeloperoxidase, respectively.

 


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  		FIGURE 5. Western blot analysis of {beta}1 Ab-immunoaffinity-purified laminin from isolated blood monocytes. Mouse IgG (mIgG), rabbit IgG (rIgG), purified rabbit Abs to laminin {alpha}4-chain (anti-LN{alpha}4), mAb to laminin {beta}1-chain (DG10), and mAb to laminin {gamma}1-chain (22) were used. Arrows to the right indicate bands of 230, 220, 180, and 130 kDa, corresponding to laminin {beta}1-, {gamma}1-, and {alpha}4- (intact and fragment) chains, respectively. Five percent acrylamide gel under reducing conditions.

 
Monoblastic JOSK-I cells adhere to laminin-8 and other laminin isoforms, but the adherence to laminin-8 is preferentially mediated by {alpha}6{beta}1 and {beta}2 integrin

Monocytic cells may interact with either secreted or cell surface-exposed endogenous laminin, or with exogenous laminin-8. To investigate whether laminin-8 was adhesive for cells of the monocytic lineage, plastic surfaces were coated with HSA, mouse laminin-1, placenta laminin-10/11, or recombinant laminin-8. Monoblastic cells were incubated for 1 h at 37°C on the protein-coated surfaces and, thereafter, nonadherent cells were removed by extensive washing. THP-1 cells adhered minimally to laminin-8 (data not shown). In contrast, all laminin isoforms were adhesive for JOSK-I cells, and the adherence to laminin-8 and laminin-10/11 were statistically significant (p < 0.01 and p < 0.001, respectively) (Fig. 6GoA). The most adhesive was laminin-10/11, followed by laminin-8 and laminin-1. To identify the cell surface receptors involved in this constitutive adhesive process, adhesion-blocking mAbs to integrins were used (Fig. 6GoB). Previous studies have demonstrated that {alpha}6{beta}1 integrin (CD49f/CD29) is a receptor for laminin-8 (25, 31). In accordance to these findings, mAbs to integrin {beta}1- and {alpha}6-chains inhibited largely, but not completely, the cell adherence. Interestingly, mAb IB4 to integrin {beta}2-chain (CD18) blocked nearly 50% of the adherence, and together with mAb 13 to integrin {beta}1-chain, abolished the cell binding (data not shown). The blocking effect of the mAbs was statistically highly significant (p < 0.001). Though almost completely inhibited by mAb 13, adherence to laminin-1 and -10/11 was minimally blocked by mAb GoH3 to integrin {alpha}6-chain, and only to a minor extent by the anti-{beta}2 integrin Ab. Cell surface expression of the integrin subunits CD29 ({beta}1), CD49f ({alpha}6), and CD18 ({beta}2) in JOSK-I cells was confirmed by immunofluorescence flow cytometry (data not shown).



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  		FIGURE 6. Adherence of monoblastic JOSK-I cells to laminin-8 and other laminin isoforms and identification of the integrin receptors. A, Adherence of JOSK-I cells to plastic surfaces coated with HSA, mouse laminin-1 (LN-1), recombinant laminin-8 (LN-8), and placenta laminin-10/11 (LN-10/11). B, Effect of mAbs to integrins in adhesion of JOSK-I cells to laminin-1, -8, and -10/11. IgG, control IgG; 13, mAb to integrin {beta}1-chain (CD29); GoH3, mAb to integrin {alpha}6-chain (CD49f); IB4, mAb to integrin {beta}2-chain (CD18). Plastic surfaces were coated with laminins at 20 µg/ml. Adhesion blocking Abs were used at 20 µg/ml. Mean and SD of four experiments are shown in A and B (*, p < 0.05; **, p < 0.01; ***, p < 0.001). Adherence was measured after incubating the cells (105 cells/well) in the coated plates for 1 h at 37°C and shown as OD (A) and % adherence (B).

