©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Integrin-dependent Cell Adhesion Is Regulated by a Low Affinity Receptor Pool That Is Conformationally Responsive to Ligand (*)

(Received for publication, August 9, 1995)

Ted A. Yednock (§) Catherine Cannon Christopher Vandevert Erich G. Goldbach Gray Shaw (1) Debra K. Ellis (1) Chen Liaw Lawrence C. Fritz Laura I. Tanner

From the From Athena Neurosciences, Inc., South San Francisco, California 94080 and Genetics Institute, Cambridge, Massachusetts 02140

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

alpha(4)beta(1) integrin (VLA-4) appears to be unique among the leukocyte integrins in that it can initiate the adhesion of circulating lymphocytes without cellular activation. It is not known how lymphocytes or other cell types maintain constitutive levels of alpha(4)beta(1) integrin activity. The current report describes a monoclonal antibody, 15/7, that recognizes a high affinity or ligand-occupied conformation of beta(1) integrin. Studies with 15/7 revealed that alpha(4)beta(1) integrin-dependent adhesion of leukocytic cell lines is mediated by a population of low affinity receptors that is conformationally responsive to ligand; the 15/7 epitope could be induced by nanomolar concentrations of soluble VCAM-1 or by micromolar concentrations of a peptide derived from the type III connecting segment domain of fibronectin (as ligands for alpha(4)beta(1) integrin). The same receptors were also responsive to adhesion activating reagents, such as Mn, activating anti-beta(1) integrin antibodies, and phorbol myristate acetate, which induced the 15/7 epitope directly and/or decreased the concentration of ligand required for epitope induction. In addition to the responsive receptor pool, cells expressed a second population of alpha(4)beta(1) integrin that was conformationally restrained, failing to respond to ligand or to any of the activating reagents. The relative size of the responsive and inactive receptor pools, as well as the affinity of the responsive receptors, represented a stable phenotype of different cell types and played important roles in defining the cells' adhesive capacity and ligand specificity. Similar receptor populations were measured on lymphocyte subsets in whole blood. These studies provide insight into how cells maintain different constitutive levels of alpha(4)beta(1) integrin activity, and how the activity of beta(1) integrin can be modulated by activators of cell adhesion.


INTRODUCTION

Integrins are heterodimeric adhesion molecules that contribute to the specificity of cellular interactions through the recognition of numerous matrix and cell-associated ligands(1) . Importantly, the ligand binding activity of integrins can be modulated rapidly, allowing cells to specify the timing and location of integrin-mediated adhesive interactions. On circulating leukocytes, for example, the beta(2) integrins LFA-1 and Mac-1 are thought to be activated by site-specific factors (cytokines or other adhesive interactions) during transient cell interactions with the vascular wall; the receptors then establish firm adhesive contacts and mediate leukocyte extravasation(2, 3) . Likewise, platelets are stimulated at sites of vascular injury allowing alphaII(b)beta(3) integrin to bind to fibrinogen and initiate thrombosis(4) .

The regulation of alpha(4) integrin activity on circulating immune cells appears to be different from that of the other leukocyte integrins described above. alpha(4)beta(1) and alpha(4)beta(7) integrin can mediate leukocyte adhesion to their endothelial ligands VCAM-1 (5) and MAdCAM-1(6) , respectively, without cellular activation, and can do so even in the presence of the shear forces encountered in normal blood flow(7, 8, 9, 10) . These results suggest that circulating leukocytes maintain a constitutive level of alpha(4) integrin activity. alpha(4)beta(1) and alpha(4)beta(7) integrin also bind to the alternatively spliced connecting segment III (CS1) (^1)domain of fibronectin (FN; 11-13), but freshly isolated blood lymphocytes require activation in order to adhere to this ligand(7) . Cell lines maintained in culture exhibit a range of alpha(4)beta(1) integrin activity; some cells bind to both VCAM-1 and the CS1 domain of fibronectin, others bind VCAM-1 but require activation to bind FN CS1, while others cannot bind FN CS1 even in the presence of activating reagents(14) .

Similar differences in activity and ligand specificity have also been described for other integrins, such as alphaII(b)beta(3) integrin and its interaction with fibrinogen and fibronectin(15) , as well as for alpha(2)beta(1) integrin and its interaction with collagen and laminin(16) . Distinct subsets of integrin can also exist simultaneously on the cell surface that exhibit different affinities for ligand. Interestingly, alpha(5)beta(1) integrin-dependent cell adhesion appears to be mediated by a large subset of low affinity receptors(17) , while Mac-1-dependent adhesion is mediated by a small subset of high affinity receptors(18) . Subsets of LFA-1 have also been described that differ not only in their state of activation but also in their ability to be activated(19, 20) .

Changes in integrin activity can be induced by several types of reagents in vitro, and are associated with changes in receptor conformation. Antibodies against beta(1) integrin, such as TS2/16 and 8A2, bind to the receptor regardless of its state of activation and induce a more active form(21, 22, 23) . The divalent cation Mn also interacts directly with integrins to induce an active conformation(24) . Although integrin-dependent adhesion can be enhanced by cell stimulating agents such as PMA, the effects of PMA are more complex than those of the direct integrin activators; in some systems PMA stimulation enhances integrin affinity(25, 26) , while in others, it causes cell spreading without inducing receptor activation(27) . Changes in receptor conformation have been characterized by anti-integrin antibodies that recognize activation-dependent epitopes. These antibodies can either inhibit adhesive function by engaging the active binding site of the receptor(18, 28, 29) , or promote adhesion by stabilizing an active integrin conformation(30, 31, 32) . Another class of antibodies recognize ligand-induced binding sites associated with alphaII(b)beta(3) integrin(33) , and document changes that occur in receptor conformation upon ligand occupation.

Although much is known about integrin activity and changes in receptor conformation, there is no clear understanding of how cells modulate both integrin activity and ligand specificity, and how cells maintain stable differences in integrin activation states. In this report we describe a monoclonal antibody, 15/7, against an activation/ligand-induced epitope on beta(1) integrin. Studies with 15/7 provided a model for the regulation of alpha(4)beta(1) integrin activity that differs from the conventional view of integrin-mediated cell adhesion. In this model, receptors that mediate cell adhesion under resting conditions undergo a change in conformation in response to ligand, rather than a change in conformation that triggers ligand binding. The ligand-responsive receptors were also sensitive to Mn and to activating antibodies against beta(1) integrin, as well as to PMA. These receptors generally exhibited a low affinity conformation (interacting reversibly with soluble ligand), but supported cell adhesion through a multivalent interaction with immobilized ligand. However, not all surface beta(1) integrin could respond to ligand or to activating agents, and this subset constituted an inactive receptor pool. The size of the inactive pool was found to be a stable phenotype of different cells and played an important role in determining the capacity of the cells to bind ligand, as well as the specificity of ligands that the cells could bind.


MATERIALS AND METHODS

Monoclonal Antibodies

The monoclonal antibodies 15/1, 15/7, and 15/10 (all mouse IgG(1)) were raised against immunopurified alpha(4)beta(1) integrin. A lysate of 10^9 U937 cells (in 1% Triton X-100, with 1 mM phenylmethylsulfonyl fluoride, 3.6 µg/ml E-64, and 0.5 µg/ml leupeptin) was exposed to 0.3 ml of Sepharose conjugated with the anti-alpha(4) integrin antibody, HP2/1 (Immunotech, Inc.; 3 mg of antibody/ml gel). alpha(4)beta(1) integrin was released from the column at pH 3 in 1% octyl glucoside. The eluate, containing approximately 10 µg of alpha(4)beta(1) integrin, was neutralized, dialyzed into phosphate-buffered saline with 1% octyl glucoside, and concentrated to 120 µl. 2.5 µg of alpha(4)beta(1) was emulsified in Freund's complete adjuvant and injected subcutaneously into three mice on day 0. The immunization was repeated with Freund's incomplete adjuvant on day 3, and again on day 6. Draining lymph nodes were isolated on day 9 and the lymphocytes were fused with the mouse myeloma SP2/0. Antibodies were screened for cell surface reactivity with U937. Supernatants from positive hybridomas were then rescreened for differential reactivity with Jurkat and THP-1 cells, as described in Fig. 2. Reactivity of 15/1 and 15/7 with human beta(1) integrin was confirmed by Western analysis (Fig. 2B); alpha(4)beta(1) integrin was purified as described above from U937 cells or from U937 cells that had been surface biotinylated (0.1 mg/ml NHS-LC-biotin (Pierce), 10^7 cells/ml, 40 min at room temperature). Purified alpha(4)beta(1) integrin was run on a 4-12% SDS-PAGE gel under nonreducing conditions, and was then transferred to Immobilon-P (Millipore). The blot was probed with primary antibody, washed, and then incubated with horseradish peroxidase-conjugated sheep anti-mouse (Amersham) or horseradish peroxidase-conjugated streptavidin (Calbiochem) for detection by chemiluminescence (ECL, Amersham).


