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4
1
1)1
*
Microbiology and Tumor Biology Center, and
Division of Matrix Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden;
Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia; and
Institute of Biomedicine, Department of Anatomy, University of Helsinki, Helsinki, Finland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1-,
1-, and 
4-chains were detected by RT-PCR.
Following immunoaffinity purification on a laminin 1
Ab-Sepharose column, laminin 1- (220 kDa),
1- (200 kDa), and 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 1-chain reacted with
practically all blood monocytes by immunofluorescence flow cytometry,
and laminin-8 (411) could
be isolated also from these cells. Monoblastic JOSK-I cells adhered
constitutively to immobilized recombinant laminin-8, less than to
laminin-10/11
(511/521)
but to a higher level than to laminin-1
(111). Compared with the
other laminin isoforms, adhesion to laminin-8 was preferentially
mediated by 61 and 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 |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Laminins are a growing family of large heterotrimeric proteins composed
of -, -, and -chains (7, 8, 9). Five -, three
-, and three -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 -chains, is cell and tissue specific
(7, 8, 9). The prototype laminin-1
(111),
originally isolated from a mouse tumor in 1979, has been well
characterized in vitro, and its -chain is recognized by several
integrin receptors (7, 8, 9). However, laminin
1-chain, in contrast to the other laminin
-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 -chains can be either full-length
(1, 2,
3B, and 5, 
400
kDa) or truncated (3A and
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 -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 2- and
3-chains, respectively. Laminin-8
(411)
and -10
(511)
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
61 (CD49f/CD29) and
2 (CD11/CD18) integrins, and promotion of
monocyte migration by laminin-8.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 1-chain,
and 2E8, LN-41 (Takara Shuzo, Kyoto, Japan), and 22 (Transduction
Laboratories, Lexington, KY) to human laminin
1, as confirmed by reactivity with recombinant
human laminin 1- and
1-chains (29), were used. Rabbit
Abs to recombinant human laminin 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 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
(L or CD11a, kindly provided by Prof. Hans
Wigzell, Karolinska Institutet), 13 (1 or
CD29, generous gift from Prof. Kenneth Yamada, National Institutes of
Health, Bethesda, MD), GoH3 (6 or CD49f,
Immunotech, Marseille, France), and IB4 (32)
(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
(111,
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 5-chain and recently
classified as laminin-10/11
(511/521)
(33). Full-length recombinant laminin-8
(411)
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 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 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 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
4-chain with 5 µg of total RNA for cDNA
synthesis. PCR 1 included the 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
4 antisense strand primer was combined with
the 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 1- and
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
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; 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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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4, 1, and
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
4-, 1-, and
1-chains. After 35 cycles of PCR with reversed
transcribed THP-1 mRNA, amplified products with the expected size were
detected with primers for 1 (687 bp, strong)
and 1 (679 bp, weak), but not for
4 (Fig. 1
A). In contrast to the
1 product, the 1
product was not always found. Similar results were obtained with Mono
Mac 6, whereas only the 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
4 mRNA in THP-1 cells, the products amplified
with the 4 primers were further amplified with
an additional 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. 1B).
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1 and
1 polypeptides and overproduction of the
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 1- or
1-chains (Fig. 2). Immunoprecipitation with Abs to
laminin 1 revealed a single band of 200 kDa,
whereas no radiolabeled polypeptides were immunoprecipitated with mAbs
to laminin 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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1-chain and mAb DG10 to laminin
1-chain recognized polypeptides with the
expected size of 200 (strong) and 220 (weak), respectively (data not
shown). Because rabbit Abs to laminin 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 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 1 (data not shown). This component had
a migration slightly faster than that of fibronectin (450 kDa), and
corresponds to a disulfide-bonded
1/1 heterodimer. The
laminin 1- and
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 1-chain than
1-chain, the majority of
1-chain was present apparently as monomer,
whereas most 1-chain was associated to
1-chain.
