From the Interdisciplinary Center for Clinical
Research and the § Institute for Experimental
Medicine, Nikolaus Fiebiger Center, University of Erlangen-Nuremberg,
91054 Erlangen, Germany, and the ¶ Dermatology Department,
University of Cologne, 50931 Cologne, Germany
Received for publication, December 4, 2000, and in revised form, March 5, 2001
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ABSTRACT |
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Regulated adhesion of leukocytes to the
extracellular matrix is essential for transmigration of blood vessels
and subsequent migration into the stroma of inflamed tissues. Although
One of the main functions of endothelial cells is to
prevent leukocyte emigration from the blood vessel into the underlying tissues, and only in cases of inflammation is this barrier function physiologically removed. During such extravasation processes emigrating cells not only have to rapidly traverse the tight endothelial cell
monolayer but also the basement membrane of the blood vessel endothelium and migrate into the underlying interstitial extracellular matrix. Considerable effort has been made to understand the initial steps in this extravasation process, i.e. rolling along the
blood vessel endothelium and firm adhesion, revealing the function of specific cell adhesion molecules of the selectin, immunoglobulin, and
One of the major components of basement membranes is the laminin family
of molecules, heterotrimers composed of Endothelial cells express two laminin isoforms, depending on their
tissue of origin and state of growth or activation (2, 16). Laminin 8 (composed of laminin Among the first cells present at a site of inflammation are
polymorphonuclear granulocytes (PMN) and monocytes (24). Depending on
the type of inflammation, it is thought that these cells provide the
necessary signals for subsequent extravasation of lymphocytes into the
site of inflammation. It cannot be disputed that the Cells--
PMN were isolated from femurs of
ECM and Integrin Antibodies--
The antibodies to extracellular
matrix proteins employed were rat monoclonal antibodies (mAbs) to mouse
laminin
The antibodies to cell surface adhesion molecules employed were chicken
polyclonal antibody against the RGD-cell binding site of human
fibronectin (36); hamster anti-rat Flow Cytometry Analysis--
Flow cytometry analysis was carried
out as previously described (27).
Isolation and Characterization of Laminins 8 and 10--
Mouse
laminin 8 (composed of ECM Proteins and Arginine-Glycine-Aspartic (RGD)
Peptides--
Other extracellular matrix molecules employed in
in vitro assays were laminin 1, collagen type IV, and
perlecan isolated from the Engelbreth-Holm-Swarm mouse tumor (40);
laminin 2 from mouse hearts (41); mouse fibronectin isolated from
plasma by gelatin-Sepharose chromatography (42); rat tail collagen type
I (Roche Molecular Biochemicals); mouse vitronectin (Life Technologies,
Inc.); recombinant human tenascin-C supplied by H. Erickson (Duke
University, Durham, NC). Peptides tested for inhibitory effects in
attachment assays included linear RGDS (Life Technologies, Inc.);
cyclic RGDfV (EMD66203) specific for Attachment Assays--
In vitro cell attachment
assays were performed as previously described (27) using colorimetric
analysis of lysosomal hexosaminidase (A405) to quantitate the number of
adherent cells. Because leukocytes are activated by chemokines and
cytokines are released at sites of inflammation in vivo,
attachment assays were performed with nonactivated and activated cells.
Activation was achieved either with a physiological activator, fMLP (10 ng/ml), or with the general leukocyte activator, PMA (100 ng/ml), that
bypasses ligand-receptor interactions and simulates a strong activation
signal (27). The effect of divalent cations was tested by performing
attachment assays in the presence of 1 mM EDTA. Inhibition
studies assessed the effects of linear and cyclic RGD peptides or
antibodies against integrin subunits and extracellular matrix proteins
on cell-matrix interactions and involved a 30-min preincubation of
cells prior to addition to protein-coated microtiter plates with
varying concentrations of antibodies (10-300 µg/ml) or peptides
(10-100 µM for cyclic and linear RGD peptides).
Experiments were carried out using a single concentration of
extracellular matrix protein (30 µg/ml) at which cell binding was
saturated, in the presence of varying concentrations of inhibitory
antibodies or peptides. The experimental procedure was otherwise as
described previously (27). The percentage of cells that bound
specifically to the coating substrate was determined as follows:
((A405 of total bound cells Migration Assays--
Transmigration assays were performed using
Costar Transwells (polycarbonate filter, 5-µm pore size). Membranes
were coated overnight at 4 °C with 10-15 µg/ml of laminins 1, 8, or 10 prepared in our laboratory, commercial laminin 10/11,
fibronectin, or vitronectin and subsequently blocked with 1 mg/ml BSA
for 1 h at 37 °C. Only Statistical Analyses--
Cell adhesion and migration assays
were carried out at least six times with triplicates performed in each
experiment. Student's t test was used to test statistical
significance of differences in Bmax (maximal adhesion) values measured
with activated and nonactivated PMN; differences in mean rates of
migration across all substrates was compared pairwise using the Student
Newman Keuls method (i.e comparisons were made between fMLP-induced
transmigration across laminin 10 and 8, laminin 10 and 1, laminin10 and
fibronectin, laminin 10 and vitronectin, and laminin 1 and 8.
