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The Journal of Immunology, 2001, 166: 7534-7542.
Copyright © 2001 by The American Association of Immunologists

Blockade of {alpha}5{beta}1 Integrins Reverses the Inhibitory Effect of Tenascin on Chemotaxis of Human Monocytes and Polymorphonuclear Leukocytes Through Three-Dimensional Gels of Extracellular Matrix Proteins1

John D. Loike2,*, Long Cao*, Sadna Budhu*, Stanley Hoffman{dagger} and Samuel C. Silverstein*

* Department of Physiology and Cellular Biophysics, Columbia University College of Physicians and Surgeons, New York, NY 10032; and {dagger} Department of Medicine, Division of Rheumatology and Immunology, Medical University of South Carolina, Charleston, SC 29425


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Tenascin is an extracellular matrix protein found in adults in T cell-dependent areas of lymphoid tissues, sites of inflammation, and tumors. We report here that it inhibited chemotaxis of chemoattractant-stimulated human monocytes and chemoattractant-stimulated polymorphonuclear leukocytes (PMN) through three-dimensional gels composed of collagen I or Matrigel, and chemotaxis of leukotriene B4-stimulated PMN through fibrin gels. The inhibitory effect of tenascin on monocyte or PMN chemotaxis through these matrices was reversed by Abs directed against {alpha}5{beta}1 integrins or by a peptide (GRGDSP) that binds to {beta}1 integrins. Tenascin did not affect leukotriene B4- or fMLP-stimulated expression of {beta}1 or {beta}2 integrins, but did exert a small inhibitory effect on PMN adhesion and closeness of apposition to fibrin(ogen)-containing surfaces. Thus, {alpha}5{beta}1 integrins mediate the inhibitory effect of tenascin on monocyte and PMN chemotaxis, without promoting close apposition between these leukocytes and surfaces coated with tenascin alone or with tenascin bound to other matrix proteins. This contrasts with the role played by {alpha}5{beta}1 integrins in promoting close apposition between fMLP-stimulated PMN and fibrin containing surfaces, thereby inhibiting chemotaxis of fMLP-stimulated PMN through fibrin gels. Thus, chemoattractants and matrix proteins regulate chemotaxis of phagocytic leukocytes by at least two different mechanisms: one in which specific chemoattractants promote very tight adhesion of leukocytes to specific matrix proteins and another in which specific matrix proteins signal cessation of migration without markedly affecting strength of leukocyte adhesion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Tenascin-C (1) is a member of a family of three homologous glycoproteins found in extracellular matrices. It is composed of six polypeptides joined together at the N terminus to form a hexameric structure that resembles the spokes of a wheel projecting from a central hub. Each tenascin polypeptide contains an N-terminal series of epidermal growth factor-like domains, a series of fibronectin III-like repeats, and a C-terminal fibrinogen-like globular domain.

Tenascin is widely expressed in embryonic tissues where it is synthesized by many cell types including fibroblasts (2). It regulates cell migration during organogenesis (1, 3) and is thought to play an important role in the development of many tissues (i.e., muscle, tendon, bone, cartilage, hair follicles, teeth, mammary gland, bone marrow, hemopoietic cells, and kidney; Refs. 1, 2, 3, 4). It is largely absent from normal adult tissues except for brain and bone marrow (2), and the T cell-dependent areas of lymphoid organs (4, 5). However, tenascin is re-expressed in adult tissues in a variety of pathological situations. For example, it is expressed at sites of endothelial cell damage (6), in granulation tissue (4, 7), in association with proliferating and migrating epidermal (8) and endothelial cells (9), in lungs undergoing fibrosis (10), in inflamed and scarred human corneas (11), and in macrophage-rich atherosclerotic plaques (12). It is also expressed in the stroma of many tumors (e.g., gliomas, mammary carcinomas, Wilms’s tumor, basal and squamous cell carcinomas, and melanomas) where it is produced by both the tumor and stromal cells (7, 13, 14).

Tenascin-C knockout mice exhibit abnormal neural development (e.g., decreased axon outgrowth, myelination, and synapse formation), decreased fibronectin deposition in wounded skin and cornea, prolonged influx and retention of polymorphonuclear leukocytes (PMN) in dinitro-chlorobenzene (DNCB)-sensitized skin (15), and increased numbers of monocytes/macrophages in the stroma of spontaneously arising mammary tumors (16). The findings that absence of tenascin-C alters PMN and monocyte/macrophage accumulation led us to examine the effects of tenascin-C on PMN and monocyte chemotaxis. We report here that tenascin inhibits chemotaxis of PMN and monocytes through three-dimensional matrices formed of collagen I, reconstituted basement membrane proteins (Matrigel; Collaborative Research, Bedford, MA), or fibrin in response to all chemoattractants tested (i.e., leukotriene B4 (LTB4), IL-8, fMLP, TNF-{alpha}, and monocyte chemoattractant protein 1 (MCP-1)), that arginine-glycine-aspartic acid (RGD)-containing peptides and Abs that block {alpha}5{beta}1 integrins reverse the inhibitory effect of tenascin on PMN and monocyte chemotaxis through these matrices, and that tenascin exerts its migration-inhibitory effects without detectably affecting the expression of {beta}1 or {beta}2 integrins or inducing close apposition between PMN or monocytes and surfaces coated with tenascin alone or in combination with other matrix proteins.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells

PMN were isolated from heparinized human blood as described (17). Contaminating red blood cells were removed by hypotonic lysis. The purity of PMN isolated by this method was >95% as determined by Wright-Giemsa staining. Mononuclear cells were isolated by centrifugation of heparinized human blood on Ficoll-Hypaque gradients as described (18, 19). The mononuclear cell fraction was resuspended in RMPI 1640 medium supplemented with 10% pooled human serum (HS) or autologous serum (AS) and used immediately for monocyte migration studies. For some experiments, monocytes were obtained by centrifugation of whole blood or from a white blood cell concentrate (Leukopak; New York Blood Center, NY) on Nycodenz gradients (Accurate Chemical & Scientific, Westbury, NY) as described (20). Greater than 90% of nucleated cells obtained using Nycodenz were monocytes, as assessed by their phagocytosis of IgG-coated sheep red blood cells (data not shown) and uptake of DiI-labeled acetylated low density lipoprotein (21). Monocytes cultured for 24 h: 107 total blood mononuclear cells, isolated as described above (18, 19), were suspended in 10 ml of RPMI 1640 + HS and incubated in Falcon T-150 tissue culture flasks (BD Biosciences, Franklin Lakes, NJ) for 1 h at 37°C. Nonadherent cells were removed by washing, leaving an adherent cell population consisting of >90% monocyte-derived macrophages as measured by phagocytosis of IgG-coated sheep red blood cells (data not shown) and endocytosis of DiI-labeled acetylated low density lipoprotein (21). These cells were maintained in culture for 24 h in RPMI 1640 + HS or AS, and detached by gentle pipetting of 10 ml of ice-cold PBS containing 1.0 mM Ca2+ and 0.5 mM Mg2+ (without Ca2+ or Mg2+) containing 5.0 mM EDTA. The cells recovered were resuspended in RPMI 1640 + HS as described (19, 21).

Protein-coated inserts

Cell culture inserts composed of polyethylene terephthalate filters, 8-µm pore size (BD Biosciences, Franklin Lakes, NJ), were used as chemotaxis chambers. Where indicated, cell culture inserts were overlaid with 0.1 ml of Matrigel (200–250 µg protein/filter) and maintained overnight at room temperature. Following gelation of the Matrigel, the inserts were washed with PBS containing 1.0 mM Mg2+ and 1.0 mM Ca2+. Inserts coated with rat tail collagen I (ICN Pharmaceuticals, Costa, Mesa, CA) (400 µg/ml in the PBS mixture mentioned above) were prepared by adding 0.1 ml of this solution to each insert and incubating it at room temperature for 24 h. Inserts containing fibrin gels were prepared as described (22, 23). Where indicated, gels were overlaid with 0.1 ml of a PBS solution (pH 7.2) containing the specified amounts of purified chick brain or human tenascin and maintained at room temperature for 2 h to allow adsorption of the tenascin to the gels. All protein-coated gels were washed again with PBS and used within 12 h for cell migration studies.

Cell migration

PMN. PMN (106) in 100 µl of PBS supplemented with 5.5 mM glucose (PBSG) and 0.1% HS albumin (HSA) were placed in the upper compartment of each cell culture insert. PBSG-HSA (500 µl) with or without chemoattractant, as indicated, was added to the bottom compartment. Inserts containing PMN were incubated for 4 h at 37°C in a humidified atmosphere containing 95% air/5% CO2. Maximal PMN migration into the lower chamber of the insert occurred within 4 h (data not shown). It was measured as described (23).

Monocytes and monocyte-derived macrophages. Mononuclear phagocytes (2–10 x 105) in 0.5 ml of RPMI 1640 + HS or AS were placed into a cell culture insert coated with the indicated matrix protein. The same medium (0.5 ml) with or without the indicated chemoattractant was placed in the bottom compartment. The inserts were incubated in a humidified CO2 incubator at 37°C for 24 h. The number of cells that migrated into the lower chamber of the insert was measured using either a Coulter counter (Coulter Pharmaceutical, Seattle, WA) or a hemocytometer. Maximal monocyte migration occurred within 24 h (data not shown).

Tenascin

Chick tenascin was extracted from 14-day embryonic chick brains by homogenization in the presence of protease inhibitors. Extracts were clarified as previously described (24), brought to a final concentration of 0.5 g/ml CsCl, and centrifuged (20 h, 45,000 rpm, 20°C) in a Beckman VAC 50 rotor. Five 8-ml fractions were collected from these gradients. The third and fourth fractions containing partially purified tenascin were pooled, dialyzed vs 10 mM Tris pH 8.0, and incubated with chondroitin ABC lyase (Seikagaka America, Rockville, MD), in the presence of protease inhibitors to degrade contaminating proteoglycans (24). This sample then was lyophilized, resuspended in 4 M guanidine-hydrochloride/0.1 M Tris (pH 7.6), and fractionated on Sephacryl S-500 (Pharmacia, Piscataway, NJ) that had been equilibrated in the same buffer. Tenascin-rich fractions were pooled, dialyzed, lyophilized, resuspended in a small volume of guanidine buffer, and dialyzed extensively vs PBS. This procedure yielded large amounts of purified tenascin that migrated as characteristic 220-, 200-, and 190-kDa polypeptides when analyzed by SDS-PAGE under reducing conditions (25). Human tenascin was obtained from Life Technologies (Grand Island, NY). To remove detergents in this preparation, tenascin was run over a gel filtration column in the presence of 4 M guanidine hydrochloride; the tenascin-containing fractions were recovered and dialyzed vs PBS.

Measurement of matrix-bound tenascin

125I-labeled chick tenascin was prepared using chloramine T (26), and mixed with unlabeled tenascin at a 1:100 ratio. PBS (250 µl) containing varying amounts of this mixture was added to cell culture inserts coated with Matrigel or collagen I. The inserts were incubated at room temperature for 4 h at 37°C in a humidified 95% air/5% CO2 atmosphere and washed with PBS. The filters were cut from the inserts with a scalpel and assayed for 125I in a Beckman gamma counter.