Laminin 4-, 1-, and
1-chains are physically associated as laminin-8 in
monoblastic THP-1, U-937, and Mono Mac 6 cells
To identify the -chain and to demonstrate its physical
association to 1- and
1-chains, laminin was isolated from THP-1
lysates by immunoaffinity chromatography on a laminin
1 (DG10) Ab column. The isolated material was
then analyzed by Western blotting (Fig. 3). Under reducing conditions, mAbs to
1- and 1-chains
strongly reacted with 220- and 200-kDa polypeptides, respectively.
Affinity purified rabbit Abs to laminin
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. 3).
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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. 4).
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 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 1- and
4-chains to form laminin-8, the cell lysate of
monocytes isolated by plastic adherence was immunopurified on the
laminin 1 Ab column, and the isolated material
was analyzed by Western blotting (Fig. 5). Similar to the observations with
monoblastic cell lines, mAb DG10 to laminin
1-chain and mAb 22 to laminin
1-chain recognized polypeptides of 230 and 220
kDa, respectively, whereas rabbit Abs to 4
reacted with bands of 180 and 130 kDa, under reducing conditions.
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61 and 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. 6A). 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. 6B). Previous studies have demonstrated that
61 integrin
(CD49f/CD29) is a receptor for laminin-8 (25, 31). In
accordance to these findings, mAbs to integrin
1- and 6-chains
inhibited largely, but not completely, the cell adherence.
Interestingly, mAb IB4 to integrin 2-chain
(CD18) blocked nearly 50% of the adherence, and together with mAb 13
to integrin 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
6-chain, and only to a minor extent by the
anti-2 integrin Ab. Cell surface
expression of the integrin subunits CD29 (1),
CD49f (6), and CD18
(2) in JOSK-I cells was confirmed by
immunofluorescence flow cytometry (data not shown).
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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. 7). 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 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.
Laminin-8 also promoted migration of blood lymphocytes, but the effect
was less evident than on monocytes (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-, -, and
-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
61 (CD49f/CD29) and
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
1-chain, synthesize laminin
4-, 1-, and
1-chains and assemble them to form laminin-8.
Difficulties in detecting laminin 1 and
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 1 Ab column indicate physical
association of the chains and major enrichment of the heterotrimer. The
unbalance in laminin chain biosynthesis explains why most
1-chain is present in a monomeric form, but
the biological significance of this phenomenon is presently unknown.
Electrophoretic mobility of laminin 1- and
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
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 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
(111)
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 4-chain was localized in the
intersinusoidal spaces, in large arteries, and in small arterioles,
whereas no laminin 1-chain was found. Laminin
2- and 5-chains were
immunolocalized in arterioles, and 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 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 1 mAb column, because
1- (230 kDa), 1- (220
kDa), and 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
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
1-chain protein and mRNA by a murine
neutrophil precursor cell line, but no laminin - or -chains
(protein or mRNA) were found. In another study, Thompson et al.
(39) reported laminin 1- and
1-chain protein and mRNA, but no -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 -chain, namely
1, was known. We have been unable to
demonstrate presence of full-length laminin -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- and LPS (18, 41). In
other studies, monocytes were found to adhere constitutively to
immobilized laminin (unspecified isoform) (42) and,
following stimulation with TGF-, 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 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 61
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
1-chain, in contrast to laminin
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
61 integrin.
Interestingly, mAb to 1 (CD29), but not to
6 (CD49f), integrin chains largely inhibited
the adhesion to laminin-1 and -10/11. Because
61 integrin is known
to recognize all three laminin isoforms (13, 24, 25, 31),
the monocytic cells appear to use simultaneously
61 and other
1 integrins to bind laminin-1 and -10/11.