 
Laminin-8, but not laminin-10/11, promotes migration of monocytes

To determine the effect of laminin-8 on monocyte migration, transmigration of blood monocytes through laminin-8-coated filters was studied in insert assays. Compared with HSA, laminin-8 efficiently promoted monocyte migration in either absence or presence of chemoattractant, and this effect was statistically highly significant (p < 0.001) (Fig. 7Go). Laminin-1 also enhanced cell migration, but to a lower extent, whereas laminin-10/11 was inhibitory. For each substrate, excluding laminin-10/11, SDF-1{alpha} stimulated a major increase in monocyte migration compared with cells without chemoattractant. More than 40% of the monocyte population transmigrated on the laminin-8 substrate in presence of SDF-1{alpha}. Laminin-8 also promoted migration of blood lymphocytes, but the effect was less evident than on monocytes (data not shown).



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  		FIGURE 7. Laminin-8, but not laminin-10/11, promotes monocyte migration. Monocyte migration assays were performed by using 5-µm pore insert filters precoated with HSA, laminin-1 (LN-1), laminin-8 (LN-8), or laminin-10/11 (LN-10/11). Number of transmigrated cells was determined microscopically, and the percentage of monocytes in the cell population was established by flow cytometry. Mean and SD of four experiments are shown (**, p < 0.01; ***, p < 0.001). Starting with 5 x 105 blood mononuclear cells (45% monocytes) in the upper chamber, migration to the lower chamber was measured after incubating the cells for 3 h at 37°C in absence or presence of SDF-1 (500 ng/ml) in the lower chamber.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we demonstrate that monoblastic cells synthesize laminin-8 and that blood monocytes contain this laminin isoform. For the first time, expression of laminin {alpha}-, {beta}-, and {gamma}-chains, and their assembly into a complete laminin molecule is shown in leukocytes. We also demonstrate that cells of the monocytic lineage adhere to laminin-8 via {alpha}6{beta}1 (CD49f/CD29) and {beta}2 (CD11/CD18) integrins, and that laminin-8 promotes monocyte migration.

THP-1, U-937, and Mono Mac 6 cells represent monoblast/promonocytes and, hence, are less differentiated than monocytes (6, 27, 35). Following treatment with inducers such as phorbol esters and retinoids, these cell lines differentiate along the monocyte/macrophage pathway (6, 35). Altogether, RT-PCR, immunoprecipitation, immunoaffinity purification, and Western blotting studies indicated that THP-1 cells, while overproducing {gamma}1-chain, synthesize laminin {alpha}4-, {beta}1-, and {gamma}1-chains and assemble them to form laminin-8. Difficulties in detecting laminin {beta}1 and {alpha}4 transcripts, as also observed in blood monocytes, and the corresponding metabolically labeled polypeptides may be explained by low turnover of the chains. In contrast, detection of the three chains following immunoaffinity purification by using the laminin {beta}1 Ab column indicate physical association of the chains and major enrichment of the heterotrimer. The unbalance in laminin chain biosynthesis explains why most {gamma}1-chain is present in a monomeric form, but the biological significance of this phenomenon is presently unknown. Electrophoretic mobility of laminin {beta}1- and {gamma}1-chains of the monoblastic cells under nonreducing conditions indicated that the chains were disulfide bonded to each other and, most likely, noncovalently associated to laminin {alpha}4 chain, as found in platelets, erythromegakaryocytic cells and lymphocytes (Refs. 25, 26 , and our unpublished data). The 180kDa polypeptide appears to be the mature laminin {alpha}4-chain, whereas the minor components of 200 and 130 kDa may correspond to precursor and large fragment, respectively. As similar results were obtained with U-937 and Mono Mac 6 cells, we conclude that monoblastic cells are able to synthesize laminin-8.