Figure 2: Antibody reactivity by FACS and Western analysis. A, FACS analysis: Jurkat and THP-1 cells were exposed to the indicated antibody for 30 min at room temperature, washed, and exposed to PE-conjugated goat anti-mouse IgG Fc for FACS analysis (``Materials and Methods''). In a separate experiment it was found that alpha(4) integrin accounts for the majority of beta(1) integrin expressed on both THP-1 and Jurkat cells. The cells were exposed to alpha-chain-specific monoclonal antibodies (see ``Materials and Methods'') and then to FITC-conjugated anti-mouse Ig for FACS analysis. Fluorescence intensity Jurkat/THP-1: IgG, 3/5; anti-alpha(1), 3/3; anti-alpha(2), 5/6; anti-alpha(3), 9/25; anti-alpha(4), 204/193; anti-alpha(5), 14/62; anti-alpha(6), 25/49; anti-beta(1) integrin, 200/276. &cjs2108;, Jurkat cells; &cjs2110;, THP-1 cells. B, Western analysis. alpha(4)beta(1) integrin was isolated from a lysate of U937 cells with an anti-alpha(4) integrin affinity column, subjected to SDS-PAGE (non-reduced), and transferred to Immobilon. A separate sample of alpha(4)beta(1) integrin was isolated from a lysate of U937 cells that had been surface biotinylated. Individual strips of the blot were probed with control IgG(1), 15/1, 15/7, and TS2/16 (control anti-beta(1) integrin) at 10 µg/ml. Bound antibody was detected with sheep anti-mouse horseradish peroxidase (``Materials and Methods''). The strip derived from the biotinylated cells was probed with streptavidin-horseradish peroxidase (SA) to visualize the separate alpha(4) and beta(1) integrin bands. On U937 cells alpha(4) integrin is expressed as the cleaved form of the molecule, as described(48) , migrating as two bands at 70 and 80 kDa. A small amount of uncleaved alpha(4) integrin (150 kDa), as well as a 180-kDa form described (49) is also present. beta(1) integrin runs as a 130-kDa protein.



The inhibitory antibody against human alpha(4) integrin, AN100226m, has been described(34) , and F(ab`)(2) fragments of the antibody were prepared by TSD BioServices (Newark, DE). The activating antibody against beta(1) integrin, TS2/16(21, 22) , was generously provided by Dr. F. Sanchez-Madrid (University of Madrid, Spain), as a hybridoma supernatant (used at a dilution of 1:5), or as an ascites (used at 1:2000). 8A2, also an activating antibody against beta(1) integrin(23) , and its F(ab`)(2) fragments were kindly provided by Dr. N. Kovach (University of Washington, Seattle, WA) as purified reagents and were used at 1 and 10 µg/ml, respectively. The inhibitory anti-beta(1) integrin antibody, AIIB2(35) , was kindly provided by Dr. C. Damsky (University of California, San Francisco). Antibody TS2/7 (against alpha(1) integrin) was obtained from T Cell Sciences (Cambridge, MA). Antibodies Gi9 (against alpha(2) integrin), GoH3 (against alpha6 integrin), K20 (against beta(1) integrin), and FITC-conjugated K20 were purchased from Immunotech, Inc. (Westbrook, ME). Antibodies P1B5 (against alpha(3) integrin) and P1D6 (against alpha(5) integrin) were obtained from Life Technologies, Inc. (Grand Island, NY). The hybridoma cell line secreting TS1/18, against beta(2) integrin, was obtained from the ATCC (#HB203). 15/1, 15/7, AN100226m, and TS1/18 were grown as ascites, and purified by ammonium sulfate precipitation and fast protein liquid chromatography. The purified antibodies contained less than 1 unit of endotoxin/mg of protein. 15/10 was used as a hybridoma supernatant diluted 1:4. For some experiments, 15/7 was conjugated to fluorescein isothiocyanate (FITC; Sigma) according to the manufacturer's instructions and used within 30 days. FITC-conjugated goat anti-mouse IgG, FITC-conjugated goat anti-rat IgG, and FITC-conjugated mouse IgG (MOPC-21; negative control) were obtained from Sigma. FITC-conjugated goat anti-human IgG was obtained from Vector Laboratories, Inc. (Burlingame, CA), and PE-conjugated goat F(ab`)(2) anti-mouse IgG Fc was obtained from Immunotech (Westbrook, ME). FITC-conjugated CD45Ro was obtained from DAKO (Denmark).

Cells and Cell Culture

The human T cell line, Jurkat, was obtained from Dr. A. Weiss (University of California, San Francisco). U937 (monocytic; CRL 1593) and THP-1 (monocytic; TIB 202) cells were obtained from the American Type Culture Collection (Rockville, MD). A stable mouse L cell line that expressed human VCAM-1 was obtained as follows: human VCAM-1 cDNA was obtained by polymerase chain reaction, based on the published sequence (36, 37, 38) from tumor necrosis factor-stimulated human umbilical vein endothelial cells (Clonetics, San Diego, CA). The cDNA was cloned into the pR2 expression vector (39) , and transfected into mouse L cells by calcium phosphate precipitation. Stable L cell line clones expressing high levels of human VCAM-1 were selected by FACS, and maintained by culture in 600 µg/ml G418. All cell lines were grown in RPMI 1640 supplemented with 10% fetal bovine serum, penicillin, streptomycin, and glutamine.

Reagents

Human VCAM-1 was immunopurified from VCAM-1-transfected mouse L-cells, as described above for alpha(4)beta(1) integrin, by affinity chromatography on Sepharose conjugated with the anti-VCAM-1 antibody, 11/47 (3.4 mg/ml gel; 11/47 will be described in a separate report). (^2)VCAM-1 purity was confirmed by SDS-PAGE and silver staining.

Recombinant soluble VCAM-1 was expressed as a fusion protein with the heavy chain of human IgG(1). The construct carried the seven immunoglobulin domains of VCAM-1 on the N terminus (to phenylalanine 697) and the Fc domain of human IgG(1) at the C terminus (including the hinge, CH2, and CH3 regions; the hinge region cysteine, normally disulfide-bonded to the light chains, was mutated to arginine). The cDNA for the soluble construct was ligated into pVL.MCS, a derivative of pVL1393 (40) , and the plasmid was designated pVL.sVCAM.Fc. Recombinant baculovirus was generated by co-transfecting Sf9 cells with 3 µg of pVL.sVCAM.Fc plasmid DNA and 1 µg of BaculoGold viral DNA (Pharmingen) and plaque-purified by visual screening. High titer virus (10^8 plaque-forming units/ml) was used to infect Trichoplusia ni High Five cells (Invitrogen, San Diego, CA). The supernatant 72 h post-infection was collected and sVCAM-1-IgG was affinity-purified by Protein A-Sepharose chromatography. Its purity was established by SDS-PAGE and silver staining.