Cell adhesion assays

Ten microliters of deionized H2O containing 10 µg of Matrigel was placed on each 6-mm-diameter spot of a Multispot glass slide (Shandon, Pittsburgh, PA) and allowed to gel. Where indicated, 2–5 µg of tenascin was added to these Matrigel-coated spots. The slides were air dried in a laminar flow tissue culture hood. Fifty microliters of serum-free RPMI 1640 supplemented with 1 mg/ml BSA and containing 50,000 monocytes was placed on each spot, and the slides were placed in a humidified CO2 incubator at 37°C for 1 h to allow cells to adhere. The slides then were washed three times with serum-free RPMI 1640, and the number of cells adherent to each spot was determined as described (21, 27). Fibrin(ogen)- or fibronectin-coated surfaces were prepared as described (22). Where indicated, tenascin was added to these surfaces as described above, and the adhesion of leukocytes was measured as described above. Cell adhesion studies with PMN were performed as described above except that the cells were suspended in PBS supplemented with 0.1% HSA.

FACS analysis: flow cytometric analysis.

Unstimulated, fMLP- or LTB4-stimulated PMN (105 cells/200 µl of PBSG-HSA) were incubated in suspension at 37°C for 15 min in the presence or absence of 10 µg tenascin, transferred to 96-well polystyrene tissue culture microtiter plates (Corning Glass, Corning, NY), incubated for 30 min at 4°C in 200 µl PBSG-HSA containing the indicated primary Ab (2 µg/ml), washed three times with PBSG-HSA at 4°C, further incubated for 30 min at 4°C with either Alexa-488-conjugated or phycoerythrin-conjugated rabbit anti-mouse F(ab')2 in 200 µl of PBSG-HSA, washed three times again with PBSG-HSA at 4°C, and resuspended at 4°C in 300 µl PBS containing 2% BSA and 0.3 mg/ml propidium iodide to determine cell viability. The contribution of dead cells (usually <2%) was removed from the final data analysis. The mean fluorescence intensity of 3–5 x 103 cells was determined using a BD Biosciences FACSCalibur.

Reagents: mAbs

P4C10 (anti-{beta}1 integrin) was obtained from Life Technologies, LeuM5 (anti-{alpha}x integrin) was obtained from Organon Teknika (Malvern, PA), P1D6 (anti-{alpha}5 integrin) was obtained from Oncogene Sciences (Cambridge, MA), G0H3 (anti-{alpha}6 integrin) was obtained from Immunotech (Westbrook, ME), and IB4 (anti-{beta}2 integrin) was as described (28). Gifts of Abs Y9A2 (anti-{alpha}9 integrin) and 15/7 (directed against an activation epitope on {beta}1 integrins) were obtained from T. Yednock (Elan Pharmaceuticals, San Francisco, CA), anti-{alpha}v{beta}3 was obtained from B. Hendey (Rush Medical College, Chicago, IL), and Ab 24 (directed against an activation epitope of the {alpha}m chain of {beta}1 integrins), was obtained from (N. Hogg, Imperial Cancer Research Fund, London, U.K.) F(ab')2 of anti-tenascin Abs were prepared as described (29). Chondroitin sulfate proteoglycan monomers were purified and Abs were prepared against them as described (30). The F(ab')2 used in this study were prepared from total anti-proteoglycan IgG. These Abs recognize both 400- and 250-kDa proteoglycan core proteins (30). Fibrinogen was obtained from Enzyme Research Laboratories (South Bend, IL).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Rationale for use of LTB4, IL-8, fMLP, and TNF{alpha}

LTB4, IL-8, fMLP, and TNF{alpha} were used in these studies because they are representative of two groups of chemoattractants that differentially affect leukocyte adhesion to, and migration through, fibrin-containing matrices. Previous studies showed that PMN stimulated by LTB4 or IL-8 adhere loosely to, and migrate efficiently through, three-dimensional gels containing fibrin. In contrast, PMN stimulated by fMLP or TNF{alpha} adhere tightly to, and cease migrating when they interact with, fibrin-containing matrices (22, 23).

Chick and human tenascin block chemotaxis of monocytes and PMN through Matrigel

Approximately 15–20% of freshly isolated monocytes (Fig. 1GoA), ~15% of monocytes cultured for 24 h (Fig. 1GoB), and 10–20% of PMN (Fig. 2Go), migrated through Matrigel-coated culture inserts in response to each of the chemoattractants (LTB4, fMLP), chemokines (IL-8, MCP-1; data not shown), and cytokines (TNF-{alpha}) tested. In response to these substances, freshly isolated monocytes and cultured monocytes first appeared in the lower compartment at 6 h, and reached maximum numbers by 24 h, whereas PMN first appeared in the lower compartment at 1 h, and reached maximum numbers by 4 h. Control experiments revealed that fewer than 1.5% of added monocytes or PMN migrated into the lower compartment in the absence of chemoattractant/chemokine/cytokine stimulation (Figs. 1Go and 2Go). The presence of chick tenascin in the Matrigel dramatically reduced the number of freshly isolated monocytes, 24-h cultured monocytes, and PMN that migrated through the gels in response to chemoattractants (Figs. 1Go and 2Go). In addition, tenascin inhibited monocyte or PMN chemotaxis whether it was added together with Matrigel to form the gel or whether it was added after the Matrigel had gelled (data not shown). The presence of tenascin had no significant effect on the small number of monocytes that migrated in the absence of chemoattractant (Fig. 1Go), but it reduced even further the number of PMN that migrated in the absence of chemoattractant (Fig. 2Go).



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  		FIGURE 1. Chick tenascin inhibits chemotaxis of monocytes and monocyte-derived macrophages through Matrigel-coated inserts. Chemotaxis chambers containing inserts coated with either Matrigel or Matrigel and chick tenascin (TN) were prepared as described in Materials and Methods. Freshly harvested monocytes (106, A) or 106 monocytes cultured for 24 h (B) were added to the upper compartment, and the indicated chemoattractant (LTB4, 10-7 M; fMLP, 10-7 M) or cytokine (TNF{alpha}, 5 x 10-7 M) was added to the lower compartment. The inserts were incubated for 24 h at 37°C, and cells that migrated into the lower compartment were counted using a Coulter Counter. Values represent the average ± SEM of three separate experiments each performed in duplicate.