Laminin 8 and Laminin 10 Purification
Using ion exchange and affinity chromatography employing laminin
chain-specific mAbs produced in our laboratory, intact mouse laminin 8 and human laminin 10 were purified from conditioned media of mouse 3T3
and MC3T3-G2/PA6 cells, and human placenta, respectively. Laminin 8 was
prepared using a rat mAb specific for mouse laminin 2-integrins play an indisputable role in adhesion
of polymorphonuclear granulocytes (PMN) to endothelium, we show here
that
1- and
3-integrins but not
2-integrin are essential for the adhesion to and
migration on extracellular matrix molecules of the endothelial cell
basement membrane and subjacent interstitial matrix. Mouse wild type
and
2-integrin null PMN and the progranulocytic cell
line 32DC13 were employed in in vitro adhesion and
migration assays using extracellular matrix molecules expressed at
sites of extravasation in vivo, in particular the
endothelial cell laminins 8 and 10. Wild type and
2-integrin null PMN showed the same pattern of ECM
binding, indicating that
2-integrins do not mediate
specific adhesion of PMN to the extracellular matrix molecules tested; binding was observed to the interstitial matrix molecules, fibronectin and vitronectin, via integrins
5
1 and
v
3, respectively; to laminin 10 via
6
1; but not to laminins 1, 2, and 8, collagen type I and IV, perlecan, or tenascin-C. PMN binding to
laminins 1, 2, and 8 could not be induced despite surface expression of functionally active integrin
6
1, a major
laminin receptor, demonstrating that expression of
6
1 alone is insufficient for ligand
binding and suggesting the involvement of accessory factors.
Nevertheless, laminins 1, 8, and 10 supported PMN migration, indicating
that differential cellular signaling via laminins is independent of the
extent of adhesion. The data demonstrate that adhesive and nonadhesive
interactions with components of the endothelial cell basement membrane
and subjacent interstitium play decisive roles in controlling PMN
movement into sites of inflammation and illustrate that
2-integrins are not essential for such interactions.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2- and
4-integrin families (1). However,
little is known of the subsequent interaction of leukocytes with the
underlying extracellular matrix
(ECM)1 proteins of the blood
vessel basement membrane and of the interstitium. A fact frequently
ignored is that blood vessel basement membranes are biochemically
unique and that growth factors and cytokines that affect other aspects
of endothelial cell physiology also alter ECM expression (2-7).
,
, and
chains. To
date 5
, 4
, and 3
laminin chains (2, 8-10) have been
identified that may combine to form at least 12 different isoforms that
have tissue-specific and developmentally regulated expression patterns
(2, 3, 11-14). The data suggest that laminins play significant roles
in adhesion, migration, and differentiation of several cell types.
Further, the occurrence of diseases involving laminin gene defects,
such as epidermolysis bullosa and congenital muscular dystrophies and
the generation of mice lacking particular laminin chains, suggests that
this family of basement membrane molecules play crucial roles in
vivo (reviewed in Ref. 15). Nevertheless, the information
available on the functional significance of individual isoforms remains limited.
4,
1, and
1 chains) is expressed by all endothelial cells
regardless of their stage of development, and its expression is
strongly up-regulated by cytokines and growth factors that play a role
in inflammatory events, such as interleukin-1 or tumor necrosis
factor-
(TNF-
) (2).2
Laminin 10 (composed of laminin
5,
1, and
1 chains) is detectable primarily in basement membranes
of capillaries and venules commencing 3-4 weeks after birth (3, 18).
In contrast to laminin 8, endothelial cell expression of laminin 10 is
up-regulated only by strong proinflammatory signals and, in addition,
angiostatic agents such as progesterone and its derivatives
(19).2 Other extracellular matrix molecules are also
differentially expressed by endothelium, varying with the endothelium
type and/or activation state. Several endothelial cell-specific
proteoglycans and extracellular molecules such as BM40 (SPARC),
fibrinogen, thrombospondin, and fibronectin have been reported to be
regulated by proinflammatory cytokines (20-23). The data speak for a
dynamic endothelial cell extracellular matrix that presents different molecular information depending on the type of endothelium and/or physiological situation.