Whole human fibronectin, rat laminin, and the peptides GRGDSP and GRGESP were purchased from Life Technologies, Inc. An eight amino acid peptide (EILDVPST), derived from the CS1 region of fibronectin, was prepared (430 ABI peptide synthesizer) and purified by high performance liquid chromatography. For adhesion studies, the same peptide was synthesized with a linker sequence on the N terminus (CGGG) and was conjugated to rabbit serum albumin as described by Wayner and Kovach (41) . Recombinant soluble human ICAM-1 was a generous gift from Dr. Mary Perez (Wyeth-Ayerst, Princeton, NJ).

Cell Adhesion Assays

Purified membrane VCAM-1 was plated onto wells of a 96-well RIA plate (#3590 Costar; Cambridge, MA) in 50 µl of phosphate-buffered saline with 0.1% octyl glucoside (Fig. 5, 8, and 9). Adhesion to immobilized recombinant soluble VCAM-1 was examined in Fig. 1; in this instance wells of the RIA plate were coated with Protein A (Calbiochem) at 0.5 µg/ml, blocked with 1% bovine serum albumin (Calbiochem Fraction V) in phosphate-buffered saline/Ca/Mg for 60 min at room temperature, and then exposed to recombinant soluble VCAM-1 at the indicated concentration. Fibronectin, laminin, and the fibronectin CS1 (LDV) peptide-albumin conjugate were plated in 50 µl of phosphate-buffered saline at the concentration specified. Typically, proteins were adsorbed to the 96-well RIA plates for 16 h at 4 °C, and then the coated plates were blocked with bovine serum albumin. Cells were prelabeled with the fluorescent dye, PKH26 (Sigma) according to the manufacturer's instructions. 100,000 cells in Hepes (20 mM)-buffered saline containing 1 mM Ca, 1 mM Mg, and 0.5% bovine serum albumin, were added to each well. After a 30-min incubation at room temperature (unless otherwise specified), the plates were washed four times and the adherent cells were extracted with 0.1% Triton X-100. Fluorescence was quantified on a 96-well fluorescent plate reader (Pandex Screen Machine; Baxter, Mundelein, IL). As a measure of the starting number of cells added to each well (total input cells), 100,000 cells were extracted and measured in parallel. Values for adhesion were expressed as the percentage of the cells added to each well that remained bound after four rounds of washing (% input), and were expressed as the average of triplicate samples ± S.D.


Figure 5: The effect of 15/7 on THP-1 cell adhesion to different substrates. Cells were treated with the indicated alpha-chain-specific antibodies for 30 min on ice, then added to wells coated with purified laminin, fibronectin, ICAM-1 (each at 500 ng/well), or with membrane VCAM-1 (3 ng/well). The binding assay was carried out in the presence of no additions, 1 mM Mn, or 5 µg/ml 15/7 (which were added at the time of the adhesion incubation) for 30 min at room temperature.




Figure 1: THP-1 and Jurkat adhesion to VCAM-1. A, THP-1 and Jurkat cells were added to wells that had been coated with recombinant VCAM-1, and allowed to adhere for 30 min at room temperature. box, Jurkat cells; circle, THP-1. B, THP-1 cells were pretreated with 8A2, Mn (1.5 mM), or with no additions for 30 min on ice and then added to recombinant VCAM-1-coated wells. Another set of cells was added to wells containing PMA (50 nM). The cells were allowed to adhere for 30 min at 37 °C. It was found in separate experiments that adhesion of both cell types was completely inhibited by anti-alpha(4) integrin at all densities of VCAM-1 examined. circle, no stimulation; box, PMA; , Mn; Delta, 8A2.



FACS Analysis

After treatment with antibody, as indicated in the figure legends, cells were washed twice. The cells were then exposed to the appropriate secondary reagent (conjugated with either PE or FITC), for 30 min on ice in Hepes/saline containing Ca, Mg, 5% fetal bovine serum, and 5% mouse or human serum. The cells were washed twice and analyzed on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA). In the experiments described in Fig. 10, blood was collected from healthy human volunteers in heparinized collection tubes and used immediately. After exposure to primary antibody, cells were washed twice and incubated with PE-conjugated goat anti-mouse IgG Fc. The cells were washed again and exposed to FITC-conjugated mouse anti-CD45Ro in the presence of 5% mouse serum. Red blood cells were lysed with Becton-Dickinson lysing solution and the samples were examined by FACS analysis as above. Data was collected for 10,000 events, and values were expressed as the mean fluorescence intensity of the gated cell population.


Figure 10: Induction of the 15/7 epitope on whole blood lymphocytes by LDV peptide. Samples of freshly isolated whole human blood (containing heparin as an anticoagulant) were incubated for 5 min at 37 °C with or without PMA (50 nM). Samples of the blood were then exposed to 15/7 (10 µg/ml) in the presence of LDV peptide (at the indicated concentration) for 30 min at room temperature and processed for double color FACS analysis with anti-CD45Ro (``Materials and Methods''). -box-, CD45Ro; -circle-, CD45Ro; bulletbulletbulletboxbulletbulletbullet, PMA, CD45Ro; bulletbulletbulletcirclebulletbulletbullet, PMA, CD45Ro.




RESULTS

Identification of an Antibody Against an Activation Epitope Associated with beta(1) Integrin

Jurkat and THP-1 cells expressed similar levels of alpha(4)beta(1) integrin (see Fig. 2), but exhibited marked differences in alpha(4)beta(1) integrin activity. The experiments shown in Fig. 1compared the ability of Jurkat and THP-1 cells to bind to purified VCAM-1 that had been coated on assay wells over a range of densities. A 10-fold higher level of VCAM-1 was required to support THP-1 cell adhesion at a level that was comparable to that of Jurkat cells (half-maximal binding occurred at 20 and 2 ng of VCAM-1/well respectively; Fig. 1A). Binding of both cell lines was increased by adhesion activation reagents. As shown in Fig. 1B, THP-1 cells exhibited the highest levels of adhesion following exposure to 8A2, an activating antibody against beta(1) integrin. Adhesion was also strongly stimulated by either PMA or Mn. Under all conditions VCAM-1 binding was completely inhibited by anti-alpha(4) integrin (not shown). Comparable results were obtained with Jurkat cells, although the activating reagents did not produce as great of a change in the already high levels of Jurkat cell adhesion (not shown).

THP-1 and Jurkat cells were used as the basis of a screen to identify potential activation epitopes associated with alpha(4)beta(1) integrin; it was expected that such an epitope would be expressed at higher levels on Jurkat cells in correspondence to their higher level of VCAM-1 binding activity (both cell types expressed similar levels of alpha(4)beta(1) integrin). Antibodies were raised against purified alpha(4)beta(1) integrin (see ``Materials and Methods'') and their ability to recognize THP-1 and Jurkat cells was examined by FACS analysis; the reactivity of three of these antibodies is shown in Fig. 2(15/1, 15/7, and 15/10) compared to that of control reagents against alpha(4) and beta(1) integrin (all antibodies were mouse IgG(1)). Jurkat and THP-1 cells expressed similar levels of alpha(4) integrin, while THP-1 cells expressed higher levels of the beta(1) integrin subunit. Of the 70 reactive antibodies raised against purified alpha(4)beta(1) integrin, only 15/7 produced a staining profile that was markedly different than that of the control reagents. 15/7 reacted at low levels with Jurkat cells, and even lower levels with THP-1 (fluorescence intensity of 24 and 2 units above control IgG background, respectively). Interestingly, addition of the integrin activating reagent Mn (1.5 mM) caused a 10-fold increase in the expression of the 15/7 epitope on both cell types (fluorescence intensity of 253 and 22 for Jurkat and THP-1 cells, respectively), but did not affect the reactivity of the other antibodies (not shown). Western analysis indicated that 15/7 recognized the beta(1) integrin subunit (Fig. 2B). Even though THP-1 cells expressed higher levels of beta(1) integrin than Jurkat, they expressed much lower levels of the 15/7 epitope (in the presence or absence of Mn); this difference could not be explained by expression of different alpha-chain subunits, since both cell types expressed similar levels of alpha(4) integrin, and expression of alpha(4) integrin could account for the majority of beta(1) integrin on both cell types (see Fig. 2, legend). Neither THP-1 nor Jurkat cells expressed beta(7) integrin (not shown).