 


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  		FIGURE 2. Chick tenascin inhibits chemotaxis of PMN through Matrigel-coated inserts. Chemotaxis chambers containing inserts coated with either Matrigel or Matrigel and chick tenascin (TN) were prepared as described in Fig. 1Go. Freshly isolated PMN (106) were added to the upper compartment, and the indicated chemoattractant (LTB4, 10-7 M; fMLP, 10-7 M) or cytokine (TNF{alpha}, 5 x 10-7 M) was added to the lower compartment. The inserts were incubated for 4 h at 37°C, and the cells that migrated into the lower compartment were counted as in Fig. 1Go. Values represent the average ± SEM of three separate experiments each performed in duplicate.

 
To confirm that human tenascin, also inhibited monocyte and PMN chemotaxis we examined the effect of human tenascin obtained from cultured human glioma cells. Five micrograms of human tenascin reduced by 75% the number of LTB4-stimulated monocytes, and by ~70% the number of LTB4- or fMLP- stimulated PMN, that migrated through Matrigel in response to these chemoattractants (Fig. 3Go). Thus, both chick and human tenascin had similar inhibitory effects on PMN and monocyte chemotaxis.



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  		FIGURE 3. Human tenascin inhibits chemotaxis of monocytes or PMN through Matrigel-coated inserts. Chemotaxis chambers containing inserts coated with either Matrigel or Matrigel and human tenascin (TN) were prepared as described in Materials and Methods. Freshly isolated monocytes (5 x 105), or 106 PMN were added to the upper compartment, and the indicated chemoattractant (LTB4, 10-7 M; fMLP, 10-7 M) was added to the lower compartment. Migration of monocytes and PMN proceeded for 24 and 6 h, respectively, at 37°C. Cells that migrated into the lower compartment were counted as in Fig. 1Go. Values represent the average ± SEM of three separate experiments each performed in duplicate.

 
To test the stability of tenascin binding to protein matrices we incubated Matrigel-coated filters with varying amounts of 125I-labeled chick tenascin for 4 h at 37°C, washed the inserts with PBS, and determined the amount of radiolabeled protein that remained bound to the matrix. Near maximal adsorption of 125I-labeled chick tenascin to Matrigel-coated filters was obtained with addition of ~1 µg of chick tenascin (Fig. 4Go). This resulted in binding of ~0.2 µg of tenascin per insert. Once bound, <1% of 125I-labeled chick tenascin eluted into the medium bathing either the upper or lower compartments of the chambers during a 4- to 6-h incubation at 37°C (data not shown). Thus, ~99% of tenascin remained bound to Matrigel-coated filters.



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  		FIGURE 4. Amount of matrix-bound tenascin required to inhibit monocyte chemotaxis: 125I-labeled chick tenascin (TN) mixed with unlabeled tenascin at a protein ratio of 1:100 was added to Matrigel-coated inserts in the amounts indicated, and the inserts were incubated at 37°C for 4 h. Then the inserts were washed. The protein-coated filters were cut from the inserts, counted in a LKB Instruments (Gaithersburg, MD) gamma counter, and the total amount of tenascin bound to each filter (•) was calculated. To determine the amount of chick tenascin required for half-maximal inhibition of chemotaxis, 2 x 105 monocytes cultured for 24 h were placed in the upper compartment of chemotaxis chambers containing inserts coated with Matrigel and the indicated amounts of tenascin. TNF-{alpha} (5 x 10-7 M) was added to the lower compartment, and the cells were allowed to migrate for 24 h at 37°C ({blacksquare}). Control experiments (data not shown) indicated that fewer than 1500 cells migrated in the absence of TNF-{alpha}, and ~125,000 cells migrated in the absence of tenascin. Values represent the average ± SEM of three separate experiments each performed in duplicate.

 
To determine the amount of tenascin required to give half-maximal inhibition of chemotaxis, we prepared Matrigel matrices containing varying amounts of tenascin. Addition of 0.625 µg tenascin per insert (~0.12 µg tenascin bound to the matrix) caused half-maximal inhibition of TNF{alpha}-stimulated monocyte migration (Fig. 4Go). Roughly the same amount of tenascin caused half-maximal inhibition of LTB4-stimulated PMN migration (data not shown).

Tenascin blocks chemotaxis of monocytes through collagen I gels and of PMN through fibrin gels

We examined the effects of tenascin on monocyte migration through collagen I matrices. Twelve percent of TNF{alpha}-stimulated monocytes and 18% of LTB4-stimulated monocytes migrated through collagen I gel-coated cell culture inserts (Fig. 5GoA). Addition of chick tenascin to collagen I gels reduced TNF{alpha}- or LTB4-stimulated monocyte migration by 65 and 62%, respectively (Fig. 5GoA). Binding studies with 125I-labeled tenascin revealed that similar amounts of tenascin bound to collagen I-coated inserts as to Matrigel-coated inserts (data not shown).



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  		FIGURE 5. Effects of chick tenascin on chemotaxis of monocytes through collagen I gels and of PMN through fibrin gels. Chemotaxis chambers containing inserts coated with gels formed with 40 µg of collagen I, or with 75 µg of thrombin-clotted fibrinogen were incubated where indicated with 100 µl PBS containing 5 µg of chick tenascin (TN) for 4 h, and washed. Monocytes (5 x 105), cultured for 24 h, were added to the upper compartment of inserts containing collagen I. PMN (106) were added to the upper compartment of inserts containing fibrin gels. TNF{alpha} (5 x 10-7 M), LTB4 (10-7 M), or buffer was added to the lower compartment of inserts as indicated. Inserts containing collagen I were incubated at 37°C for 24 h (A), whereas those containing fibrin were incubated at 37°C for 4 h (B). Cells that migrated into the lower compartment were counted as described in Fig. 1Go. Values represent the average ± SEM of three separate experiments each performed in duplicate.

