Cell Adhesion and Migration Properties of beta 2-Integrin Negative Polymorphonuclear Granulocytes on Defined Extracellular Matrix Molecules

RELEVANCE FOR LEUKOCYTE EXTRAVASATION*

Michael SixtDagger , Rupert Hallmann§, Olaf WendlerDagger , Karin Scharffetter-Kochanek, and Lydia M. SorokinDagger ||

From the Dagger  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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta 2-integrins play an indisputable role in adhesion of polymorphonuclear granulocytes (PMN) to endothelium, we show here that beta 1- and beta 3-integrins but not beta 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 beta 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 beta 2-integrin null PMN showed the same pattern of ECM binding, indicating that beta 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 alpha 5beta 1 and alpha vbeta 3, respectively; to laminin 10 via alpha 6beta 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 alpha 6beta 1, a major laminin receptor, demonstrating that expression of alpha 6beta 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 beta 2-integrins are not essential for such interactions.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta 2- and alpha 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).

One of the major components of basement membranes is the laminin family of molecules, heterotrimers composed of alpha , beta , and gamma  chains. To date 5alpha , 4beta , and 3gamma 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.

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 alpha 4, beta 1, and gamma 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-alpha (TNF-alpha ) (2).2 Laminin 10 (composed of laminin alpha 5, beta 1, and gamma 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.

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 beta 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 beta 1- and beta 3-integrins on their surface that are employed for specific interactions with ECM molecules of basement membranes and the interstitium (27). Integrins of the beta 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 beta 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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cells-- PMN were isolated from femurs of beta 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.

ECM and Integrin Antibodies-- The antibodies to extracellular matrix proteins employed were rat monoclonal antibodies (mAbs) to mouse laminin alpha 1 (198) (11), alpha 2 (4H8-2) (33), and alpha 5 (4G6) (3); affinity purified rabbit polyclonal antibodies to mouse laminin alpha 4 (377) (18), alpha 5 (405), and laminin 1 (308) (which recognizes laminin alpha 1, beta 1, and gamma 1 chains) (11); and mouse mAbs to human laminin beta 2 (C4), gamma 1 (D18) (34), and alpha 5 (4C7) (35) (Hybridoma Bank, Iowa City, Iowa).

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 beta 1-integrin (Ha2/5) (Pharmingen); rat anti-mouse beta 2-integrin (C71/16) (Pharmingen); hamster anti-mouse beta 3-integrin (2C9G2) (Pharmingen); rat mAbs to mouse alpha 6-integrin (GoH3) supplied by A. Sonnenberg (Amsterdam) and mouse alpha 5-integrin (5H10) (Pharmingen); rabbit polyclonal anti-human integrin alpha 3 supplied by G. Tarone (Turin); goat anti-human integrin alpha 3 (C-18) (Santa Cruz Biotechnology); rabbit polyclonal anti-human alpha 2-integrin (R218) (37); rat anti-mouse alpha 4-integrin (R1-2) (38); and rat anti-mouse L-selectin (Mel-14) (39).

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 alpha 4, beta 1, and gamma 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 gamma 1 (3E10).2 Laminin 10 was isolated from human placenta by affinity chromatography using mouse anti-human gamma 1 (D18) and, subsequently, mouse anti-human laminin alpha 5 (4C7) antibodies.2 Mouse anti-human laminin beta 2 (C4) antibody was employed to immunoabsorb laminin beta 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.

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 alpha 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).

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 - A405 of BSA-bound cells)/A405 of 50000 applied) × 100 = % Specific Binding.

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 beta 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.

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.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 gamma 1 (3E10) in affinity chromatography (Fig. 1, lane 3), whereas human laminin 10 was isolated using mouse mAbs to the human laminin gamma 1 (D18) and alpha 5 (4C7) chains (Ref. 34 and Fig. 1, lane 7). Immunoblot analysis of the isolated laminin isoforms revealed the presence of only laminin alpha 4, beta 1, and gamma 1 chains in the laminin 8 preparation, whereas the human laminin 10 preparation contained predominantly laminin alpha 5, beta 1, and gamma 1 chains (laminin 10) and trace amounts of laminin beta 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 alpha 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 alpha 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.