However, they use exclusively
61, among the
1 integrins, to adhere to laminin-8. It is
noteworthy that 1 and
5 are full-length laminin chains and contain
domains, which are missing in the truncated
4-chain (8, 12). Interestingly,
mAb to 2 integrin (CD18) inhibited 50% of the
cell adhesion to laminin-8, and together with the Ab to
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 2 integrins
than adhesion to laminin-1, and the difference could be due to the
laminin -chain. This finding, together with the presence of laminin
4-, but not 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
61 integrin
(CD49f/CD29), but the expression of the
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
21 (CD49b/CD29) and
31 (CD49c/CD29), can
also be found in mononuclear phagocytes. Small amounts of
21 are present in
monocytes (48), and expression of
31, a receptor for
laminin-5
(332)
and -10
(511)
(50), is up-regulated during differentiation of monocytes
to macrophages (6). In the literature,
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
2 integrin subfamily, also known as CD11/CD18
molecules or Leu-CAMs, is composed of four members:
L2 (CD11a/CD18,
LFA-1), M2
(CD11b/CD18, Mac-1),
X2 (CD11c/CD18, p150,
95), and d2 (3, 4, 54). Identification of the 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-stimulated migration. SDF-1 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
(332)
(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 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
4 mRNA expression following treatment with
inflammatory cytokines (14), this laminin isoform may be
most relevant for the development of immune and inflammatory
responses.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Manuel Patarroyo, MTC, Karolinska Institutet, S 171 77 Stockholm, Sweden.
3 Abbreviations used in this paper: HSA, human serum albumin; SDF, stromal cell-derived factor.
Received for publication June 7, 2000. Accepted for publication August 28, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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chains: expression, developmental transitions, and chromosomal localization of 1–5, identification of heterotrimeric laminins 8–11, and cloning of a novel 3 isoform. J. Cell Biol. 137:685.1 chain in normal adult mouse tissues. Matrix Biol. 18:557.[Medline]
1 chain shows a restricted distribution in epithelial basement membranes of fetal and adult tissues. Exp. Cell Res. 257:298.[Medline]
5, and widespread expression in adult mouse tissues. J. Biol. Chem. 270:28523.4 cDNA: expression in a subset of endothelium. Eur. J. Biochem. 246:727.[Medline]
5 chain suggests a role in epithelial and endothelial cell maturation. Dev. Biol. 189:285.[Medline]
2 integrins. Am. J. Physiol. 267:C1112.2-integrins to migrate on laminin and fibronectin. Am. J. Physiol. 269:C60.5-containing laminins as adhesive proteins for multipotent hematopoietic FDCP-mix cells. Blood 93:2533.411) and adhere to laminin-8 via 61 integrin. Exp. Cell Res. 253:723.[Medline]
411). Exp. Cell Res. 254:189.[Medline]
1 and 1 chains. Matrix Biol. 19:169.[Medline]
4 chain. J. Biol. Chem. 272:27862.411). Production, purification and interactions with integrins. J. Biol. Chem. 275:14583.
4 chain. FEBS Lett. 365:183.[Medline]
6 integrin) and CD66a (BGP) are specifically induced by retinoids during human monocytic differentiation. Leukemia 9:2034.[Medline]
and lipopolysaccharide promotes macrophage adherence to basement membrane glycoproteins. J. Exp. Med. 169:303. enhances integrin expression and type IV collagenase secretion in human monocytes. Proc. Natl. Acad. Sci. USA 90:4577.91 integrin is expressed on human neutrophils and contributes to neutrophil migration through human lung and synovial fibroblast barriers. J. Leukocyte Biol. 66:809.[Abstract]
61 integrin. J. Cell Biol. 110:2167.31 integrin: purification, processing, regulation and specific binding to laminin-5 and invasin in a mutually exclusive manner. Biochemistry 37:1094.[Medline]
show increased adherence to extracellular matrix proteins, which is mediated via the CD11b/CD18 complex. Clin. Exp. Immunol. 90:280.[Medline]
1-stimulated adhesion of human mononuclear phagocytes to fibronectin and laminin is abolished by IFN-: dependence on 51 and 2 integrins. Exp. Cell Res. 222:209.[Medline]
2-integrins and their cellular ligands. Eur. J. Biochem. 245:215.[Medline]
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