2-integrins are essential for adhesion of PMN,
monocytes, and lymphocytes to endothelium and also for traversing the
endothelial cell monolayer; however, the molecular interactions
necessary for the subsequent transmigration of the basement membrane
and entry into the subjacent interstitial extracellular matrix remain largely undefined (25, 26). We have previously shown that mouse PMN,
progranulocytic cells, and monocytes express functionally active
1- and
3-integrins on their surface that
are employed for specific interactions with ECM molecules of basement
membranes and the interstitium (27). Integrins of the
2
class, in contrast, were shown to mediate a general stickiness to many
ECM molecules and even to bovine serum albumin and plastic (27-29).
The aim of the present study is to define the role of cell-matrix
interactions in PMN migration into extravascular tissues by using
2-integrin null PMN. Comparisons have been made with
mouse wild type PMN and the progranulocytic cell line 32DC13. In
contrast to previous studies, the extracellular matrix molecules
employed here in in vitro adhesion and migration assays
reflect those expressed at sites of extravasation in vivo,
with emphasis on the recently identified endothelial cell laminin
isoforms, laminins 8 and 10.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-integrin (CD18) null mice (26) and their wild type
littermates as previously described (27). The mouse granulocyte
progenitor cell line 32DC13 (30) was supplied by G. Rovera (Wistar
Institute, Philadelphia, PA). 32DC13 and the human fibrosarcoma cell
line, HT1080 (American Type Culture Collection number CCL121), were
maintained in Dulbecco's modified Eagle's medium supplemented with
Glutamax and 10% fetal calf serum. In the case of 32DC13, 10%
conditioned medium from the murine myelomonocytic cell line, WEHI 3B
(31), was also added. 3T3 fibroblasts (American Type Culture
Collection number CCL163) and the MC3T3-G2/PA6 preadipocyte cell line
(PA6M) (32), employed for laminin 8 purification, were grown in
Dulbecco's modified Eagle's medium supplemented with Glutamax plus
10% fetal calf serum. All cells were grown at 37 °C in 5%
CO2.
1 (198) (11),
2 (4H8-2) (33), and
5 (4G6) (3); affinity purified rabbit polyclonal
antibodies to mouse laminin
4 (377) (18),
5 (405), and laminin 1 (308) (which recognizes laminin
1,
1, and
1 chains) (11);
and mouse mAbs to human laminin
2 (C4),
1
(D18) (34), and
5 (4C7) (35) (Hybridoma Bank, Iowa
City, Iowa).
1-integrin (Ha2/5) (Pharmingen); rat anti-mouse
2-integrin (C71/16)
(Pharmingen); hamster anti-mouse
3-integrin (2C9G2)
(Pharmingen); rat mAbs to mouse
6-integrin (GoH3)
supplied by A. Sonnenberg (Amsterdam) and mouse
5-integrin (5H10) (Pharmingen); rabbit polyclonal
anti-human integrin
3 supplied by G. Tarone (Turin);
goat anti-human integrin
3 (C-18) (Santa Cruz
Biotechnology); rabbit polyclonal anti-human
2-integrin
(R218) (37); rat anti-mouse
4-integrin (R1-2) (38); and
rat anti-mouse L-selectin (Mel-14) (39).
4,
1, and
1 chains) was isolated from the conditioned media of 3T3
fibroblasts and the MC3T3-G2/PA6 preadipocyte cell line using a
combination of an ion exchange chromatography (POROS 20 HQ column)
and immunoaffinity chromatography (CNBr-Sepharose) with a rat mAb to
laminin
1 (3E10).2 Laminin 10 was isolated
from human placenta by affinity chromatography using mouse anti-human
1 (D18) and, subsequently, mouse anti-human laminin
5 (4C7) antibodies.2 Mouse anti-human
laminin
2 (C4) antibody was employed to immunoabsorb laminin
2-containing complexes where necessary. The
purity of the laminin 8 and 10 preparations was assessed by
immunoblotting and enzyme-linked immunosorbent assay using laminin
chain-specific antibodies as described previously (11). For comparison,
a commercial source of human laminin 10/11 (Life Technologies, Inc.)
was also employed in adhesion and migration assays.
v-integrins; and
cyclic RbADfV (EMD69601) control peptide (43, 44). Cyclic peptides were
provided by Dr. A. Jonczyk (Merck KGaA; European Patent EPO
578.083A2).
A405 of BSA-bound cells)/A405 of 50000 applied) × 100 = % Specific Binding.
2-integrin null PMN were
used in these experiments (for reasons outlined under "Results").
1 × 105 cells in attachment buffer (Dulbecco's
modified Eagle's medium, 0.5% BSA, pH 7.5) were added to the upper
chamber, and 10
7 M fMLP, a physiological
chemotactic factor for PMN, was added simultaneously to the bottom
chamber. Control experiments were performed in the absence of fMLP.