Synthesis of laminin-8 by monoblastic cells may determine presence of this laminin isoform in monocyte/macrophages. Alternatively, monoblast laminin-8 may contribute to monopoiesis and the interaction of monocyte progenitors with matrix and stromal cells. Interestingly, laminin-1 has been reported to promote differentiation of NB4 promyelocytic leukemia cells with all-trans retinoic acid (36). Monoblasts and promonocytes are found in bone marrow in small numbers. Immunostaining of bone marrow with antiserum to laminin-1 ({alpha}1{beta}1{gamma}1) has demonstrated widespread distribution of laminins in the arteriolar walls and in the sinusoidal subendothelial basement membranes, and also in the intersinusoidal interstitial tissue (24, 37). Laminin {alpha}4-chain was localized in the intersinusoidal spaces, in large arteries, and in small arterioles, whereas no laminin {alpha}1-chain was found. Laminin {alpha}2- and {alpha}5-chains were immunolocalized in arterioles, and {alpha}5 in sinusoidal basement membranes (24). The laminins may contribute to the interaction of progenitor cells with bone marrow macrophages, other stromal cells and extracellular matrices during hemopoiesis (2).

Practically the entire population of blood monocytes reacted with mAb LN-41 to laminin {gamma}1-chain, suggesting that 100% of the cells, and not just a subpopulation, contained laminin-8. Because cell permeabilization was needed for detection, laminin appears to have an intracellular localization. However, expression of laminin epitopes was observed in nonpermeabilized cells in a few experiments, suggesting that laminin may be also expressed on the cell surface. Whether this depends on the activation state of the monocytes or on other parameters is presently unknown. As in monoblastic cell lines, laminin-8 could be immunoaffinity purified from isolated monocytes by using a laminin {beta}1 mAb column, because {beta}1- (230 kDa), {gamma}1- (220 kDa), and {alpha}4- (180/140) chains were detected and found to be physically associated. Interestingly, the 140-kDa polypeptide was more prominent than in the monoblastic cell lines, probably because of partial proteolysis of the {alpha}4 chain during monocyte purification. Synthesis of laminin-8 by monoblastic cell lines strongly suggests that blood monocyte laminin-8 originates from synthesis in bone marrow monocyte progenitors.

Attempts to demonstrate synthesis of complete laminin molecules by hemopoietic/blood cells have been reported by other groups. Tweardy et al. (38) described production of laminin {gamma}1-chain protein and mRNA by a murine neutrophil precursor cell line, but no laminin {beta}- or {alpha}-chains (protein or mRNA) were found. In another study, Thompson et al. (39) reported laminin {gamma}1- and {beta}1-chain protein and mRNA, but no {alpha}-chain protein, in murine mast cells. Moreover, Morone et al. (40) described immunoprecipitation of 200- and 400-kDa proteins from metabolically radiolabeled rat NK cells with rabbit Abs to mouse laminin-1. However, identification of the immunoprecipitated polypeptides was not reported. These three studies were performed before 1991, when only one laminin {alpha}-chain, namely {alpha}1, was known. We have been unable to demonstrate presence of full-length laminin {alpha}-chains in a human NK cell line (our unpublished data).

Following immune and/or inflammatory stimuli, blood monocytes extravasate and accumulate at inflammatory foci (1, 2). In 1983, Wicha and Huard (23) reported expression of laminin on the cell surface of thioglycollate-elicited mouse peritoneal macrophages, as detected by indirect immunofluorescence with a rabbit antiserum to mouse laminin-1. Interestingly, the percentage of immunoreactive macrophages increased with time. A week after thioglycollate stimulation, 60% of the peritoneal macrophages were positive, compared with 14% of nonstimulated resident macrophages. However, molecular characterization of the laminin or whether the laminin represented de novo synthesis by the macrophages or uptake of exogenous laminin was not established.

Several studies have described the effect of exogenous laminin on monocyte/macrophage physiology. Adherence of human blood monocytes and mouse peritoneal macrophages to tissue culture plastic was largely inhibited by laminin-1 (16, 17), and the latter cells adhered to and spread on laminin-1 substrate only after stimulation with either phorbol ester or IFN-{gamma} and LPS (18, 41). In other studies, monocytes were found to adhere constitutively to immobilized laminin (unspecified isoform) (42) and, following stimulation with TGF-{beta}, to laminin-1 (43). When compared with fibronectin and type I and IV collagens, laminin-1 supported the largest chemoattractant-induced migration of monocytes (24). Moreover, laminin-1 enhanced phagocytosis of opsonized erythrocytes by cultured human macrophages (19), facilitated the binding between peritoneal macrophages and tumor cells (44), and enhanced monocyte-mediated tumoricidal activity against human melanoma cells (20). Proliferation of bone marrow-derived mouse macrophages was also promoted by laminin-1 (45). Laminin {alpha}1 SIKVAV peptide induced production of PGE2 and matrix metalloproteinases (MMP) 1 and 9 in human monocytes (46), and laminin (unspecified isoform) induced expression of MMP-9 and urokinase-type plasminogen activator (uPA) by monocytic cell lines (47). uPA expression was also induced by the laminin peptide via {alpha}6{beta}1 integrin (47).