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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 alpha 4 antibody (377), revealing the 220-kDa alpha 4 chain, and for the two laminin 10 preparations performed with polyclonal anti-mouse laminin alpha 5 antibody (405) (lanes 6 and 8). The typical laminin alpha 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 alpha 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 alpha 2 plus a beta 1 or gamma 1 chain. The prominent 150-kDa band in lane 2 is nidogen (nd), a laminin-associated molecule. MWt, molecular mass marker.

Integrin Expression on beta 2-Integrin Null PMN

Flow cytometric analysis revealed no difference in the surface expression of beta 1- and beta 3-integrins between wild type and beta 2-integrin null PMN (Table I) and confirmed the absence of the beta 2-integrin from the surface of the latter cells (Fig. 2). Moderate levels of integrins alpha 6beta 1, alpha 5beta 1, and alpha 4beta 1 occurred on the cell surface of both wild type and beta 2-integrin null PMN, which did not change upon activation with PMA (Fig. 2); alpha 2beta 1 or alpha 3beta 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 beta 3 was expressed on wild type (Table I), beta 2-integrin null PMN (Fig. 2 and Table I), and 32DC13 cells (Table I). Activation with PMA markedly increased integrin beta 2 expression in wild type but not in beta 2-integrin null PMN (Fig. 2 and Table I). Treatment with PMA strongly reduced L-selectin expression on beta 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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Table I
Integrin subunits expressed by mouse wild type and beta 2-integrin null PMN: summary of flow cytometry analyses
PMA, cells activated with 100 ng/ml PMA; NA, nonactivated (reflects the results obtained with fMLP-treated cells); Receptor density was rated high (+++), intermediate (++), low (+), or not detectable (-).


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Fig. 2.   Flow cytometry analysis of integrin receptor expression on wild type and beta 2-integrin null PMN in nonactivated and PMA-activated conditions. Control is the binding of fluorescently labeled second layer antibody plus appropriate isotype control antibody. The vertical axis shows the cell number, and the horizontal axis shows the log fluorescence intensity. Integrin receptors investigated included beta 1, alpha 3, alpha 5, alpha 6, beta 3, beta 2, alpha 2, and alpha 4 (latter two are not shown). L-selectin expression was used as a measure of PMN activation.

Specific Cell Adhesion to ECM Molecules

We have previously reported that beta 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-beta 2-integrin-mediated adhesive processes. Ablation of the beta 2-integrin significantly reduced this "background" binding from 50-60% in activated wild type PMN to below 10% in the beta 2-integrin null PMN (Fig. 3), confirming the promiscuous nature of beta 2-integrin on PMN. The absence of this background binding in the case of beta 2-integrin null PMN permitted clear identification of adhesive and nonadhesive substrates.


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Fig. 3.   Percentage of total cell adhesion of wild type (open bars) and beta 2-integrin null PMN (shaded bars) to BSA (A) and plastic (B). Data shown are for nonactivated cells (Control) and after activated with 100 ng/ml PMA or 10 mM Mn2+. The values represent the means of at least six separate experiments ± S.E. There was no difference in the results between fMLP-treated and control cells.

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 beta 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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Table II
Mean maximal binding values of nonactivated and PMA-activated leukocytes to extracellular matrix proteins (± S.E.)
Data are the means of at least six separate experiments, expressed as the percentages of specific adhesion at substrates concentrations where adhesion is saturated. NA, nonactivated (reflects the results obtained with fMLP-treated cells); PMA, cells activated with 100 ng/ml PMA; (-), no binding.

Concentration-dependent binding of nonactivated and PMA-activated beta 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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Fig. 4.   Specific cell adhesion of nonactivated (black-diamond ) and PMA-activated () beta 2-integrin null PMN to increasing molar concentrations of fibronectin (A) and vitronectin (B) and of PMA-activated beta 2-integrin null PMN to 30 nM fibronectin (C) or 30 nM vitronectin (D) in the presence or absence (Control) of added inhibitors. Inhibitors employed included linear RGD peptide, 10 µM control cyclic RbADfV (69601), or alpha v-specific cyclic RGDfV (66203) peptide, anti-beta 3 integrin (2C9G2), a chicken antibody against the RGD-fibronectin cell binding site that is recognized by alpha 5 integrin and associated control chicken IgG, hamster anti-rat beta 1-integrin (Ha2/5) and associated control hamster IgG, anti-alpha 4-integrin (R1-2), or 10 mM EDTA. Data shown were the same for wild type PMN, and the same pattern of results as in C and D was found for nonactivated beta 2-integrin null PMN. Data shown are for one representative experiment where the mean specific adhesion is calculated from at least triplicate values per concentration ± S.D.