 
About 20% of PMN migrate across fibrin gels (22) in response to LTB4. Addition of tenascin reduced the number of PMN that migrated across these gels to 7.5% (Fig. 5GoB). Thus, the inhibitory effect of tenascin on PMN and monocyte chemotaxis is not restricted to matrices containing basement membrane-type proteins (e.g., Matrigel).

F(ab')2 of anti-tenascin IgG reverse the inhibitory effect of tenascin on monocyte chemotaxis through Matrigel

To gain insight into the mechanisms by which tenascin blocks monocyte or PMN chemotaxis, we used F(ab')2 of rabbit anti-chick tenascin IgG to block putative leukocyte binding sites on tenascin. Monocytes or PMN were added to the upper compartment of inserts coated with Matrigel alone or with Matrigel/tenascin. F(ab')2 of anti-tenascin IgG were added to the medium in the upper compartment, and LTB4 (10-7 M) to the lower compartment. F(ab')2 anti-tenascin restored monocyte and PMN chemotaxis through Matrigel-tenascin to 50–95% of control levels (Fig. 6Go). Control experiments showed that F(ab')2 anti-tenascin did not significantly affect LTB4-stimulated monocyte or PMN migration through Matrigel alone (Fig. 6Go).



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  		FIGURE 6. F(ab')2 of anti-tenascin IgG reverse the inhibitory effect of tenascin on LTB4-stimulated monocyte and PMN chemotaxis through Matrigel. Where indicated, chemotaxis chambers containing inserts coated with Matrigel-tenascin were incubated for 30 min at 4°C with 5 µg/ml of F(ab')2 of anti-tenascin IgG or F(ab')2 of anti-proteoglycan IgG. Freshly isolated monocytes (106, A) or 106 PMN (B) were then added to the upper compartment. LTB4 (10-7 M) was added to the lower compartment, and the chambers were further incubated at 37°C for 24 (A) or 4 h (B). The number of cells that migrated into the lower compartment was determined as described in Fig. 1Go. Values represent the average ± SEM of three separate experiments each performed in duplicate.

 
Proteoglycans have a high affinity for tenascin. Thus, some tenascin preparations may contain small amounts of these substances (26, 29). The methods used to prepare tenascin should have removed most, if not all, contaminating proteoglycans from the chick tenascin. Nonetheless, we examined the effects of F(ab')2 of polyclonal anti-proteoglycan Abs on monocyte migration across inserts coated with tenascin and Matrigel. Unlike the effect of F(ab')2 anti-tenascin, F(ab')2 of anti-proteoglycan IgG (10 µg/ml) did not reverse the inhibitory effect of tenascin on monocyte chemotaxis across Matrigel-tenascin-coated inserts (data not shown). Thus it is unlikely that contaminating proteoglycans are responsible for the inhibitory effect of tenascin on monocyte and PMN chemotaxis.

Abs that block {alpha}5{beta}1 integrins reverse the inhibitory effects of tenascin on monocyte and PMN chemotaxis

Tenascin contains {beta}1 integrin-binding sites in its fibronectin type III repeats and fibrinogen-like C-terminal domains (31, 32). Attachment and spreading of normal (e.g., endothelial cells) and transformed (e.g., gliomas and carcinomas) cells on surfaces containing human tenascin has been shown to be partially mediated by {beta}1 integrins (33, 34) and, particularly, by {alpha}9{beta}1 integrins (35). Because Abs against {beta}1 integrins reversed the inhibitory effects of fMLP and TNF{alpha} on PMN migration through fibrin gels (22, 23), we examined the effects of Abs directed against {beta}1 integrins on monocyte and PMN chemotaxis through Matrigel-tenascin (Fig. 7Go). Monoclonal Abs P4C10 and AIIB2, which block the ligand-binding domain of {beta}1 integrins (36, 37), reversed the inhibitory effect of tenascin on LTB4-stimulated monocyte migration through Matrigel-tenascin by ~50% (Fig. 7GoA) and ~60% (data not shown), respectively. Control experiments showed these Abs had no effect on LTB4-stimulated monocyte chemotaxis through inserts coated with Matrigel alone (Fig. 7GoA, and data not shown).



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  		FIGURE 7. Abs that block {beta}1 integrins reverse the inhibitory effect of tenascin on chemotaxis of monocyte through Matrigel. A, Monocytes (5 x 105), cultured for 24 h, were preincubated for 30 min at 4°C with medium alone or medium containing either 2 µg/ml of anti-{beta}1 integrin Ab P4C10 or 2 µg/ml of anti-{alpha}5 integrin Ab. The cells were then added in Ab-containing medium to the upper compartment of chemotaxis chambers containing inserts coated with Matrigel, without or with 5 µg of chick tenascin (TN). LTB4 (10-7 M) was added to the lower compartment, and the chambers were incubated at 37°C for 24 h at which time the number of cells in the lower compartment was counted as described in Fig. 1Go. B, PMN (106) were incubated for 30 min at 4°C with medium alone or with medium containing either 2 µg anti-{beta}1 integrin Ab P4C10 or 2 µg of anti-{alpha}5 integrin Ab. The cells were then added in Ab-containing medium to the upper compartment of chemotaxis chambers containing inserts coated with Matrigel, without or with 5 µg of chick tenascin. LTB4 (10-7 M) was added to the lower compartment, and the chambers were incubated at 37°C for 24 h, at which time the number of cells in the lower compartment was counted as in Fig. 1Go. Values represent the average ± SEM of three separate experiments each performed in duplicate.

 
mAb P4C10 had a small (~20%) inhibitory effect on MCP-1-stimulated monocyte migration through Matrigel. However, it almost completely reversed the inhibitory effect of tenascin on MCP-1-stimulated monocyte chemotaxis through Matrigel (Fig. 8Go). Similarly, P4C10 (Fig. 7GoB) and AIIB2 (data not shown) reversed the effects of tenascin on LTB4- (Fig. 7GoB) or TNF{alpha}-stimulated (data not shown) PMN chemotaxis through Matrigel.