Adherence of monoblastic JOSK-I cells to laminin-8 and -10/11 suggests that these laminin isoforms are relevant to leukocyte physiology. The modest adhesion to laminin-1 may be related to the fact that laminin {alpha}1-chain, in contrast to laminin {alpha}4-chain, is not expressed in hemopoietic cells, lymphatic organs, and most vessels (10, 24). As in platelets (25), the major receptor for laminin-8 in the monocytic cells was identified as {alpha}6{beta}1 integrin. Interestingly, mAb to {beta}1 (CD29), but not to {alpha}6 (CD49f), integrin chains largely inhibited the adhesion to laminin-1 and -10/11. Because {alpha}6{beta}1 integrin is known to recognize all three laminin isoforms (13, 24, 25, 31), the monocytic cells appear to use simultaneously {alpha}6{beta}1 and other {beta}1 integrins to bind laminin-1 and -10/11. However, they use exclusively {alpha}6{beta}1, among the {beta}1 integrins, to adhere to laminin-8. It is noteworthy that {alpha}1 and {alpha}5 are full-length laminin chains and contain domains, which are missing in the truncated {alpha}4-chain (8, 12). Interestingly, mAb to {beta}2 integrin (CD18) inhibited 50% of the cell adhesion to laminin-8, and together with the Ab to {beta}1 integrin abolished the cell adherence. Modest inhibition was observed on cell adhesion to laminin-1, and to laminin-10/11 the inhibitory effect was minimal. Thus, cell adhesion to laminin-8 is more dependent on {beta}2 integrins than adhesion to laminin-1, and the difference could be due to the laminin {alpha}-chain. This finding, together with the presence of laminin {alpha}4-, but not {alpha}1-, chain in leukocytes and hemopoietic/lymphoid tissue as well as in vascular endothelium (Refs. 10, 13, 24 , and present results), indicates that laminin-8 is more relevant for leukocyte physiology than laminin-1.

Integrin and nonintegrin laminin receptors have been identified in mononuclear phagocytes and their precursors. Monoblastic cell lines express relatively low amounts of {alpha}6{beta}1 integrin (CD49f/CD29), but the expression of the {alpha}6-chain (CD49f) is specifically increased by treatment of the cells with retinoids, which induce differentiation of the cells along the monocyte/macrophage pathway (35). Blood monocytes are CD49f-positive, and macrophages from different tissues also express this laminin receptor (6, 35, 48, 49). Other integrin laminin receptors, such as {alpha}2{beta}1 (CD49b/CD29) and {alpha}3{beta}1 (CD49c/CD29), can also be found in mononuclear phagocytes. Small amounts of {alpha}2{beta}1 are present in monocytes (48), and expression of {alpha}3{beta}1, a receptor for laminin-5 ({alpha}3{beta}3{gamma}2) and -10 ({alpha}5{beta}1{gamma}1) (50), is up-regulated during differentiation of monocytes to macrophages (6). In the literature, {beta}2 integrins are not recognized as laminin receptors, though several studies have demonstrated inhibition of PMN and monocyte adhesion to laminin-1 substrate by using blocking Abs to these leukocyte-specific integrins (21, 51, 52, 53). The {beta}2 integrin subfamily, also known as CD11/CD18 molecules or Leu-CAMs, is composed of four members: {alpha}L{beta}2 (CD11a/CD18, LFA-1), {alpha}M{beta}2 (CD11b/CD18, Mac-1), {alpha}X{beta}2 (CD11c/CD18, p150, 95), and {alpha}d{beta}2 (3, 4, 54). Identification of the {beta}2 integrin(s) participating in the cell adhesion to laminin-8 and its specific role in the process are presently under investigation. Among nonintegrin laminin-1 binding proteins, galectin-3 and 67-kDa protein have been found in monocytes and macrophages (55, 56).