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Fig. 5.   Specific binding of nonactivated (black-triangle) and PMA-activated () beta 2-integrin null PMN to increasing molar concentrations of laminin 10 (A) and laminin 8 (B). Insets in A and B show that even at low concentrations (1-5 µM) of laminin 10 cells quickly adhere and spread (5-10 min), whereas on laminin 8 cells remained rounded. C, inhibition of specific binding of nonactivated (NA), PMA-activated, and Mn2+-activated beta 2-integrin null PMN to 30 nM laminin 10 in the absence (Control) or presence 10 µg/ml anti-integrin beta 1 (Ha2/5) or anti-integrin alpha 6 (GoH3), or 10 mM EDTA. Because Ha2/5 is a hamster IgG and GoH3 is a rat IgG, control experiments were performed in the presence of hamster IgG and rat IgG in all activation states; the data for PMA-activated beta 2-integrin null PMN are shown. Specific adhesion of beta 2-integrin null PMN to 30 nM laminin 1 could not be induced by activation of cells with 100 ng/ml PMA or 10 mM Mn2+. The same pattern of results was observed for wild type PMN. D, human HT1080 binding to 30 nM laminins 1 and 10. Binding to laminin 1 but not laminin 10 was inhibited by 5 µg/ml anti-alpha 6 integrin (GoH3). Control refers to a percentage of specific adhesion in the absence of added inhibitors, whereas rat IgG refers to a percentage of specific adhesion in the presence of control rat IgG. Data shown are for one representative experiment where the mean specific adhesion is calculated from at least triplicate values per concentration (Conc.) ± S.D.

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 beta 2-integrin null PMN and 32DC13.

Fibronectin-- Cell binding to fibronectin can be mediated by alpha 5beta 1, by alpha 4beta 1, or by the alpha v series integrins (50). alpha 4beta 1 binding to fibronectin is not RGD-dependent, whereas both alpha 5beta 1 and the alpha v series integrins bind to the RGD sequence in fibronectin. However, alpha v-mediated interactions with fibronectin are very sensitive to inhibition by linear RGD peptides (<10 µM), and alpha 5beta 1-dependent binding is inhibited only at high concentrations of linear RGD (>100 µM) (27, 43). Because function-blocking antibodies exist only for mouse alpha 4- and beta 1-integrins but not alpha 5- and alpha 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 beta 2-integrin null PMN and 32DC13 to fibronectin was measured in the presence of anti-mouse alpha 4 mAb (R1-2), anti-beta 1 polyclonal antibody (Ha2/5), a chick antibody against the RGD-cell binding site in fibronectin that is specifically recognized by the alpha 5beta 1 integrin, linear RGD, and cyclic RGD peptides (EMD66203 and EMD69601).

Binding of wild type and beta 2-integrin null PMN and 32DC13 to fibronectin was significantly inhibited only by high concentrations of linear RGD (>100 µM) (27), the anti-beta 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 alpha 4 antibody (R1-2) (10-50 mg/ml) had no effect on binding, suggesting alpha 5beta 1-mediated adhesion to fibronectin. An example of the results is shown for beta 2-integrin null PMN in Fig. 4C.

Vitronectin-- Binding to vitronectin is mediated by the alpha 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 alpha v-integrin interactions with vitronectin (27, 43, 51). Further, alpha 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 Rbeta ADfV peptide (EMD69601), linear RGD, and antibodies against beta 1- (Ha2/5) and beta 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 beta 1-integrin antibody (up to 100 µg/ml) and the inhibition of binding by the 10 µg/ml beta 3-integrin antibody suggested alpha vbeta 3-mediated adhesion to vitronectin. An example of the data for beta 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 beta 1-integrins, in particular alpha 6beta 1, alpha 7beta 1, and alpha 3beta 1, depending on the cell type (8, 50, 52, 53), but also alpha 6beta 4 (54). Flow cytometry analysis revealed the presence of only alpha 6beta 1 on all cells investigated here, and previous studies have shown that alpha 7beta 1 and alpha 3beta 1 do not occur on PMN (27, 55, 56). Accordingly, anti-integrin alpha 6 (GoH3) and anti-integrin beta 1 (Ha2/5) showed concentration-dependent inhibition of binding of wild type PMN, beta 2-integrin null PMN, and 32DC13 to laminin 10 with complete inhibition at antibody concentrations of 10 µg/ml (data for beta 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 beta 1 and alpha 6 antibodies (data not shown).