The sensitivity of the 15/7 epitope to Mn, and the higher expression levels of the epitope on Jurkat than on THP-1 cells suggested that 15/7 recognized an activation epitope associated with beta(1) integrin. In order to examine this possibility further, the antibody was conjugated to FITC and incubated with Jurkat and THP-1 cells in the presence of different activators of beta(1) integrin (Fig. 3). Consistent with the results above, 15/7-FITC reacted with resting Jurkat cells better than with THP-1, although reactivity with both cell types was low. Treatment of the cells with PMA produced a small increase in expression of the 15/7 epitope, while higher expression levels were induced by Mn (1.5 mM), and by two activating antibodies against beta(1) integrin, TS2/16 and 8A2 (tested at saturating concentrations). 8A2 induced the highest expression levels of the 15/7 epitope, representing a 10-fold increase above resting levels for both cell types. The inductive effects of Mn and TS2/16 were additive, resulting in an expression level of the 15/7 epitope equivalent to that produced by 8A2 alone. Mn did not affect the already high expression levels of the epitope induced by 8A2. In contrast to the activating antibodies, the inhibitory antibody against beta(1) integrin, AIIB2, completely eliminated the 15/7 epitope. A control antibody against beta(1) integrin (K20), that has no effect on beta(1) integrin adhesive function, did not significantly affect expression of the 15/7 epitope. 15/7-FITC reactivity with the cells was specific since it was effectively competed by excess unlabeled 15/7. Mn, PMA, and the activating antibodies against beta(1) integrin influence receptor activity through distinct mechanisms; therefore, these results suggest that 15/7 recognizes an epitope associated with the activation state of beta(1) integrin, rather than a conformation particular to a given activating agent.


Figure 3: Modulation of the 15/7 epitope by reagents that affect beta(1) integrin-dependent cell adhesion. THP-1 and Jurkat cells were exposed to 15/7-FITC or IgG-FITC (each at 10 µg/ml) in the presence of saturating concentrations of the indicated antibodies for 30 min at room temperature; Mn was tested at 1.5 mM. One set of samples was pretreated with PMA (50 nM) for 5 min at 37 °C, while another was preincubated with excess unlabeled 15/7 (100 µg/ml) in the presence of Mn for 30 min at room temperature (XS 15/7). After incubation with the FITC reagents, the cells were washed and examined by FACS analysis. Inset, THP-1 and Jurkat cells were treated with 15/7 in the presence of the indicated concentrations of Mn for 30 min at room temperature. The cells were washed and exposed to FITC-conjugated goat anti-mouse IgG(1) for FACS analysis.



Regardless of how the cells were stimulated, THP-1 cells exhibited 3-10-fold lower levels of the 15/7 epitope than Jurkat cells, even though THP-1 cells expressed higher levels of beta(1) integrin (Fig. 2). This result was not due to limiting quantities of the activating reagents since the stimulatory antibodies were tested at concentrations above saturation. Furthermore, the responsive receptors on the two cell types demonstrated nearly identical sensitivity to Mn (excitatory concentration for half maximal expression, or EC = 0.7 mM); higher concentrations of Mn could not compensate for low expression of the 15/7 epitope on THP-1 cells (Fig. 3, inset). These results indicate that THP-1 cells exhibited a lower number of activation-responsive receptors than Jurkat cells.

Expression of the 15/7 Epitope Is Correlated with the Activity of alpha(4)beta(1) Integrin

In addition to VCAM-1, alpha(4)beta(1) integrin binds to the CS1 domain of fibronectin(11, 12) ; however, cell adhesion to the fibronectin domain requires higher levels of alpha(4)beta(1) integrin activity (14) . As shown in Fig. 4, THP-1 cells failed to bind to a peptide derived from FN CS1, even though the peptide was plated at a saturating density. Jurkat cells bound to the peptide at a high level, consistent with their higher level of alpha(4)beta(1) integrin activity. Adhesion of THP-1 cells could be induced by the activating agents described above, and the degree of adhesion reflected closely the degree to which the activators induced the 15/7 epitope (Fig. 3). Thus, the effects of Mn and TS2/16 were additive, and together they produced a level of adhesion comparable to that of 8A2. The degree of enhancement produced by PMA was similar to that of Mn. The high level of Jurkat cell adhesion was not significantly affected by any of the activating reagents; however, when the CS1 peptide was plated at lower densities (conditions where THP-1 cells failed to bind even with activation), Jurkat cells responded to Mn and the activating antibodies in a manner identical to that of THP-1 (not shown). Thus, expression of the 15/7 epitope (as shown in Fig. 3) correlates strongly with the adhesive activity of alpha(4)beta(1) integrin.


Figure 4: THP-1 and Jurkat cell adhesion to FN CS-1 peptide. THP-1 and Jurkat cells were pre-exposed to the indicated reagent for 5 min at 37 °C and then added to wells coated with a saturating level of FN CS1 peptide-albumin conjugate (3 µg/ml). Adhesion was allowed to occur for 30 min at room temperature. &cjs2108;, Jurkat; &cjs2109;, THP-1 cells.



15/7 Promotes Cell Adhesion

In addition to recognizing an activation epitope associated with beta(1) integrin, 15/7 also promoted beta(1) integrin-dependent cell adhesion (Fig. 5). 15/7 enhanced THP-1 adhesion to VCAM-1 through alpha(4)beta(1) integrin, to whole FN through alpha(4)beta(1) and alpha(5)beta(1) integrin, and to laminin through alpha(6)beta(1) integrin. Comparable results were obtained with monovalent Fab fragments of 15/7 (not shown), indicating that the enhancing effects did not require antibody Fc and were not the result of receptor cross-linking. The degree of enhanced binding to each of the ligands was equivalent to that induced by Mn. 15/7 did not promote THP-1 cell adhesion to ICAM-1, whereas Mn induced ICAM-1 adhesion that was mediated by beta(2) integrin. Thus, 15/7 selectively enhanced beta(1) integrin-dependent cell adhesion. As shown below, 15/7 promoted cell adhesion by stabilizing an active conformation of beta(1) integrin.

Expression of the 15/7 Epitope Is Enhanced in the Presence of Ligand

The degree to which 15/7 enhanced THP-1 cell adhesion was surprising since the antibody reacted with unstimulated THP-1 cells at very low levels (Fig. 2), yet promoted adhesion as strongly as Mn. This discrepancy may be explained if expression of the 15/7 epitope was enhanced by ligand encountered during the adhesion assay. In order to address this possibility THP-1 and Jurkat cells were exposed to 15/7 in the presence of recombinant soluble VCAM-1. Fig. 6illustrates that soluble VCAM-1 enhanced expression of the 15/7 epitope in a dose-dependent fashion on both cell types, and the induction was completely prevented by an antibody against alpha(4) integrin. The conformational response of the receptors was rapid, occurring within 1 min of VCAM-1 addition, and occurred to the same extent at 4, 25, and 37 °C (although longer incubation times were required at 4 °C; not shown). Even though THP-1 and Jurkat cells expressed similar levels of alpha(4)beta(1) integrin (Fig. 2), they differed in both the number of receptors responsive to sVCAM-1 (3-fold difference in fluorescence at saturation) and in the concentration of ligand required to induce the epitope (EC for Jurkat = 20 nM sVCAM-1; THP-1 = 180 nM).