Cells were incubated at 37 °C for 2 h, subsequently collected,
and then counted microscopically.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1
(3E10) in affinity chromatography (Fig.
1, lane 3), whereas human
laminin 10 was isolated using mouse mAbs to the human laminin
1 (D18) and
5 (4C7) chains (Ref. 34 and Fig. 1, lane 7). Immunoblot analysis of the isolated laminin
isoforms revealed the presence of only laminin
4,
1, and
1 chains in the laminin 8 preparation, whereas the human laminin 10 preparation contained
predominantly laminin
5,
1, and
1 chains (laminin 10) and trace amounts of laminin
2
chain (laminin 11) as detected by immunoblots and enzyme-linked
immunosorbent assay (data not shown). For comparison, a commercially
available human laminin 10/11 (Life Technologies, Inc.) preparation
isolated from pepsin-digested placenta by affinity chromatography using
the 4C7 mAb and widely used in in vitro assays is also shown
(Fig. 1, lane 5). In contrast to laminin 10 prepared in our
laboratory, the intact 400-kDa laminin
5 chain (3) was
not detectable in the commercial preparation of laminin 10/11, and
several proteolytic fragments were present (Fig. 1, lanes 5 and 6). Immunoblots of commercial laminin 10/11 revealed the
laminin
5 chain as a major 200-220-kDa band plus minor
bands of 125 and 97 kDa (Fig. 1, lane 6). This commercial preparation of laminin 10/11 was found to be significantly more adhesive than that prepared in our laboratory. Most experiments were
therefore carried out with the laminin 10 preparation from our
laboratory, which better reflects the laminin isoform found in blood
vessel basement membranes.
View larger version (28K):
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Fig. 1.
Silver-stained, 5% acrylamide gels run under
reducing conditions showing mouse laminins 1 (LM1;
lane 1), 2 (LM2; lane
2), and 8 (lane 3), commercial human
laminin 10/11 (lane 5), and human laminin 10 isolated
in our laboratory (lane 7). Corresponding
immunoblots (IB) are shown for laminin 8 (lane 4)
performed with a polyclonal anti-mouse laminin 4
antibody (377), revealing the 220-kDa
4 chain, and for
the two laminin 10 preparations performed with polyclonal anti-mouse
laminin
5 antibody (405) (lanes 6 and
8). The typical laminin
5 doublet of slightly
under 400 kDa (marked in lane 8), which probably reflects
different glycosylation forms of the chain (see Ref. 12), is not
detectable in the commercial laminin 10/11 (asterisk in
lanes 5 and 6). In the immunoblot of commercial
laminin 10/11 the laminin
5 chain appears as a major
200-220-kDa band plus minor bands of 125 and 97 kDa (lane
6). The high molecular mass band in lane 2 (approximately 600 kDa) is typical of laminin 2 and probably reflects
dimers of the laminin
2 plus a
1 or
1 chain. The prominent 150-kDa band in lane 2 is nidogen (nd), a laminin-associated molecule.
MWt, molecular mass marker.
Integrin Expression on 2-Integrin Null PMN
Flow cytometric analysis revealed no difference in the
surface expression of 1- and
3-integrins between wild type and
2-integrin null PMN (Table
I) and confirmed the absence of the
2-integrin from the surface of the latter cells (Fig.
2). Moderate levels of integrins
6
1,
5
1, and
4
1 occurred on the cell surface of both
wild type and
2-integrin null PMN, which did not change upon activation with PMA (Fig. 2);
2
1 or
3
1 were confirmed undetectable on either
cell type either before (45) or after activation (Fig. 2) despite the
fact that the antibodies employed are functional in flow cytometric
analysis on other cell types (37, 46). Similar results are obtained
with 32DC13 cells, as previously described (27). Integrin
3 was expressed on wild type (Table I),
2-integrin null PMN (Fig. 2 and Table I), and 32DC13
cells (Table I). Activation with PMA markedly increased integrin
2 expression in wild type but not in
2-integrin null PMN (Fig. 2 and Table I). Treatment with
PMA strongly reduced L-selectin expression on
2-integrin
null PMN (Fig. 2 and Table I), wild type PMN, and 32DC13 (27),
indicating effective activation of the cells. Treatment of cells with
fMLP had no effect on the surface expression of integrins (Table I)
(27).
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Specific Cell Adhesion to ECM Molecules
We have previously reported that 2-integrins confer
a general stickiness to PMN resulting in strong adhesion to many
substrates, including BSA and plastic (27), and also extracellular
matrix molecules, such as collagen types I and IV (47, 48). This strong
adhesion makes it impossible to identify
non-
2-integrin-mediated adhesive processes. Ablation of
the
2-integrin significantly reduced this
"background" binding from 50-60% in activated wild type PMN to
below 10% in the
2-integrin null PMN (Fig.