Integrin alpha 6beta 1 is considered to be a specific receptor for laminin 1 (8). However, despite the constitutive expression of integrin alpha 6beta 1 on the cell surface of wild type and beta 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 beta 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 alpha 6beta 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 alpha 6beta 1 (Fig. 5D) (57, 58) and non-integrin alpha 6beta 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 alpha 6beta 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 beta 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, beta 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 beta 1 (Ha2/5) and alpha 6 (GoH3) at concentrations of 10 µg/ml (Fig. 6), implicating the alpha 6beta 1 integrin in the transmigration process. However, on laminin 1 or anti-integrin beta 1 or alpha 6 antibodies had no statistically significant effect (Fig. 6).


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Fig. 6.   Transmigration data for beta 2-integrin null PMN. Number of cells transmigrated/min ± S.E. across Transwell filters (5-µm pore size) coated with 15 µg/ml laminins 10, 8, and 1 prepared in our laboratory, commercial laminin 10/11 (LM 10/11), fibronectin (FN), vitronectin (VN), or BSA in the presence (open bars) or absence (gray bars) of fMLP in the lower chamber. 1 × 105 cells were added per filter and allowed to transmigrate for 2 h at 37 °C; the total number of cells was then counted in the lower chamber. Control is transmigration in the absence of added inhibitors; anti-alpha 6 is 5 µg/ml anti-integrin alpha 6 (GoH3); anti-beta 1 is 10 µg/ml hamster anti-integrin beta 1 (Ha2/5); and IgG is 10 µg/ml control hamster. All values represent the means of at least six separate experiments ± S.E.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta 1-integrins to PMN extravasation processes as compared with beta 2-integrins. Although the expression of functionally active beta 1-integrin on the PMN surface is gaining acceptance (27, 28, 61), it still remains difficult to investigate beta 1-integrin function on the PMN without the complication of beta 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 beta 2-integrin-mediated interactions through the use of PMN isolated from beta 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 alpha 5 chain and several digestion products. In view of the fact that the laminin alpha  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 gamma 1 and alpha 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 alpha 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 (alpha 5beta 1gamma 1) and 11 (alpha 5beta 2gamma 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 beta 2-integrin null PMN did not differ significantly, indicating that the beta 2-integrins do not mediate specific binding to any of the extracellular matrix molecules tested here. Transmigration experiments also clearly showed that beta 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 alpha 4 and gamma 1 and mouse laminin beta 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 alpha 6beta 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 alpha 6beta 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 beta 2-integrin null PMN can bind to and migrate on laminin substrates using beta 1-integrins also demonstrates that beta 2-integrin is not essential for this function. The absence of PMN in inflamed tissues in the beta 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 alpha 6beta 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 alpha 6beta 1 binding activity. Indeed, alpha 6beta 1 (and alpha 3beta 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 alpha 6beta 1-laminin interactions in the case of PMN is currently under investigation.

Our data confirm the promiscuous nature of beta 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 beta 1- and beta 3-integrins but not beta 2-integrins. It has been shown that beta 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 beta 1- and beta 3-integrin activity predominates in the subsequent ECM transmigration steps.

    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.