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  		FIGURE 8. Anti-{beta}1 Abs reverse the inhibitory effect of tenascin on monocyte chemotaxis through Matrigel. Monocytes (5 x 105), cultured for 24 h, were preincubated for 30 min at 4°C with medium alone or medium containing either 2 µg/ml anti-{beta}1 integrin Ab (P4C10) or 2 µg/ml anti-{beta}2 integrin Ab (IB4) and added to the upper compartment of chemotaxis chambers containing inserts coated with Matrigel, without or with 5 µg of chick tenascin (TN). MCP-1 (10-8 M) was added to the lower compartment. The chambers were incubated at 37°C for 24 h, at which time the number of cells in the lower compartment was counted as in Fig. 1Go. Values represent the average ± SEM of three separate experiments each performed in duplicate.

 
To identify whether a specific {beta}1 integrin mediated these effects we tested various Abs against integrin {alpha}-chains on monocyte and PMN chemotaxis through Matrigel-tenascin. Anti-{alpha}5 chain IgG reversed the inhibitory effect of tenascin on LTB4-stimulated monocyte migration through Matrigel-tenascin. It had no effect on LTB4-stimulated monocyte migration through Matrigel alone (Fig. 7GoA). Control experiments showed that anti-{alpha}5 chain IgG had a small (~20%) inhibitory effect on LTB4-stimulated monocyte migration through Matrigel (Fig. 7GoA). Anti-{alpha}5 chain IgG also reversed the inhibitory effect of tenascin on LTB4-stimulated PMN migration through Matrigel (Fig. 7GoB). In contrast, Abs that block the specific {alpha}-chains of {alpha}6{beta}1, {alpha}9{beta}1, or {alpha}v{beta}3 integrins, or that block the {alpha}x-chain of {beta}2 (38), had no effect on tenascin’s capacity to inhibit chemotaxis of LTB4-stimulated PMN through Matrigel-tenascin (data not shown).

As expected, monoclonal Ab IB4, directed against {beta}2 integrins, inhibited MCP-1-stimulated chemotaxis of monocytes through Matrigel (Fig. 8Go) (36), and did not reverse the inhibitory effect of tenascin on MCP-1-stimulated monocyte chemotaxis through Matrigel-tenascin (Fig. 8Go). Thus, only Abs that block {alpha}5{beta}1 integrins reversed the inhibitory effect of tenascin on monocyte and PMN chemotaxis.

RGD peptides reverse the inhibitory effects of tenascin on PMN and monocyte chemotaxis

Tenascin contains an RGD sequence in its third fibronectin III repeat (39) and integrin binding domains in its fibrinogen-like C-terminal domains (32). RGD-containing peptides block interactions of {alpha}5{beta}1 integrins with matrix proteins (40), and reverse the inhibitory effects of fMLP and TNF-{alpha} on PMN migration through fibrin (22, 23). GRGDSP peptide (1 mg/ml) reversed the inhibitory effect of tenascin on monocyte chemotaxis by ~80% (Table IGo). Control peptide GREDSP was without effect (Table IGo). Thus, a peptide predicted to block interactions of monocyte or PMN {beta}1 integrins with RGD ligands on tenascin reversed by 70–100% the inhibitory effect of tenascin on monocyte and PMN chemotaxis.


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  		Table I. RGD-containing peptides reverse the inhibitory effect of tenascin on monocyte chemotaxis through Matrigel1

 
Efficiency and strength of adhesion of monocytes or PMN to surfaces containing Matrigel, fibronectin, or fibrinogen without or with tenascin

It is important to re-emphasize that when tenascin bound to, or was incorporated into, a matrix it did not elute (Fig. 4Go and data not shown). Thus the findings reported in Tables IIGo and IIIGo reflect the effects of matrix-bound tenascin, not soluble tenascin, on the quantity and quality of adhesion of monocytes and PMN.


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  		Table II. Adhesion of PMN and monocytes to surfaces coated with Matrigel, Matrigel and tenascin, fibronectin, or fibronectin and tenascin1

 

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  		Table III. Adhesion and formation of zones of close apposition (ZCA) by PMN and monocytes plated on surfaces coated with fibrinogen vs fibrinogen and tenascin1

 
We performed two types of experiments to examine the effects of tenascin on PMN and monocyte adhesion. First, we compared the relative efficiency with which LTB4, fMLP, and TNF{alpha} promoted monocyte adhesion to surfaces coated with Matrigel vs Matrigel-tenascin (Table IIGo) or with fibronectin vs fibronectin-tenascin (Table IIGo), and the relative efficiency with which LTB4 and fMLP stimulated monocytes and PMN to adhere to surfaces coated with fibrinogen vs fibrinogen-tenascin (Table IIIGo).

Tenascin did not affect the number of chemoattractant-stimulated monocytes or PMN that adhered to Matrigel- or fibronectin-coated surfaces (Table IIGo), or of chemoattractant-stimulated PMN that adhered to fibrinogen-coated surfaces (Table IIIGo). However, tenascin did reduce by ~40% the number of unstimulated and LTB4-stimulated monocytes that adhered to fibrinogen-coated surfaces (Table IIIGo).