3), confirming the promiscuous nature of
2-integrin on PMN. The absence of this background
binding in the case of
2-integrin null PMN permitted clear identification of adhesive and nonadhesive substrates.
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To best assess the role of PMN-matrix interactions in the extravasation process, the following ECM molecules were employed in all in vitro adhesion assays: (i) typical endothelial cell basement membrane components: laminins 8 and 10, collagen type IV, and perlecan, (ii) basement membrane components that do not occur in endothelial cell basement membranes: laminins 1 and 2, and (iii) interstitial matrix molecules that are up-regulated in the interstitial matrix surrounding blood vessels during inflammation: collagen type I, tenascin-C, fibronectin, and vitronectin.
In the nonactivated or the fMLP-treated state, wild type and
2-integrin null PMN showed the same qualitative pattern
of specific binding to the interstitial matrix molecules, fibronectin
and vitronectin, and the basement membrane molecule laminin 10 (Table II). No specific binding to laminins 1, 2, and 8, collagen types I and IV, tenascin-C, or perlecan was measured
(Table II). Coatings of the basement membrane heparan sulfate
proteoglycan, perlecan, or of the basement membrane collagen type IV
repelled cells from coated surfaces, as previously reported for U937
and human erythroleukemic cells lines (49) and for wild type PMN
(27).
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Concentration-dependent binding of nonactivated and
PMA-activated 2-integrin null PMN to fibronectin (Fig.
4A), vitronectin (Fig.
4B) and laminin 10 (Fig.
5A) is shown and revealed
statistically significant increased maximal binding values
(Bmax) after PMA stimulation (p < 0.005 for all three substrates, Student's t test). However, no significant difference was observed in the approximate binding affinities (1/Bmax) for these three
substrates before and after PMA activation (data not shown). Wild
type PMN binding to fibronectin, vitronectin, and laminin 10 was
slightly reduced after PMA activation (Table II), as previously
reported for fibronectin and vitronectin (27).
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The progranulocytic cell line 32DC13 showed significant binding only to fibronectin (35%) and laminin 10 (32%) in the nonactivated or fMLP-treated state (Table II). Upon PMA activation the extent of binding to fibronectin and laminin 10 increased significantly, and an additional binding to vitronectin was induced (Table II). In contrast to fully mature PMN, PMA activation of 32DC13 also induced low level and low affinity binding to laminins 1 (18%), 2 (10%), and 8 (24%) (Table II). Binding to all three laminin isoforms was statistically significantly above background binding to BSA. No binding of 32DC13 to collagen types I and IV, perlecan, or tenascin-C was detectable before or after activation (Table II).
Integrin Receptors Involved in ECM Binding
To determine the cellular receptors mediating adhesion to the
extracellular substrates, inhibition studies were carried out using
nonactivated, fMLP-treated, and PMA-activated wild type or
2-integrin null PMN and 32DC13.
Fibronectin--
Cell binding to fibronectin can be mediated by
5
1, by
4
1,
or by the
v series integrins (50).
4
1 binding to fibronectin is not
RGD-dependent, whereas both
5
1 and the
v series
integrins bind to the RGD sequence in fibronectin. However,
v-mediated interactions with fibronectin are very
sensitive to inhibition by linear RGD peptides (<10 µM),
and
5
1-dependent binding is inhibited only at high concentrations of linear RGD (>100
µM) (27, 43). Because function-blocking antibodies exist
only for mouse
4- and
1-integrins but not
5- and
v-integrins, we exploited the
differential sensitivity to linear RGD peptides to characterize the
interaction of the mouse cells with fibronectin. Specific binding of
wild type and
2-integrin null PMN and 32DC13 to
fibronectin was measured in the presence of anti-mouse
4
mAb (R1-2), anti-
1 polyclonal antibody (Ha2/5), a chick
antibody against the RGD-cell binding site in fibronectin that is
specifically recognized by the
5
1
integrin, linear RGD, and cyclic RGD peptides (EMD66203 and EMD69601).
Binding of wild type and 2-integrin null PMN and 32DC13
to fibronectin was significantly inhibited only by high concentrations of linear RGD (>100 µM) (27), the
anti-
1-integrin antibody (Ha2/5), and the antibody
against the RGD-fibronectin cell binding site. The chicken antibody
inhibited specific binding to fibronectin only at concentrations of 300 µg/ml; however, similar concentrations of purified preimmune chick
IgG did not inhibit binding to fibronectin. Cyclic RGD peptides
(EMD6603/EMD69601) at concentrations up to 100 µM and the
anti-integrin
4 antibody (R1-2) (10-50 mg/ml) had
no effect on binding, suggesting
5
1-mediated adhesion to fibronectin. An
example of the results is shown for
2-integrin null PMN
in Fig. 4C.