    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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. von Andrian, U. H., and Mackay, C. R. (2000) N. Engl. J. Med. 343, 1021-1034
2. Frieser, M., Nöckel, H., Pausch, F., Röder, C., Hahn, A., Deutzmann, R., and Sorokin, L. M. (1997) Eur. J. Biochem. 246, 727-735[Abstract]
3. Sorokin, L. M., Frieser, M., Pausch, F., Kröger, S., Ohage, E., and Deutzmann, R. (1997) Dev. Biol. 189, 285-300[CrossRef][Medline] [Order article via Infotrieve]
4. O'Reilly, M. S., Boehm, T., Shing, Y., Fukai, N., Vasios, G., Lane, W. S., Flynn, E., Birkhead, J. R., Olsen, B. R., and Folkman, J. (1997) Cell 88, 277-285[Medline] [Order article via Infotrieve]
5. Sasaki, T., Fukai, N., Mann, K., Gohring, W., Olsen, B. R., and Timpl, R. (1998) EMBO J. 17, 4249-4256[Abstract/Free Full Text]
6. Bazzoni, G., Dejana, E., and Lampugnani, M. G. (1999) Curr. Opin. Cell Biol. 11, 573-581[CrossRef][Medline] [Order article via Infotrieve]
7. Vaday, G. G., and Lider, O. (2000) J. Leukocyte Biol. 67, 149-159[Abstract]
8. Delwel, G. O., and Sonnenberg, A. (1996) in Adhesion Receptors as Therapeutic Targets (Horton, M. A., ed) , pp. 9-36, CRC Press, London
9. Galliano, M.-F., Aberdam, D., Aguzzi, A., Ortonne, J.-P., and Meneguzzi, G. (1995) J. Biol. Chem. 270, 21820-21826[Abstract/Free Full Text]
10. Koch, M., Olson, P., Albus, A., Jin, W., Hunter, D. D., Brunken, W., Burgeson, R. E., and Champliaud, M.-F. (1999) J. Cell Biol. 145, 605-617[Abstract/Free Full Text]
11. Sorokin, L. M., Conzelmann, S., Ekblom, P., Aumailley, M., Battaglia, C., and Timpl, R. (1992) Exp. Cell Res. 201, 137-144[Medline] [Order article via Infotrieve]
12. Sorokin, L. M., Pausch, F., Durbeej, M., and Ekblom, P. (1997) Dev. Dynamics 210, 446-462[CrossRef][Medline] [Order article via Infotrieve]
13. Lefebvre, O., Sorokin, L., Kedinger, M., and Simon-Assmann, P. (1999) Dev. Biol. 210, 135-150[CrossRef][Medline] [Order article via Infotrieve]
14. Miner, J. H., Patton, B. L., Lentz, S. I., Gilbert, D. J., Snider, W. D., Jenkins, N. A., Copeland, N. G., and Sanes, J. R. (1997) J. Cell Biol. 137, 685-701[Abstract/Free Full Text]
15. Aumailley, M., and Smyth, N. (1998) J. Anat. 193, 1-21[CrossRef][Medline] [Order article via Infotrieve]
16. Sorokin, L. M., Girg, W., Gopfert, T., Hallmann, R., and Deutzmann, R. (1994) Eur. J. Biochem. 223, 603-610[Abstract]
17. Werb, Z., and Chin, J. R. (1998) Ann. N. Y. Acad. Sci. 857, 110-118[Abstract/Free Full Text]
18. Ringelmann, B., Röder, C., Hallmann, R., Maley, M., Davies, M., Grounds, M., and Sorokin, L. M. (1999) Exp. Cell Res. 246, 165-182[CrossRef][Medline] [Order article via Infotrieve]
19. Tokida, Y., Aratani, Y., Morita, A., and Kitagawa, Y. (1990) J. Biol. Chem. 265, 18123-18129[Abstract/Free Full Text]
20. Kramer, M. D., Gissler, H. M., Weidenthaler-Barth, B., and Preissner, K. T. (1993) in Biology of Vitronectins and Their Receptors (Preissner, K. T. , Rosenblatt, S. , Kost, C. , Wegerhoff, J. , and Mosher, D. F., eds) , pp. 295-301, Elsevier Science Publishers B.V., Amsterdam
21. Nelimarkka, L., Kainulainen, V., Schönherr, E., Moisander, S., Jortikka, M., Lammi, M., Elenius, K., Jalkanen, M., and Järveläinen, H. (1997) J. Biol. Chem. 272, 12730-12737[Abstract/Free Full Text]