Figure 6: Induction of the 15/7 epitope by soluble VCAM-1. Jurkat and THP-1 cells were treated with F(ab`)(2) fragments of anti-alpha(4) integrin (AN100226m; 10 µg/ml), or with no additions for 30 min on ice. The cells were then exposed to 15/7 (10 µg/ml) in the presence of recombinant soluble VCAM-1 (at the indicated concentration) for 30 min at room temperature, washed, and exposed to PE-conjugated goat anti-mouse Fc (in the presence of human serum) to detect 15/7 by FACS analysis. -box-, Jurkat; bulletbulletbulletboxbulletbulletbullet, Jurkat + anti-alpha(4) integrin; -circle-, THP-1; bulletbulletbulletcirclebulletbulletbullet, THP-1 + anti-alpha(4) integrin.



Expression of the 15/7 Epitope Is Induced by Peptide Ligand and Potentiated by Activators of Cell Adhesion

The expression of the 15/7 epitope could also be enhanced on Jurkat and THP-1 cells by a peptide derived from the CS1 domain of fibronectin, as a soluble ligand for alpha(4)beta(1) integrin (Fig. 7). This peptide was an unconjugated, monovalent form of the peptide used for the adhesion assay presented above (Fig. 4), and contained the alpha(4) integrin-specific recognition sequence LDV. As with VCAM-1, induction of the 15/7 epitope by LDV peptide was dose-dependent (Fig. 7), occurred immediately upon peptide addition, required divalent cations, and occurred to the same extent at either 4 or 37 °C (not shown). At saturation, the peptide induced a 4-fold higher level of the 15/7 epitope on Jurkat than on THP-1 cells, even though both cell types express similar levels of alpha(4)beta(1) integrin. Furthermore, the responsive receptors on Jurkat cells exhibited a higher apparent affinity than those on THP-1 cells for the peptide ligand (EC Jurkat = 20 µM; THP-1 = 120 µM). These results, and those presented in Fig. 6, indicate that 15/7 not only recognizes an active form of alpha(4)beta(1) integrin (in the presence of Mn, 8A2, or PMA), but also recognizes the receptor when it is occupied by ligand (either VCAM-1 or LDV peptide).


Figure 7: Induction of the 15/7 epitope by LDV peptide. THP-1 (circle) and Jurkat (box) cells were treated with 15/7 (10 µg/ml) and LDV peptide (at the indicated concentration) for 30 min at room temperature in the presence of F(ab`)(2) fragments of 8A2 (-bullet-bullet-bullet, 5 µg/ml), Mn (- - -, 1 mM), PMA (bulletbulletbullet, 50 nM), or no additions(-). The cells with PMA were preincubated for 5 min at 37 °C. The cells were washed and exposed to PE-conjugated goat anti-mouse Ig Fc for FACS analysis.



Also shown in Fig. 7, 15/7 could be used to measure the relative number and affinity of receptors that were available for ligand occupancy in the presence of three different activators of cell adhesion. While Mn induced the 15/7 epitope by itself (as shown in Fig. 3), it further potentiated the response of receptors to LDV peptide (EC Jurkat with Mn = 2 µM; THP-1 = 5 µM). Interestingly, Mn did not change the overall number of receptors responsive to LDV peptide, suggesting that Mn and the peptide interact with the same receptor population. In contrast, 8A2 increased both the sensitivity of receptors to LDV peptide, as well as the number of receptors that expressed the 15/7 epitope at peptide saturation; in the presence of 8A2, receptors on both Jurkat and THP-1 cells exhibited an EC of 2 µM for the peptide, while fluorescence at saturation increased 30 and 100% on the two cell types, respectively. PMA increased the apparent affinity of receptors on THP-1 cells by 5-fold, but did not affect receptor affinity on Jurkat cells. Thus, in the presence of PMA, both cell types exhibited the same apparent affinity for the peptide ligand (EC = 20 µM). PMA did not significantly increase the number of responsive receptors on either cell type; Jurkat cells continued to express 4-fold higher levels of the 15/7 epitope than THP-1 cells in the presence of PMA. Table 1summarizes the EC for induction of the 15/7 epitope on Jurkat and THP-1 cells in the presence of the activating reagents.



15/7 Locks beta(1) Integrin in an Active Conformation Induced by Mn or Peptide Ligand

Since THP-1 cells express the 15/7 epitope in the presence of ligand, 15/7 may promote THP-1 cell adhesion (as in Fig. 5) by stabilizing the active or ligand-occupied conformation of beta(1) integrin. This possibility was examined by taking advantage of the reversible nature of the interactions of alpha(4)beta(1) integrin with LDV peptide (as a ligand) and with Mn (as an activating reagent). Fig. 8A demonstrates that while LDV peptide and Mn both induced the 15/7 epitope on THP-1 cells, neither reagent produced a lasting effect on epitope expression if they were removed from the cells prior to 15/7 exposure. In contrast, if THP-1 cells were exposed to the reagents in the presence of 15/7, then the antibody remained bound to beta(1) integrin when the reagents were removed. Fig. 8B demonstrates that 15/7 stabilized an active conformation of beta(1) integrin, induced by either Mn or LDV peptide, to promote THP-1 cell adhesion to VCAM-1. Exposure of THP-1 cells to 15/7, by itself, did not promote adhesion if the antibody was washed away prior to the binding assay. This result is consistent with the low expression level of the 15/7 epitope on resting THP-1 cells (Fig. 3). Likewise, Mn, by itself, induced cell adhesion only when it was present during the adhesion assay. Pretreatment of THP-1 cells with the combination of 15/7 and Mn, followed by washing, promoted cell adhesion to the same extent as if Mn or 15/7 were present during the adhesion incubation. This result indicates that 15/7 stabilized the active conformation of alpha(4)beta(1) integrin that had been induced by Mn. Pretreatment of THP-1 cells with 15/7 and the LDV peptide also induced strong adhesion to VCAM-1; however, if the reagents were left present throughout the assay, the LDV peptide completely inhibited adhesion. These results demonstrate that 15/7 recognized and stabilized the ``ligand-occupied'' conformation of alpha(4)beta(1) integrin as an ``active'' conformation to promote cell adhesion to VCAM-1 when the low affinity peptide was washed away. The fact that 15/7, by itself, promoted adhesion when present throughout the assay supports the idea that THP-1 cells express the 15/7 epitope when they encounter immobilized VCAM-1 during the adhesion incubation. Treatment of THP-1 cells with TS2/16 promoted cell adhesion to the same extent regardless of whether unbound antibody was left present during or removed prior to the adhesion assay. This result indicates that, unlike 15/7, TS2/16 reacted with beta(1) integrin to directly induce an active conformation of the receptor.


Figure 8: 15/7 stabilizes an active conformation of alpha(4)beta(1) integrin induced by Mn or by LDV peptide. A, FACS analysis. THP-1 cells were treated with Mn (1.5 mM), LDV peptide (500 µM), or no additions for 30 min on ice. Half of the cells in each sample were washed (&cjs2108;) free of the reagents, while the remaining cells were left unwashed (&cjs2110;). Both sets of cells were exposed to 15/7 (10 µg/ml) for 30 min on ice, and then to PE-conjugate goat anti-mouse Ig Fc for FACS analysis. B, cell adhesion. THP-1 cells were treated with the following reagents either individually or in combination (as indicated in the figure) for 30 min on ice: 15/7 (10 µg/ml), Mn (1.5 mM), LDV peptide (500 µM), anti-alpha(4) integrin (AN100226m; 5 µg/ml), or TS2/16 (5 µg/ml). The cells were washed quickly or left unwashed, as in A, and immediately tested for their ability to bind to purified membrane VCAM-1 (3 ng/well) as described under ``Materials and Methods.''