Second, we compared the effect of tenascin on the strength of monocyte and PMN adhesion. Strength of cell adhesion to a surface or matrix is an important determinant of whether and how fast cells migrate on a surface or through a matrix (22, 23, 41, 42). For example, the capacity of fMLP and TNF{alpha} to stimulate close apposition (i.e., strong adhesion) between PMN and fibrin-coated surfaces is highly correlated with the capacity of these substances to inhibit PMN migration through fibrin gels (22). Therefore, as described in Materials and Methods and Table IIIGo, we compared the effect of tenascin on the formation of zones of close apposition by LTB4- or fMLP-stimulated monocytes and PMN on surfaces coated with Matrigel or fibrinogen. Neither unstimulated nor LTB4-, IL-8-, fMLP-, or TNF{alpha}-stimulated PMN formed zones of close apposition on Matrigel (data not shown). fMLP-stimulated PMN did not form zones of close apposition when they adhered to surfaces containing Matrigel-tenascin or tenascin alone (data not shown). As expected, 87% of fMLP-stimulated PMN formed zones of close apposition on fibrinogen-coated surfaces (22). Tenascin reduced the percentage of LTB4- or fMLP-stimulated PMN, respectively, that formed zones of close apposition on fibrinogen-coated surfaces (Table IIIGo). Although a reduction in formation of zones of close apposition by LTB4-stimulated PMN on fibrinogen was not significant, reduction in formation of zones of close apposition by fMLP-stimulated PMN was (p < 0.05). Taken together, these findings suggest that in some instances tenascin reduces the efficiency (e.g., reduced number of unstimulated and LTB4-stimulated monocytes that adhered to fibrinogen) and strength (e.g., reduced percentage of fMLP-stimulated PMN forming zones of close apposition on fibrinogen) of monocyte and PMN adhesion.

Tenascin does not affect the expression of activation epitopes by {beta}1 or {beta}2 integrins

Although all chemoattractants (e.g., LTB4, fMLP), chemokines (IL-8, MCP-1), and cytokines (TNF{alpha}) tested have been shown to up-regulate surface expression of {beta}2 integrins (23, 43), only those that inhibit PMN migration through fibrin gels (e.g., fMLP and TNF{alpha}) induce the appearance of activation epitopes on {beta}1 integrins (23, 44). Therefore, we examined by FACS the effect of tenascin, alone or in combination with fMLP or LTB4, on PMN surface expression of {beta}1 and {beta}2 integrins and on expression of activation epitopes by PMN {beta}1 and {beta}2 integrins. We chose LTB4 as representative of chemoattractants that do not promote expression of activation epitopes of either {beta}1 or {beta}2 integrins, and fMLP as representative of ones that do. PMN incubated in suspension with tenascin (10 µg/ml) for 15 min at 37°C were not stimulated to express {beta}1 or {beta}2 integrins. Conversely, tenascin did not inhibit the capacity of LTB4 (10-7 M) or fMLP (10-7 M) to stimulate PMN to upregulate expression of these integrins (data not shown). Similarly, tenascin alone did not promote expression of the activation epitope recognized by mAb 15/7 on PMN {beta}1 integrins or of the activation epitope recognized by mAb 24 on PMN {beta}2 integrins. In addition, it did not inhibit the capacity of fMLP to stimulate expression of both activation epitopes on PMN {beta}1 and {beta}2 integrins (data not shown).


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Interaction between {alpha}5{beta}1 integrins and tenascin is required to inhibit leukocyte migration

Tenascin inhibited monocyte and PMN chemotaxis through different matrices in response to a variety of chemoattractants, chemokines, and cytokines ( Figs. 1–8GoGoGoGoGoGoGoGo and Table IGo). These inhibitory effects on monocyte and PMN chemotaxis were reversed by pretreatment of tenascin containing matrices with F(ab')2 of anti-tenascin IgG (Fig. 6Go, A and B), by incubating monocytes or PMN with Abs that block the ligand-binding domains of the {beta}1 or {alpha}5 chains of {beta}1 integrins (Fig. 7GoA), or by addition of RGD-containing peptides (Table IGo). We interpret these findings as indicative of a requirement for interaction between {alpha}5{beta}1 integrins and/or molecules associated with {alpha}5{beta}1 integrins with tenascin. Indeed, they are consistent with the hypothesis that {alpha}5{beta}1 integrins interact directly with RGD ligands on the third fibronectin type III repeat of tenascin (45) and/or on its fibrinogen-like C terminus, and that these interactions signal monocytes and PMN to stop migrating.

Although there is compelling evidence that tenascin binds to several integrins including {alpha}v{beta}3 (46) and {alpha}9{beta}1 (35, 47), Abs directed against these integrins did not reverse the inhibitory effect of tenascin on chemotaxis (data not shown). The ability of Abs that block {alpha}5{beta}1 integrins (Fig. 7Go), and of RGD-containing peptides (Table IGo), to reverse the effect of tenascin on chemotaxis may reflect either their ability to block interactions of {alpha}5{beta}1 integrins with RGD ligands on tenascin, or their ability to generate a qualitatively different intracellular signal than the one that is generated when {alpha}5{beta}1 integrins interact with tenascin itself. Interestingly, Andersen et al. (48) reported that addition of tenascin to fibronectin-containing matrices significantly reduced migration of corneal fibroblasts through these matrices. Furthermore, they found that anti-{alpha}5{beta}1 IgG blocked attachment of corneal fibroblasts to fibronectin/tenascin-coated surfaces more effectively than to surfaces coated with only fibronectin. These findings are consistent with those reported in Table IIIGo, suggesting that tenascin reduces the efficiency of adhesion of LTB4-stimulated monocytes, and the strength of adhesion of fMLP-stimulated PMN to fibrinogen-coated surfaces.

Unanticipated inhibitory effect of Abs vs {alpha}5{beta}1 integrins on monocyte chemotaxis

A finding that was surprising and will require further study is that treatment of monocytes with Abs vs {alpha}5{beta}1 integrins inhibited partially the migration of these cells through Matrigel (Fig. 7GoA), whereas RGD-containing peptides did not (Table IGo). This suggests that binding of Abs to {alpha}5{beta}1 integrins may initiate migration-inhibitory signals. Gresham et al. (49) reported that anti-{beta}2 integrin Abs inhibit PMN chemotaxis, and provided convincing evidence that the inhibitory effects of these Abs were due to their capacity to stimulate a rise in PMN cAMP levels. Elevated levels of cAMP are known to inhibit chemotaxis (43, 50). Although further work is needed to determine whether Abs vs {beta}1 integrins affect monocyte and PMN cAMP levels, the experiments reported here show that, in general, Abs vs {beta}1 integrins promoted monocyte and PMN chemotaxis. Thus, the ability of Abs vs {beta}1 integrins to reverse the inhibitory effects of tenascin on monocyte and PMN chemotaxis is unlikely to be due to stimulation of monocyte and PMN cAMP levels.