22. Kaji, T., Yamada, A., Miyajima, S., Yamamoto, C., Fujiwara, Y., Wight, T. N., and Kinsella, M. G. (2000) J. Biol. Chem. 275, 1463-1470[Abstract/Free Full Text]
23. Rosenblatt, S., Bassuk, J. A., Alpers, C. E., Sage, H., Timpl, R., and Preissner, K. T. (1997) Biochem. J. 324, 311-319[Medline] [Order article via Infotrieve]
24. Springer, T. A. (1994) Cell 76, 301-314[Medline] [Order article via Infotrieve]
25. Smith, C. W., Marlin, S. D., Rothlein, R., Toman, C., and Anderson, D. C. (1989) J. Clin. Invest. 83, 2008-2017[Medline] [Order article via Infotrieve]
26. Scharffetter-Kochanek, K., Lu, H., Norman, K., van Nood, N., Munoz, F., Grabbe, S., McArther, M., Lorenzo, I., Kaplan, S., Ley, K., Smith, C. W., Montgomery, C. A., Rich, S., and Beaudet, A. L. (1998) J. Exp. Med. 188, 119-131[Abstract/Free Full Text]
27. Frieser, M., Hallmann, R., Johansson, S., Vestweber, D., Goodman, S. L., and Sorokin, L. (1996) Eur. J. Immunol. 26, 3127-3136[Medline] [Order article via Infotrieve]
28. Kubes, P., Niu, X. F., Smith, C. W., Kehrli, M. E., Reinhardt, P. H., and Woodman, R. C. (1995) FASEB. J. 9, 1103-1111[Abstract/Free Full Text]
29. Hansell, P., Berger, E., Chambers, J. D., and Arfors, K. E. (1994) J. Leukocyte Biol. 56, 464-468[Abstract]
30. Valtieri, M., Tweardy, D. J., Caracciolo, D., Johnson, K., Mavilio, F., Altman, S., Santoli, D., and Rovera, G. (1987) J. Immunol. 138, 3829-3835[Abstract/Free Full Text]
31. Warner, N. L., Moore, M. A., and Metcalf, D. (1969) J. Natl. Cancer Inst. 43, 963-982[Medline] [Order article via Infotrieve]
32. Gu, Y., Sorokin, L., Durbeej, M., Hjalt, T., Jönsson, J.-I., and Ekblom, M. (1999) Blood 93, 1-11[Free Full Text]
33. Schuler, F., and Sorokin, L. M. (1995) J. Cell Sci. 108, 3795-805[Abstract/Free Full Text]
34. Sanes, J. R., Engvall, E., Butkowski, R., and Hunter, D. D. (1990) J. Cell Biol. 111, 1685-1699[Abstract]
35. Leivo, I., and Engvall, E. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 1544-1548[Abstract]
36. Johansson, S., and Hook, M. (1984) J. Cell Biol. 98, 810-817[Abstract]
37. Dürr, J., Goodman, S., Potocnik, A., von der Mark, H., and von der Mark, K. (1993) Exp. Cell Res. 207, 235-244[CrossRef][Medline] [Order article via Infotrieve]
38. Holzmann, B., McIntyre, B. W., and Weissman, I. L. (1989) Cell 56, 37-46[Medline] [Order article via Infotrieve]
39. Gallatin, W. M., Weissman, I. L., and Butcher, E. C. (1983) Nature 303, 30-34
40. Paulsson, M., Aumailley, M., Deutzmann, R., Timpl, R., Beck, K., and Engel, J. (1987) Eur. J. Biochem. 166, 11-19[Abstract]
41. Paulsson, M., and Saladin, K. (1989) J. Biol. Chem. 264, 18726-18732[Abstract/Free Full Text]
42. Vuento, M., and Vaheri, A. (1979) Biochem. J. 183, 331-337[Medline] [Order article via Infotrieve]
43. Aumailley, M., Gurrath, M., Muller, G., Calvete, J., Timpl, R., and Kessler, H. (1991) FEBS Lett. 291, 50-54[CrossRef][Medline] [Order article via Infotrieve]
44. Pfaff, M., Tangemann, K., Muller, B., Gurrath, M., Muller, G., Kessler, H., Timpl, R., and Engel, J. (1994) J. Biol. Chem. 269, 20233-20238[Abstract/Free Full Text]
45. Werr, J., Johansson, J., Eriksson, E. E., Hedqvist, P., Ruoslahti, E., and Lindblom, L. (2000) Blood 95, 1804-1809[Abstract/Free Full Text]
46. Gullberg, D., Gehlsen, K. R., Turner, D. C., Ahlen, K., Zijenah, L. S., Barnes, M. J., and Rubin, K. (1992) EMBO J. 11, 3865-3873[Abstract]