Vitronectin--
Binding to vitronectin is mediated by the
v series integrins that interact with high affinity to
the RGD sequence in this molecule. Cyclic RGD peptides have been
described that specifically inhibit murine
v
integrin
interactions with vitronectin (27, 43, 51). Further,
v-mediated interactions are very sensitive to inhibition
by linear RGD peptides that abolish cell binding at concentrations of
<50 µg/ml (27, 43, 51). The effects of 10-100 µM
cyclic RGDfV peptide (EMD66203), control cyclic R
ADfV peptide
(EMD69601), linear RGD, and antibodies against
1-
(Ha2/5) and
3-integrins (2C9G2) were therefore tested
for their ability to inhibit cell adhesion to vitronectin. The cyclic
RGDfV peptide (EMD66203) and linear RGD significantly inhibited binding
of all three cell types to vitronectin at concentrations of 10 µM. The absence of effect of the
1-integrin antibody (up to 100 µg/ml) and the
inhibition of binding by the 10 µg/ml
3-integrin
antibody suggested
v
3-mediated adhesion
to vitronectin. An example of the data for
2-integrin
null PMN is shown in Fig. 4D.
Laminin--
Binding to laminins 1, 2, 8, and 10/11 has been shown
to occur principally via the 1-integrins, in particular
6
1,
7
1, and
3
1, depending on the cell type (8, 50,
52, 53), but also
6
4 (54). Flow cytometry
analysis revealed the presence of only
6
1
on all cells investigated here, and previous studies have shown that
7
1 and
3
1
do not occur on PMN (27, 55, 56). Accordingly, anti-integrin
6 (GoH3) and anti-integrin
1 (Ha2/5)
showed concentration-dependent inhibition of binding of
wild type PMN,
2-integrin null PMN, and 32DC13 to
laminin 10 with complete inhibition at antibody concentrations
of 10 µg/ml (data for
2-integrin null PMN are shown in
Fig. 5C). In the case of PMA-activated 32DC13, binding to
laminin 8 and, as previously shown (27), to laminin 1 was also
inhibited by both anti-integrin
1 and
6
antibodies (data not shown).
Integrin 6
1 is considered to be a
specific receptor for laminin 1 (8). However, despite the constitutive
expression of integrin
6
1 on the cell
surface of wild type and
2-integrin null PMN, binding to
laminins 1, 2, or 8 could not be induced either by cellular activation
by PMA or maximal activation of integrins with 10 mM
Mn2+. An example of these data is shown for
2-integrin null PMN on laminin 1 in Fig. 5C.
The absence of cell binding was not related to the ability of the
different laminins to bind plastic or due to degradation of the
laminins, because 32DC13 bound to laminins 1 and 8 in an
6
1-dependent manner (data not
shown). This was investigated further by control experiments performed
with the human fibrosarcoma cell line, HT1080, which showed extensive
binding (90% specific adhesion) to laminin 1 via integrin
6
1 (Fig. 5D) (57, 58) and
non-integrin
6
1-dependent
binding to laminin 10 (Fig. 5D). This suggests that other
factors or accessory molecules on the surfaces of cells are involved in
regulating
6
1-dependent recognition of different laminin isoforms.
Migration Assays
Transmigration across laminin-, fibronectin-, or
vitronectin-coated filters in response to a chemotactic gradient of
fMLP permitted quantification of rates of migration. Only
2-integrin null PMN could be used in these experiments,
because the high background binding of wild type PMN masked differences
between substrates, and 32DC13 could not be induced to migrate.
Significantly more transmigration was measured in the presence of fMLP
on all substrates, except commercial laminin 10/11 and BSA (Fig.
6), both of which supported only minimal
rates of migration. In the presence of a fMLP gradient,
2-integrin null PMN migrated most efficiently and at
similar rates across laminin 10- (~200 cells/min), fibronectin- (220 cell/min), and vitronectin-coated filters (219 cells/min). Student
Newman Keuls post hoc test revealed no statistically significant
difference in the rates of migration across these three substrates,
whereas significantly lower rates of migration (p < 0.005) were measured across laminin 8- (65 cells/min) and laminin
1-coated (40 cells/min) filters (Fig. 6). Rates of PMN transmigration
across laminin 1- or 8-coated filters were not significantly different.