Mn Activates Receptors That Normally Mediate Cell Adhesion

Resting THP-1 cells did not express the 15/7 epitope, yet bound to VCAM-1 when the ligand was plated at sufficient densities (as shown in Fig. 1). Since a subpopulation of receptors on THP-1 cells were responsive to Mn or to ligand for expression of the 15/7 epitope, we wanted to determine if these responsive receptors normally mediated cell adhesion under resting conditions. In the experiment shown in Fig. 9, THP-1 cells were left untreated or were treated with 15/7 and Mn to stabilize the Mn-responsive beta(1) integrin receptors in an active conformation (as in Fig. 8). The cells were then examined for their ability to bind to immobilized VCAM-1 in the presence or absence of soluble VCAM-1 (30 nM). Resting THP-1 cells bound to high densities of VCAM-1 as expected, and adhesion was not significantly affected by the presence of soluble ligand. These results suggest that the receptors on THP-1 cells that mediate adhesion to VCAM-1 exhibit a low affinity, and are unable to establish a stable association with the soluble molecule. Presumably, the low affinity receptors can support adhesion through multivalent (high avidity) interactions with immobilized VCAM-1. As expected, pretreatment of THP-1 cells with 15/7 and Mn enhanced adhesion to the immobilized ligand; however, adhesion of these cells, even to high densities of VCAM-1, was completely eliminated in the presence of soluble VCAM-1. These results indicate that 15/7 stabilized the Mn-responsive receptors in a high affinity conformation and allowed stable association of the receptors with soluble VCAM-1. Nearly identical results were obtained with Jurkat cells (not shown). These results indicate that the Mn-responsive receptors are required for cell adhesion; when these receptors are occupied by soluble VCAM-1 the cells are no longer able to bind to the immobilized ligand.


Figure 9: Mn activates receptors that normally mediate cell adhesion. THP-1 cells were treated with a combination of 15/7 (10 µg/ml) and Mn (1.5 mM), or with no additions for 15 min at room temperature. The cells were washed rapidly, resuspended in buffer with or without recombinant soluble VCAM-1 (30 nM), and tested immediately for their ability to bind to purified membrane VCAM-1 (plated at the indicated concentration). The adhesion incubation was allowed to occur for 15 min at room temperature. circle, no treatment; Delta, rsVCAM-1; box, 15/7 + Mn; , 15/7 + Mn with rsVCAM-1.



Ligand Responsive alpha(4)beta(1) Integrin Receptors on Circulating Lymphocytes

Since it has been reported that lymphocytes in the circulation can bind to VCAM-1 without activation(7) , 15/7 was used to probe the state of beta(1) integrin activity on lymphocytes and the responsiveness of lymphocyte receptors to LDV peptide. The measurements were performed in freshly drawn whole blood to minimize the potential for receptor activation through cell handling; heparin, as an anticoagulant, did not affect expression of the 15/7 epitope, nor its ability to be induced by LDV peptide (as determined by control measurements with THP-1 and Jurkat cells added to whole blood; not shown). CD45Ro (memory) and CD45Ro (naive) lymphocyte subsets were examined independently by double color FACS analysis. As shown in Fig. 10, both exhibited low resting levels of the 15/7 epitope and the epitope was induced by LDV peptide in a dose-dependent fashion (reaching saturation at 600 µM). Perhaps reflecting higher expression levels of alpha(4)beta(1) integrin, CD45Ro cells exhibited a higher level of peptide-responsive receptors than CD45Ro cells (mean fluorescence 75 ± 9 and 32 ± 7, respectively, at peptide saturation). Furthermore, the CD45Ro cells exhibited slightly higher affinity for the LDV peptide than the Ro cells (EC = 148 ± 46 and 222 ± 74, respectively). These results suggest that lymphocytes in the circulation exhibit a level of alpha(4)beta(1) integrin activity that is similar to that of THP-1 cells, expressing low resting levels of the 15/7 epitope and exhibiting receptors with similar responsiveness to peptide ligand. The effect of PMA on the lymphocytes was also similar to that with THP-1 cells; treatment with PMA for 5 min increased the apparent affinity of the lymphocyte receptors for LDV peptide by 10-fold (EC CD45Ro = 15 ± 4 µM; CD45Ro = 25 ± 9 µM peptide). While PMA induced low levels of the 15/7 epitope in the absence of ligand, it had little impact on the number of receptors that expressed the 15/7 epitope at peptide saturation.


DISCUSSION

Several conclusions can be drawn from the studies with 15/7, and are illustrated in the model shown in Fig. 11. 1) 15/7 recognized an activation epitope on beta(1) integrin that was strongly associated with the adhesive capacity of cells. The antibody promoted beta(1) integrin-dependent cell adhesion by recognizing and directly stabilizing an active conformation of the receptor. 2) 15/7 identified both high affinity and ligand-occupied beta(1) integrin, and provided evidence that these receptor populations share a similar conformation. 3) Studies with 15/7 indicated that there are at least three subsets of beta(1) integrin on the surface of resting cells: constitutively active or high affinity, transiently active or low affinity, and receptors that are inactive or resistant to activation. 4) The low affinity receptors were available for interaction with ligand, were responsive to Mn and activating antibodies, and were the receptors that normally mediated cell adhesion when ligand was present at sufficient density. 5) The size of the low affinity receptor pool, and its relative affinity for ligand, was a distinct phenotype of THP-1 and Jurkat cells and represents a mechanism by which alpha(4)beta(1) integrin-mediated cell adhesion is likely to be regulated by cells in the circulation.


Figure 11: Subsets of alpha(4)beta(1) integrin: a model. Three subsets of alpha(4)beta(1) integrin were identified in studies with 15/7. One receptor population (shown in the middle) interacted transiently with ligand to express the 15/7 epitope, and mediated cell adhesion to immobilized ligand when it was present at sufficient density. Receptors within this pool also responded to Mn and to activating antibodies against beta(1) integrin to exhibit the 15/7 epitope and a high affinity conformation. Stimulation of cells with PMA increased the sensitivity of these receptors to ligand, but did not induce the highest affinity form. It is possible that receptors within this pool normally exhibit a rapid equilibrium between active and inactive conformations, as suggested for alphabeta(3) integrin(32) . In the presence of Mn or LDV peptide, 15/7 stabilized the conformation of the responsive receptors in a high affinity form (shown on the right). Receptors that constitutively expressed the 15/7 epitope (in the absence of ligand or activating reagents) were present at only low levels on Jurkat cells, and were almost absent on THP-1 cells and on whole blood lymphocytes. Inactive receptors (shown on the left) resisted expression of the 15/7 epitope in the presence of saturating concentrations of peptide ligand, soluble VCAM-1, Mn, activating antibodies against beta(1) integrin, or PMA. The large percentage of inactive receptors on THP-1 cells appeared to limit their adhesive capacity, even when the affinity of receptors in the responsive pool was increased by Mn or PMA.



15/7 Recognizes an Activation Epitope Associated with beta(1)Integrin

15/7 Recognized the beta(1) integrin chain (Fig. 2B) and its epitope could be induced on cells by three distinct types of beta(1) integrin activators: Mn, PMA, and the anti-beta(1) integrin activating antibodies, TS2/16 and 8A2 ( Fig. 2and Fig. 3). Expression of the epitope on THP-1 and Jurkat cells in the presence of the stimulating reagents strongly correlated with alpha(4)beta(1) integrin activity, as measured by cell adhesion to VCAM-1 and to a peptide derived from the CS1 domain of fibronectin ( Fig. 1and Fig. 4).

15/7 Enhances Cell Adhesion by Stabilizing an Active Conformation of beta(1)Integrin

15/7 promoted THP-1 cell binding to beta(1) integrin ligands, even though the cells did not express a significant level of the 15/7 epitope (Fig. 5). However, in order to promote adhesion, it was important that 15/7 was present during the adhesion incubation (Fig. 8). These results suggested that expression of the 15/7 epitope was facilitated by the presence of ligand in the assay well, and that 15/7 stabilized the ligand interaction. Consistent with this idea was the observation that soluble peptide ligand and VCAM-1, itself, promoted expression of the 15/7 epitope in a dose-dependent fashion ( Fig. 6and Fig. 7). Once 15/7 had bound beta(1) integrin in the presence of peptide, the antibody interaction was stable when the peptide was removed, and the integrin remained locked in the ligand-occupied, or active, conformation to promote cell adhesion in a subsequent assay (Fig. 8). Thus, it cannot be argued that 15/7 is simply a low affinity antibody that must be present during an adhesion assay in order to promote ligand interaction. Instead, these results imply that beta(1) integrin ligands, such as VCAM-1, fibronectin, or laminin, facilitate expression of the 15/7 epitope by engaging low affinity, or transiently active receptors. During a cell adhesion assay with resting THP-1 cells, 15/7 promoted cell attachment by engaging beta(1) integrin in the presence of ligand, and by stabilizing what would have otherwise been a transient ligand interaction.