47. Walzog, B., Schuppan, D., Hempel, C., Hafezi-Moghadam, A., Gaethgens, P., and Ley, K. (1995) Exp. Cell Res. 218, 28-38[CrossRef][Medline] [Order article via Infotrieve]
48. Monboisse, J. C., Garnotel, R., Randoux, A., Dufer, J., and Borel, J. P. (1991) J. Leukocyte Biol. 50, 373-380[Abstract]
49. Klein, G., Conzelmann, S., Beck, S., Timpl, R., and Muller, C. A. (1995) Matrix Biol. 14, 457-465[CrossRef][Medline] [Order article via Infotrieve]
50. Eble, J. A. (1997) in Integrin-Ligand Interaction (Eble, E. A. , and Kühn, K., eds) , pp. 1-40, Springer-Verlag, Heidelberg
51. Brooks, P. C., Montgomery, A. M., Rosenfeld, M., Reisfeld, R. A., Hu, T., Klier, G., and Cheresh, D. A. (1994) Cell 79, 1157-1164[Medline] [Order article via Infotrieve]
52. Talts, J. F., Andac, Z., Göhring, W., Brancaccio, A., and Timpl, R. (1999) EMBO J. 18, 863-870[Abstract/Free Full Text]
53. Kortesmaa, J., Yurchenco, P., and Tryggvason, K. (2000) J. Biol. Chem. 275, 14853-14859[Abstract/Free Full Text]
54. Kikkawa, Y., Sanzen, N., Fujiwara, H., Sonneberg, A., and Sekiguchi, K. (2000) J. Cell Sci. 113, 869-876[Abstract/Free Full Text]
55. Eble, J. A. (1997) in Integrin-Ligand Interaction (Ebel, J. A. , and Kühn, K., eds) , pp. 123-139, Springer-Verlag, Heidelberg
56. Stewart, M., Thiel, M., and Hogg, N. (1995) Curr. Opin. Cell Biol. 7, 690-696[CrossRef][Medline] [Order article via Infotrieve]
57. Aumailley, M., Nurcombe, V., Edger, D., Paulsson, M., and Timpl, R. (1987) J. Biol. Chem. 262, 11532-11538[Abstract/Free Full Text]
58. Aumailley, M., Timple, R., and Sonnenberg, A. (1990) Exp. Cell Res. 188, 55-60[Medline] [Order article via Infotrieve]
59. Ekblom, P. K., G., Ekblom, M., and Sorokin, L. M. (1991) in The Development of the Vascular System (Feinberg, R. N. , Sherer, G. K. , and Auerbach, R., eds), Vol. 14 , pp. 81-92, Karger, Basel
60. Sorokin, L. M., Maley, M., Moch, H., von der Mark, H., von der Mark, K., Karosi, S., Davies, M. J., McGeachie, J. K., and Grounds, M. D. (2000) Exp. Cell Res. 256, 500-514[CrossRef][Medline] [Order article via Infotrieve]
61. Werr, J., Xie, X., Hedqvist, P., Ruoslahti, E., and Lindblom, L. (1998) J. Exp. Med. 187, 2091-2096[Abstract/Free Full Text]
62. Timpl, R., and Brown, J. (1994) Matrix Biol. 14, 275-281[CrossRef][Medline] [Order article via Infotrieve]
63. Pedraza, C., Geberhiwot, T., Ingerpuu, S., Assefa, D., Wondimu, Z., Kortesmaa, J., Tryggvason, K., Virtanen, I., and Patarroyo, M. (2000) J. Immunol. 165, 5831-5838[Abstract/Free Full Text]
64. Hangan, D., Morris, V. L., Boeters, L., von Ballestrem, C., Uniyal, S., and Chan, B. M. (1997) Cancer Res. 57, 3812-3817[Abstract]
65. Weissmann, M., Guse, A., Sorokin, L., Frieser, M., Hallmann, R., and Mayr, G. W. (1997) J. Immunol. 158, 1618-1627[Abstract]
66. Anderson, D. C., and Springer, T. A. (1987) Annu. Rev. Med. 38, 175-194[CrossRef][Medline] [Order article via Infotrieve]
67. Hemler, M. E. (1998) Curr. Opin. Cell Biol. 10, 578-585[CrossRef][Medline] [Order article via Infotrieve]
68. Maecker, H. T., Todd, S. C., and Levy, S. (1997) FASEB J. 11, 428-442[Abstract/Free Full Text]
69. Skubitz, K. M., Campbell, K. D., Iida, J., and Skubitz, A. P. N. (1996) J. Immunol. 157, 3617-3626[Abstract]
70. Canfield, S. M., and Khakoo, A. Y. (1999) J. Immunol. 163, 3430-3440[Abstract/Free Full Text]


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