PMN transmigration of laminin 10- and 8-coated filters was
significantly reduced by anti-integrin
1 (Ha2/5) and
6 (GoH3) at concentrations of 10 µg/ml (Fig. 6), implicating the
6
1 integrin in the
transmigration process. However, on laminin 1 or anti-integrin
1 or
6 antibodies had no statistically significant effect (Fig. 6).
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DISCUSSION |
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PMN interaction with extracellular matrix molecules of the endothelial cell basement membrane and the underlying interstitium is essential for their migration into inflamed tissues. Although attempts have been made to understand this process using in vitro assays similar to those employed in the present study, in the past little consideration has been given to whether the extracellular matrix molecules employed in such studies occur at sites of inflammation, either in endothelial cell basement membranes or in the interstitial matrix (3, 16, 59, 60).
A further controversy in the published data is the relative
contribution of 1-integrins to PMN extravasation
processes as compared with
2-integrins. Although the
expression of functionally active
1-integrin on the PMN
surface is gaining acceptance (27, 28, 61), it still remains difficult
to investigate
1-integrin function on the PMN without
the complication of
2-integrin-mediated background
adhesion. In the present study we have attempted to more precisely
define PMN-ECM interactions in the extravasation process (1) by
eliminating
2-integrin-mediated interactions through the
use of PMN isolated from
2-integrin null mice (26), and
(2) by employing basement membrane and interstitial matrix molecules
known to occur around blood vessels at sites of inflammation in
in vitro adhesion and migration assays. Our choice of
extracellular matrix molecules for in vitro studies was
based on occurrence in endothelial cell basement membranes (laminins 8 and 10, collagen type IV, and perlecan) and in the interstitium of
inflamed tissues (tenascin-C, fibronectin, and vitronectin), as
compared with ECM molecules that do not occur in association with blood
vessels (laminins 1 and 2).
Laminins are difficult to isolate in pure forms because of their large
size and tendency to self-aggregate. As a consequence, commercial
preparations of laminins are frequently prepared using proteolytic
digestion and subsequent affinity chromatography, resulting in
truncations of laminin chains and digestion products. Our analysis of
such a commercial laminin 10/11 preparation clearly demonstrated the
presence of severely truncated laminin 5 chain and
several digestion products. In view of the fact that the laminin
chains carry the cell-binding domains (62), such commercial preparations should be used with caution. Proteolytic digestion may
result in the exposure of epitopes that are masked in vivo and that may have different properties to the intact native molecule. Using chain-specific mAbs, we have purified biologically active laminins 8 and 10, the only two laminin isoforms to date known to occur
in blood vessel basement membranes. The laminin 10 preparation, isolated without proteolytic digestion and by affinity chromatography using anti-laminin
1 and
5 chain mAbs,
was less adhesive than commercial laminin 10/11 (data not shown) and
supported high rates of PMN transmigration. In our hands, the
commercial preparation of laminin 10/11 did not support PMN
transmigration; a similar result has been recently reported for human
monocytes (63) and is discussed in detail below. These differences may
be due to the absence of a full-length laminin
5 chain
in the commercial laminin 10/11 and/or the exposure of additional
binding sites caused by proteolytic digestion. Alternatively, these
functional differences may be related to the fact that the commercial
laminin is a mixture of laminin 10 (
5
1
1) and 11 (
5
2
1), whereas the laminin
10 preparation prepared in the present study contained principally
laminin 10 and only trace amounts of laminin 11. Studies on the
functional differences between laminin 10 and 11 are currently in progress.
Interestingly, the pattern of specific ECM binding of wild type and
2-integrin null PMN did not differ significantly,
indicating that the
2-integrins do not mediate specific
binding to any of the extracellular matrix molecules tested here.
Transmigration experiments also clearly showed that
2-integrin is not essential for migration of PMN across
extracellular matrix substrates. Our data show that components of the
nonendothelial cell basement membranes, such as laminin 1 and 2, do not
support adhesion of mature PMN regardless of the cellular activation
state. However, components of endothelial cell basement membranes and
of the interstitial matrix in inflamed tissues provide both adhesive
and nonadhesive substrates that probably control how and when PMN
migrate into inflamed tissues and whether they become arrested at such sites.