15/7 Is Different Than beta(1)Integrin ``Activating'' Antibodies

15/7 is distinct from other antibodies against beta(1) integrin that promote cell adhesion, such as TS2/16 and 8A2. These antibodies recognize beta(1) integrin regardless of the state of activation, and can induce a more active form. Thus, by FACS analysis, TS2/16 and 8A2 reacted with THP-1 and Jurkat cells at levels comparable to control antibodies against beta(1) integrin (Fig. 2), and this high level of recognition was not influenced by the activating effects of Mn (not shown). In addition, the 15/7 epitope has recently been mapped to the central region of beta(1) integrin at a site distinct from that for the activating antibodies. (^3)Since 15/7 reacted strongly with the isolated beta(1) integrin chain under the denaturing conditions of Western analysis (Fig. 2), it may recognize a linear epitope of beta(1) integrin that is exposed or masked by conformational changes associated with the receptor heterodimer.

15/7 as a ``Ligand-induced Binding Site'' Antibody versus an Antibody Against an Activation Epitope of beta(1)Integrin

Antibodies have been described that recognize ligand-occupied conformations of alphaII(b)beta(3) integrin, or ligand-induced binding sites(32, 42) . The epitopes for these antibodies can be induced on platelets by peptide ligands derived from fibrinogen, and by fibrinogen itself, when the platelets are activated. 15/7 also functions as a ligand-induced binding site antibody since its epitope on beta(1) integrin was induced by soluble VCAM-1 or by peptide ligands ( Fig. 6and Fig. 7). However, ligand was not required for expression of the 15/7 epitope since it could be induced by several distinct activators of cell adhesion in the absence of any known beta(1) integrin ligands ( Fig. 2and Fig. 3); expression of the 15/7 epitope under these conditions correlated with adhesive activity (Fig. 4). Furthermore, 15/7 stabilized the conformation of alpha(4)beta(1) integrin induced by Mn (as an activating agent) or LDV peptide (as a ligand) to enhance cell adhesion when the reagents were removed (Fig. 8). These results indicate that 15/7 recognizes beta(1) integrin when the receptor is either active or occupied by ligand, and suggests that these two receptor forms exhibit a similar conformation. Thus, 15/7 is both a ligand-induced binding site antibody for beta(1) integrin, as well as an antibody against a beta(1) integrin activation epitope.

The ligand occupied forms of other integrins may also exhibit an active conformation, since it has been shown that peptide ligand can induce the activity of purified or fixed alphaII(b)beta(3) integrin(33) . In addition, 15/7 has characteristics similar to antibody 24, which recognizes an activation epitope associated with the beta(2) integrins(30) . Following transient cell stimulation with PMA, antibody 24 engages LFA-1 in the presence of ligand (ICAM-1 on adjacent lymphocytes) and prevents the normal decline in receptor activity. However, since LFA-1 on lymphocytes cannot interact with ICAM-1 in the absence of cell stimulation(43) , its activity is likely to be regulated in a manner different than that of alpha(4)beta(1) integrin.

Subsets of beta(1)Integrin on the Cell Surface

The model depicted in Fig. 11summarizes studies with 15/7 which suggest that there are at least three subsets of beta(1) integrin on the cell surface that can be distinguished by their state of activation: 1) active or high affinity; 2) transiently active or low affinity; and 3) inactive. The first population expressed the 15/7 epitope constitutively and is likely to be comprised of ``high affinity'' receptors described in other reports(17, 27) . These receptors represented a small percentage of the total beta(1) integrin on the surface of resting Jurkat cells, and were virtually absent on THP-1 cells and on lymphocytes in the circulation.

The second population of receptors was detected by 15/7 in the presence of saturating concentrations of peptide ligand or Mn and is likely to correspond to the population of ``low affinity'' receptors that have been described in other reports(17, 27) . These receptors were required for cell adhesion to VCAM-1, as shown in Fig. 9. Resting THP-1 cells bound well to VCAM-1 when the ligand was plated at a sufficient density, and binding was not affected significantly by soluble VCAM-1. These results suggest that the affinity of the receptors that normally mediate THP-1 cell adhesion is too low to form a stable association with soluble VCAM-1. Presumably, such low affinity receptors can mediate cell adhesion to immobilized ligand through multivalent interactions. When the Mn-responsive receptors were held active by 15/7, soluble VCAM-1 was able to completely inhibit even the resting levels of cell adhesion. These results indicate that Mn affects the population of receptors that are normally available for ligand binding. These receptors do not constitutively express the 15/7 epitope, nor do they exhibit a stable high affinity conformation for interaction with ligand, and, therefore, are not normally occupied by soluble VCAM-1; however, these receptors can initiate or mediate cell adhesion when ligand is present at sufficient density. Recently, it has been shown that in vivo administration of recombinant soluble VCAM-1, at initial circulating levels of 100-200 nM, will inhibit the onset of experimental diabetes, even though interaction of the construct with lymphocytes in the circulation could not be measured(44) . Based on the results shown in Fig. 6, it is likely that the majority of low affinity alpha(4)beta(1) integrin receptors were occupied by the VCAM-1 construct at this concentration, but that the interaction of the construct with the lymphocytes could not be measured by indirect FACS analysis (which involves multiple cell washings).

The third population of receptors on the cell surface appeared to be conformationally restrained and unavailable for ligand interactions regardless of ligand concentration or density. This population represented the majority of beta(1) integrin on the surface of THP cells. In the presence of saturating concentrations of Mn, sVCAM-1, or peptide ligand, THP-1 cells expressed 3-10-fold lower levels of the 15/7 epitope than Jurkat cells (Fig. 3, Fig. 6, and Fig. 7), even though THP-1 cells expressed higher levels of beta(1) integrin than Jurkat, and equivalent levels of alpha(4)beta(1) integrin (Fig. 2). This population of receptors resisted expression of the 15/7 epitope even when directly engaged by the activating antibodies against beta(1) integrin ( Fig. 3and Fig. 7). However, in lysates of THP-1 and Jurkat cells, alpha(4)beta(1) integrin exhibited equivalent levels of activity for binding immobilized VCAM-1 (not shown). Furthermore, by Western analysis, 15/7 and the control antibody against beta(1) integrin, 15/1, reacted equally well with the isolated beta(1) integrin subunit (Fig. 2), regardless of the cell type from which the receptor was obtained (not shown). These results suggest that factors associated with the integrin heterodimer on the cell surface, or within the cytoplasm, prevent exposure of the 15/7 epitope and regulate receptor activity. Such negative regulatory factors are likely to play a pivotal role in controlling patterns of cell migration and localization by defining the capacity of cells to respond to ligand at a given density.

The regulation of alphaII(b)beta(3) integrin appears to be different than that of alpha(4)beta(1) integrin, in that subsets of alphaII(b)beta(3) integrin were not apparent on the platelet cell surface; essentially all of the platelet receptors were responsive to RGD peptide or to PMA (45) . The existence of beta(1) integrin subsets is likely to reflect the specialized function of the many different cell types that express this receptor.