Vascular laminin-8 may contribute to extravasation of blood monocytes, whereas endogenous laminin-8 may mediate monocyte migration and chemotaxis in extravascular loci. Laminin-8 of vascular basement membranes may also participate in intravasation of mononuclear phagocytes, as these cells are able to traverse endothelium in the basal to apical direction (57). Although both exogenous laminin-8 and laminin-10/11 promoted adhesion of monocytic cells, only laminin-8 enhanced monocyte migration. Indeed, laminin-10/11 appeared to inhibit migration of the cells, presumably by persistently adhering the cells to the filter. Thus, laminin-8 and laminin-10/11 have opposite effects on monocyte migration, suggesting that laminin-8 promotes the migration of mononuclear phagocytes in tissues, whereas laminin-10/11 determines their tissue localization (arrest). The degree of cell migration was higher on laminin-8 than on laminin-1, and the effect of the former laminin isoform was evident on both spontaneous and SDF-1{alpha}-stimulated migration. SDF-1{alpha} is a potent chemoattractant for mononuclear leukocytes and binds to the chemokine receptor CXCR4. Because monocytes were able to migrate to some extent on the albumin-substrate, it is tempting to speculate that endogenous laminin-8 participates in the process. Keratinocyte migration is known to depend on endogenously secreted laminin-5 ({alpha}3{beta}3{gamma}2) (58). Recently, stimulated blood lymphocytes and PMNs were found to secrete laminin-8 (our unpublished data; Z. Wondimu et al., unpublished data). It is noteworthy that monocyte migration on laminin-1 and fibronectin stimulated by monocyte chemoattractant protein-1 is preferentially mediated by {beta}2 integrins (22). Participation of endogenous laminin-8 in leukocyte migration and the role of integrin receptors are presently under investigation.

Laminin-8 may contribute to other function/activities of monocytes and macrophages, including Ag presentation and stimulation of T lymphocytes. In recent studies, laminin-8 together with a mAb to CD3 induced considerable proliferation of T cells, whereas either reagent alone was inactive (our unpublished data). Participation of laminin-8 in phagocytosis, cytotoxicity of infected and tumor cells, production of inflammatory mediators, and tissue remodeling is also likely, and laminin-8 secreted by macrophages may modulate angiogenesis and wound healing. Furthermore, laminin-8 may promote improved phagocytosis of those microorganisms that are capable of binding laminins, such as Staphylococcus aureus, Escherichia coli, Streptococcus pyogenes, and Treponema pallidium (38). In contrast, the macrophage-matrix interaction may contribute to the development of atherosclerosis and other chronic inflammatory lesions.

Considering that leukocytes synthesize, secrete and interact with laminin-8 and that vascular endothelial cells up-regulate laminin {alpha}4 mRNA expression following treatment with inflammatory cytokines (14), this laminin isoform may be most relevant for the development of immune and inflammatory responses.


   Acknowledgments
 
We thank Dr. E. Engvall, Dr. K. Yamada, and Dr. A. Iivanainen for providing Abs, and Dr. George Klein and Dr. Olof Rådmark for providing cell lines. We also thank Dr. K. Nilsson for comments on the manuscript and Dr. B. Chambers for revising the English text. C.P. was holder of a stipendium from the Swedish Institute.


   Footnotes
 
1 This project was supported by Cancerfonden and the Karolinska Institute. Back

2 Address correspondence and reprint requests to Dr. Manuel Patarroyo, MTC, Karolinska Institutet, S 171 77 Stockholm, Sweden. Back

3 Abbreviations used in this paper: HSA, human serum albumin; SDF, stromal cell-derived factor. Back

Received for publication June 7, 2000. Accepted for publication August 28, 2000.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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