Tenascin shares with factor XIII-cross-linked fibronectin and fibrinogen, and with fibrillar {beta} amyloid, the capacity to inhibit monocyte chemotaxis

Unlike fMLP- or TNF{alpha}-stimulated PMN, monocytes do not form zones of tight adhesion on fibrinogen- or fibronectin-containing surfaces in response to any of the chemoattractants used in these studies (Table IIIGo). Nonetheless, monocytes migrate very inefficiently into fibrin gels (J. Loike, unpublished observations), through gels formed by factor XIII-cross-linked fibrinogen and fibronectin (51, 52), or through filters coated with collagen IV and fibrillar {beta} amyloid (27). Like tenascin and fibrin(ogen) (Table IIIGo), fibrillar {beta} amyloid does not induce formation of zones of tight adhesion (J. Loike, unpublished observation). Whether fibrillar {beta} amyloid shares the capacity of tenascin to reduce the strength of monocyte adhesion to matrices, and whether reduced strength of adhesion plays any role in its inhibitory effect on monocyte chemotaxis, remain to be explored.

Two general mechanisms by which cells are signaled to stop migrating

There are two general mechanisms by which soluble or matrix-bound substances signal cells to cease migrating. First, cessation of chemotaxis occurs when cells are exposed to a very high concentration of agonist for their chemoattractant receptors. Under these conditions their chemoattractant receptors are desensitized and/or down-modulated, and thereby prevented from generating signals that promote directed migration (53, 54). Nonetheless, the cells may still be capable of random and/or directed migration in response to an unrelated chemoattractant (54).

Second, cessation of chemotaxis occurs when cells expressing specific adhesion promoting receptors interact with matrices or other cells bearing a high concentration of ligands of one of these receptors. Examples of such situations include: a) {alpha}5{beta}1 integrins on epidermal cells (37) or on fMLP-stimulated PMN (22, 23) and surfaces or matrices containg high concentrations of fibronectin or Fg, respectively, b) Receptors for Advanced Glycation Endproducts (RAGE) on monocytes and filters coated with advanced glycation endproducts (55), c) class A scavenger receptors on monocytes and macrophages and matrices containing oxidized or acetylated low density lipoproteins or glycated collagen IV (19), and d) T cells expressing receptors for Ags on cognate MHC proteins on antigen presenting cells (56, 57). The cells cited in these examples are thought to adhere so forcefully to the substrate or to other cells that they become immobilized.

Tenascin exemplifies a third mechanism by which matrix proteins signal cessation of cell migration

There are two major differences between the effects of fibrin and those of tenascin on chemoattractant-stimulated leukocytes. fMLP stimulates increased surface expression of PMN {beta}1 (23) and {beta}2 integrins (M. Berger, S. Budhu, E. Lu, Y. Li, D. Loike, S. C. Silverstein, and J. D. Loike, manuscript in preparation), expression of activation neo-epitopes on PMN {beta}1 integrins (23), and formation of zones of close apposition (tight adhesion) between PMN and fibrinogen-containing matrices (Refs. 22, 23 and Table IIIGo). In contrast, tenascin does not by itself promote a change in surface expression of {beta}1 or {beta}2 integrins in unstimulated PMN. It also does not affect the capacity of these cells to increase surface expression of {beta}1 or {beta}2 integrins in response to LTB4 or fMLP, to express activated neo-epitopes of {beta}1 integrins in response to fMLP, or to form zones of close apposition on fibrinogen (Table IIIGo). However, tenascin does exert a modest but reproducible inhibitory effect on adhesion of LTB4- or fMLP-stimulated monocytes to fibrinogen-containing surfaces (Table IIIGo), and a significant inhibitory effect on formation of zones of close apposition by fMLP-stimulated PMN on these surfaces (Table IIIGo). The effects of tenascin are opposite to those expected of an agent that inhibits migration by increasing adhesive forces. These results lead us to suggest that the experiments reported here identify a third mechanism whereby leukocytes are stimulated to cease migrating by matrix-associated ligands. In the case of tenascin, signals resulting from interactions of tenascin with unactivated or activated {alpha}5{beta}1 integrins, and perhaps with other as yet unidentified receptors, appear to generate outside-in signals that reduce monocyte and PMN adhesion to extracellular matrix proteins and thereby block chemoattractant-, chemokine-, and/or cytokine-stimulated migration of these cells through three-dimensional gels composed of collagen I, fibrin, or Matrigel without inducing tight adhesion. To our knowledge, this is the first identification of a matrix protein (i.e., tenascin) that exerts such effects, and of the involvement of a specific integrin (i.e., {alpha}5{beta}1) in such a signaling pathway. Studies are now in progress to test whether the decreases in adhesion and in formation of zones of close apposition induced by tenascin are clues to the underlying cellular mechanisms involved.


   Footnotes
 
1 This work was supported in part by National Institutes of Health Grant AI 20516 (to S.C.S.) and RO1 HL37641, by grants from the RGK Foundation, the Scleroderma Federation/United Scleroderma Foundation, and the Arthritis Research Fund of the Health Science Foundation of the Medical University of South Carolina (to S.H.). Back

2 Address correspondence and reprint requests to Dr. John D. Loike, Columbia University College of Physicians and Surgeons, Department of Physiology, 630 West 168th Street, New York, NY 10032. E-mail address: jdl5{at}columbia.edu Back

3 Abbreviations used in this paper: LTB4, leukotriene B4; PMN, polymorphonuclear leukocyte(s); MCP-1, monocyte chemoattractant protein 1; RGD, arginine-glycine-aspartic acid; HS, human serum; AS, autologous serum; PBSG, PBS supplemented with 5.5 mM glucose; HSA, HS albumin. Back

Received for publication January 2, 2001. Accepted for publication April 11, 2001.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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