Activators of beta(1)Integrin Can Affect Either the Apparent Affinity of beta(1)Integrin or the Size of the Responsive Receptor Pool

Mn and soluble peptide ligand appear to interact with the same subset of low affinity receptors to induce the 15/7 epitope since the effects of the reagents were not additive when peptide was present at a saturating level (Fig. 7). However, Mn increased receptor sensitivity to LDV peptide by 10-fold on Jurkat cells, and by 25-fold on THP-1 (see Table 1), so that receptor saturation was reached at a lower concentration of peptide. These results suggest that Mn facilitates cell adhesion by increasing the affinity of the available receptors, favoring an active conformation of beta(1) integrin and expression of the 15/7 epitope, but does not increase the number of receptors that are available for cell adhesion.

The activating antibody against beta(1) integrin, 8A2, affected the same receptor population that was responsive to Mn since their inductive effects were not additive (Fig. 3); however, 8A2 also recruited additional receptors from the otherwise inactive pool. In the presence of 8A2, maximal expression of the 15/7 epitope was achieved with the same low concentrations of peptide ligand as with Mn (IC = 2 µM) but the level of response to peptide was significantly higher (100% increase on THP-1 cells). Thus, in contrast to Mn, 8A2 increased both the affinity and the number of receptors available for ligand binding. These results are consistent with the finding that 8A2 induced higher levels of cell adhesion than Mn ( Fig. 1and Fig. 4).

PMA produced a small increase in expression of the 15/7 epitope, indicating a conformational response, or activation, of a small number of receptors (Fig. 3A). Of more significance, PMA increased the apparent affinity of the ligand responsive receptors by up to 10-fold, as measured by the concentration of LDV peptide required to induce the 15/7 epitope (Table 1). Regardless of the cell type and the resting level of receptor activity, the apparent affinity of the responsive receptors in the presence of PMA was the same; THP-1 cells, memory, and naive lymphocytes all demonstrated an EC of 20 µM LDV peptide. It is interesting that the receptors on nonstimulated Jurkat cells already exhibited this level of apparent affinity and showed minimal response to PMA. Mn or the activating antibodies against beta(1) integrin increased the apparent affinity of alpha(4)beta(1) integrin on both THP-1 and Jurkat cells to a level that was 10-fold higher than that induced by PMA (EC = 2 µM peptide). These results indicate that although PMA induced low levels of the 15/7 epitope and increased receptor affinity to a certain level, the receptors still exhibited a low affinity conformation; separate experiments have demonstrated that these receptors will not form stable association with soluble VCAM-1 (not shown). Mn and the activating antibodies directly induced high levels of the 15/7 epitope as well as a high affinity receptor conformation, and were found to promote stable association with soluble VCAM-1 ( Fig. 9and not shown). It is possible that the differing effects of PMA that have been reported for different integrin systems (see Introduction) may reflect its ability to increase receptor affinity without inducing the highest affinity receptor form.

Ligand Specificity of alpha(4)beta(1)Integrin-mediated Cell Adhesion

Different cells that express alpha(4)beta(1) integrin can differ greatly in their ability to bind VCAM-1 and FN CS1. In general, cell adhesion to FN CS1 requires higher levels of alpha(4)beta(1) integrin activity than adhesion to VCAM-1; however, the fact that some cells demonstrate absolute preference for VCAM-1 (lacking the ability to bind FN CS1) has lead to the idea that alpha(4)beta(1) integrin exhibits binding sites for VCAM-1 and for FN CS1 that can be regulated independently(14, 46) . In contrast, there is evidence that VCAM-1 and FN CS1 occupy the same ligand binding site on alpha(4)beta(1) integrin, but that the affinity for CS1 is simply lower than that for VCAM-1(47) . In the current study, THP-1 cells bound strongly to VCAM-1 when the ligand was plated at a high density (Fig. 1), but could not bind to a peptide derived from FN CS1 even when the peptide was plated at a saturating level (Fig. 4). This difference could not be explained by differential regulation of independent ligand-binding sites, since the apparent affinity of alpha(4)beta(1) integrin on THP-1 cells for both VCAM-1 and CS1 peptide was much less than that on Jurkat cells ( Table 1and Fig. 6text). The low affinity of alpha(4)beta(1) integrin on THP-1 cells for ligand, in general, appears to have precluded cell adhesion to the lower affinity CS1 peptide. Even in the presence of Mn or PMA, when receptors on THP-1 and Jurkat cells exhibited similar affinity for soluble CS1 peptide (Table 1), THP-1 cell adhesion to the immobilized peptide continued to be lower than that of Jurkat cells (Fig. 4). A likely explanation for the difference between these two cells types is that in the presence of Mn or PMA, THP-1 cells continued to exhibit a lower number of receptors that were available for ligand interaction than Jurkat cells (Fig. 7). Stimulation of THP-1 cells with 8A2, where both receptor affinity and the number of available receptors was increased, allowed THP-1 cells to bind to the CS1 peptide at a level approaching that of resting Jurkat cells (Fig. 4). Therefore, regulation of receptor number, as well as receptor affinity, is likely to contribute to differences in ligand binding specificity of alpha(4)beta(1) integrin expressed on different cells.

Conclusions

The most novel proposal to emerge from the studies with 15/7 is that alpha(4)beta(1) integrin-dependent cell adhesion can be regulated by the size and affinity of a ligand-responsive population of receptors; a population that is available for ligand interactions, but with low affinity or transient activity. Under resting conditions, the receptors that mediate cell adhesion change conformation in response to ligand, and do not require an exogenous stimulus to trigger ligand binding. These findings are of particular interest since it has recently been demonstrated that alpha(4)beta(1) integrin can support lymphocyte rolling and adhesive interactions on VCAM-1 in the presence of shear forces encountered within normal blood flow (see Introduction). Based on the findings of the current study with 15/7, activating signals or cytokines would not be required to initiate the interaction of cells with VCAM-1 at inflammatory sites. Instead, the ability of cells to roll on VCAM-1 may reflect their stable expression of a low affinity receptor population; the larger the receptor pool, the lower the density of VCAM-1 required to initiate cell binding. In this regard, it is interesting that memory cells in the circulation were found to express a greater number of receptors that were responsive to alpha(4)beta(1) integrin ligand than naive cells, and the receptors exhibited a higher apparent affinity. Chemokines or other activating signals would play an important role following initial cell contact with the vessel wall, and may act to increase alpha(4)beta(1) integrin affinity in a manner similar to that observed in the current study with PMA. The size of the ligand responsive pool, and relative affinity of the receptors within it, are likely to represent the major differences between cell types with different adhesive capacities under resting conditions. The regulation of beta(1) integrin on cells in the circulation appears to be distinct from that of other integrins, such as alphaII(b)beta(3) integrin on platelets, LFA-1 on lymphocytes, or Mac-1 on neutrophils, which require activation to initiate adhesive contact. Just as antibodies that recognize activation and ligand-dependent epitopes have been invaluable tools for understanding the regulation of beta(2) and beta(3) integrins, antibodies such as 15/7 will greatly help to understand the complex regulation of beta(1) integrin activity.


FOOTNOTES

*
This work was funded, in part, by Wyeth-Ayerst Research, Princeton, NJ. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: 800 Gateway Blvd., South San Francisco, CA 94080. Tel.: 415-877-7622; Fax: 415-877-8370.

(^1)
The abbreviations used are: CS1, type III connecting segment region 1; EC, excitatory concentration for half-maximal expression; FN, fibronectin; FITC, fluorescein isothiocyanate; PE, phycoerythrin; PMA, phorbol myristate acetate; PAGE, polyacrylamide gel electrophoresis; FACS, fluorescence-activated cell sorter.

(^2)
L. I. Tanner and T. A. Yednock, unpublished observations.

(^3)
W. Puzon, T. A. Yednock, and Y. Takada, manuscript submitted for publication.


ACKNOWLEDGEMENTS

We thank Robin Barbour for her excellent guidance during the production of the monoclonal antibodies described in this study. We also thank Dr. Louis Picker, Dr. Kevin Tomaselli, and Dr. Lloyd Stoolman for valuable discussions.


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