The data also demonstrate that despite the absence of strong adhesion,
integrins can still signal specific information to cells. Even though
PMN bound strongly to laminin 10, fibronectin, and vitronectin and not
at all to laminins 1 and 8, all substrates supported fMLP-induced
transmigration, albeit at significant reduced rates in the case of the
latter two substrates. Monocytes have recently been shown to
transmigrate across laminin 8-coated but not laminin 10-coated filters
(63). The difference from our data is very likely to be related to the
source of laminin 10/11 employed because Pedraza et al. (63)
used the commercial laminin 10/11, which we also found to be a poor
substrate in transmigration studies. It cannot be excluded, however,
that the migratory behavior of monocytes differs from that of PMN, as
implied by the fact that monocytes show significant rates of migration
across laminin-coated filters even in the absence of a chemotactic
gradient (63), whereas PMN do not. It is interesting to note that the
rate of monocyte transmigration across recombinant laminin 8, composed of human laminin 4 and
1 and mouse
laminin
1 chains (53), employed in the monocyte study
was comparable with that measured in the present study using native
mouse laminin 8 and PMN. Whether these two laminin 8 preparations
support cell adhesion equally is difficult to assess because Pedraza
et al. (63) used toluidine blue staining of adherent cells
as a measure of relative adhesion rather than the quantitative cell
adhesion assays used here.
The laminin cell adhesion and migration data presented here demonstrate
that different laminin isoforms probably all bind and signal through
6
1, and it may be that the number of
laminin molecules bound, the differential strength of adhesion, and/or the involvement of accessory molecules determines the cellular response. A similar observation has been made with mouse melanoma cells
that migrate on laminin 1 using
6
1
without strong adhesion (64) and Jurkat T-cells, which do not bind to
laminin 1 but respond to interactions with this molecule with distinct
intracellular Ca2+ signals (65). Whether differential
intracellular signals are induced in PMN by adhesive and nonadhesive
substrates is currently under investigation. The fact that
2-integrin null PMN can bind to and migrate on laminin
substrates using
1-integrins also demonstrates that
2-integrin is not essential for this function. The
absence of PMN in inflamed tissues in the
2-integrin
null mouse (26) and in LAD-1 patients (66) is, therefore, exclusively
due to the inability of these cells to penetrate the endothelial
monolayer and not the underlying basement membrane.
Integrin 6
1 has been described as a
specific receptor for laminin 1 (reviewed in Ref. 8) and laminin 8 (52,
53, 63) on several cell types, including HT1080 and the progranulocytic cell line 32DC13 as shown here. Nevertheless, despite ubiquitous expression of significant levels of this receptor on the PMN surface, binding to laminin 1 or 8 could not be induced even under maximal cell
(PMA) or integrin (Mn2+) activation conditions. This
suggests that the ability to distinguish between laminin isoforms is
dependent upon other factors/accessory molecules that affect
6
1 binding activity. Indeed,
6
1 (and
3
1)
has been shown to be associated with the tetraspan family of molecules,
which have been implicated in cell adhesion and migration (reviewed in
Refs. 67 and 68) and which occur on PMN cell surfaces (69, 70). Whether
such integrin-associated molecules are involved in the
6
1-laminin interactions in the case of
PMN is currently under investigation.
Our data confirm the promiscuous nature of 2-integrins,
which, when activated, mediate nonspecific binding to several
substrates. Such high affinity, broad specificity adhesion may be
advantageous for the adhesion to and migration across the endothelial
monolayer but not for the regulated adhesion to ECM molecules necessary for migration across the basement membrane and into the interstitium. The data presented here suggest that the differential extent and strength of PMN adhesion to different ECM molecules provides the balance of adhesive and nonadhesive forces necessary for penetration of
basement membranes at sites of inflammation and that such interactions are mediated by the
1- and
3-integrins
but not
2-integrins. It has been shown that
1-integrins are up-regulated on PMN after transmigration
of endothelial cell monolayers both in vitro and in
vivo (17, 28). It is therefore possible that PMN surface integrin
expression and/or activity is altered during transmigration of the
endothelial cell monolayer, such that
1- and
3-integrin activity predominates in the subsequent ECM
transmigration steps.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. T. Winkler for carrying out flow cytometry analysis, Dr. Simon Goodman for providing cyclic RGD peptides and helpful discussions, and Stefanie Karosi and Thomas Samson for critical review of the manuscript. This work would not have been possible without the expert technical assistance of Friederike Pausch.
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FOOTNOTES |
---|
* This work was supported by Deutsche Forschungsgemeinschaft Grants So285/1-2 and So285/1-3 (to L. M. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Interdisciplinary
Center for Clinical Research, Nikolaus Fiebiger Center,
Glückstr. 6, 91054 Erlangen, Germany.
Published, JBC Papers in Press, March 14, 2001, DOI 10.1074/jbc.M010898200
2 Sixt, M., Hallmann, R., Engelhardt, B., Wendler, O., and Sorokin, L. M. (2001) J. Cell Biol., in press.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: ECM, extracellular matrix; PMN, polymorphonuclear granulocyte(s); PMA phorbol 12-myristate 13-acetate, BSA, bovine serum albumin; mAb, monoclonal antibody; fMLP, formylmethionylleucylphenylalanine.
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