©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Activated Conformations of Very Late Activation Integrins Detected by a Group of Antibodies (HUTS) Specific for a Novel Regulatory Region(355425) of the Common 1 Chain (*)

(Received for publication, June 13, 1995; and in revised form, October 31, 1995)

Alfonso Luque (1) Manuel Gómez (2) Wilma Puzon (3) Yoshikazu Takada (3) Francisco Sánchez-Madrid (2) Carlos Cabañas (1)(§)

From the  (1)Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad Complutense, 28040 Madrid, Spain, the (2)Servicio de Inmunología, Hospital de la Princesa-U.A.M., 28006 Madrid, Spain, and the (3)Department of Vascular Biology, The Scripps Research Institute, La Jolla, California 92037

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The very late activation antigens (VLA) or beta1 integrins mediate cell attachment to different extracellular matrix proteins and intercellular adhesions. The ligand binding activity of these adhesion receptors is not constitutive and can be regulated by temperature, presence of extracellular divalent cations, stimulatory monoclonal antibodies (mAbs), and cellular activation. We have generated three novel mAbs, HUTS-4, HUTS-7, and HUTS-21, recognizing specific epitopes on the common beta1 subunit (CD29) of VLA integrins whose expression correlates with the ligand binding activity of these heterodimeric glycoproteins. This correlation has been demonstrated for several integrin heterodimers in different cell systems using a variety of extracellular and intracellular stimuli for integrin activation. Thus, the presence of micromolar concentrations of extracellular Mn, preincubation with the activating anti-beta1 mAb TS2/16, and cell treatment with phorbol esters or calcium ionophores, induced the expression of the HUTS beta1 epitopes on T lymphoblasts. Using a panel of human-mouse beta1 chimeric molecules, we have mapped these epitopes to the 355-425 sequence of the beta1 polypeptide. This segment represents therefore a novel regulatory region of beta1 that is exposed upon integrin activation. Interestingly, binding of HUTS mAbs to partially activated VLA integrins results in maximal activation of these adhesion receptors and enhancement of cell adhesion to beta1 integrin ligands collagen, laminin, and fibronectin.


INTRODUCTION

The beta1 or VLA (^1)integrins are a subgroup within the integrin family comprising at least 10 members, each with a distinct alpha subunit non-covalently associated with the common beta1 subunit (reviewed by Giancotti and Mainiero(1994), Hemler (1990), Hynes(1992), and Sánchez-Madrid and Corbi(1992)). The VLA integrins function as cellular receptors for extracellular matrix proteins such as different types of collagen, laminin, and fibronectin. However, VLA-4 not only acts as a cellular receptor for fibronectin but also mediates the interaction with its cellular ligand vascular cell adhesion molecule-1 expressed on cytokine-activated endothelial cells (Elices et al., 1990), and has been implicated in homotypic cell interactions as well (Campanero et al., 1990).

The alpha subunits of all integrins, including the VLA subfamily, are transmembrane glycoproteins that contain in the N-terminal region seven homologous domains of approximately 50 residues. Three or four of these domains are putative divalent cation binding sites with homology to the EF-hand Ca binding motif (Sánchez-Madrid and Corbi, 1992; Tuckwell et al., 1992). The common beta1 subunit of VLA integrins is also a transmembrane glycoprotein with a large extracellular domain containing a 4-fold repeat of a cysteine-rich segment and a highly conserved N-terminal segment of 200 amino acids which contains an EF-hand-like cation binding motif (Kühn and Eble, 1994). Divalent cations are essential for regulation of integrin ligand specificity and binding affinity. General features of the control by divalent cations of integrin functional activity are currently emerging. Most data indicate that Mg and Mn induce activation of integrins resulting in effective interaction with corresponding ligands (Arroyo et al., 1993; Dransfield et al., 1992a; Kirchhofer et al., 1990; Masumoto and Hemler, 1993). Conversely, Ca generally exerts inhibitory effects on integrin function (Dransfield et al., 1992a; Grzesiak et al., 1992; Hemler et al., 1990; Masumoto and Hemler, 1993; Staatz et al., 1989; Weitzman et al., 1993). In addition, an intact active cellular metabolism has been shown to represent a second type of requirement for integrin function (Campanero et al., 1990; Dransfield et al., 1990; Marlin and Springer, 1987; Rothlein and Springer, 1986; van de Wiel-van Kemenade et al., 1992).

Functional activation of integrins can also be experimentally induced from outside the cells with one of the several existing stimulatory mAbs directed against a specific alpha or the common beta integrin subunits, which seem to act by imposing a conformation of these molecules with high affinity for ligand binding (Keizer et al., 1988; Melero et al., 1993; van Kooyk et al., 1991). In particular, activation of VLA integrins has been reported by treatment with different mAbs specific for the beta1 subunit (Arroyo et al., 1992; Arroyo et al., 1993; Faull et al., 1994; Kovach et al., 1992; Luque et al., 1994; Masumoto and Hemler, 1993; van de Wiel-van Kemenade et al., 1992).

Intracellular signals generated as a result of cell activation also regulate the affinity and conformation of integrins in many cellular systems. For instance, monocyte and neutrophil activation by inflammatory mediators, such as tumor necrosis factor-alpha, C5a, or fMet-Leu-Phe, is required for beta1 and beta2 integrin-mediated adhesion to the endothelium and subsequent transmigration across the endothelial lining (Zimmerman et al., 1992). Thrombin-induced activation of platelets, which involves G-proteins and protein kinase C, is accompanied by an increase in the affinity of the alphaIIbeta3 integrin for soluble fibrinogen (Phillips et al., 1991). Similarly, the beta2 integrin leukocyte function-associated antigen-1 only mediates adhesion of T lymphocytes to intercellular adhesion molecule-1-expressing target cells after intracellular activation signals are induced through the TcR/CD3 or CD2 molecules (Dustin and Springer, 1989; van Kooyk et al., 1989). Phorbol esters such as phorbol 12-myristate 13-acetate or phorbol 12,13-dibutyrate, which are potent and sustained activators of protein kinase C, have been widely used in a number of cellular systems for induction of integrin activation. At least two different mechanisms have been implicated in the up-regulation of integrin-mediated adhesion induced by treatment with phorbol esters. The first one consists in the induction of transitions to a high affinity state in a small fraction of integrin receptors, probably as a consequence of conformational changes of these molecules. This seems to be the case for the leukocyte integrin leukocyte function-associated antigen-1 on T lymphocytes (Lollo et al., 1993). The second type of mechanism by which phorbol esters stimulate integrin-mediated cell adhesion is by altering events that occur after integrin occupancy such as the induction of cytoskeleton-driven cell spreading (Danilov and Juliano, 1989; Faull et al., 1994). Cell spreading as well as the induction of other post-receptor morphological changes can potentially favor integrin-mediated adhesion in some instances without affecting receptor affinity. The relative contribution of these two types of phorbol ester effects on integrin-mediated adhesion may well depend on the cell system under consideration. In addition to protein kinase C-mediated signaling, an increase in cytosolic free calcium has been recently demonstrated to play a major role in integrin activation through the use of calcium ionophores such as A23187 and ionomycin (van Kooyk et al., 1993; Hartfield et al., 1993).

Several ``reporter'' mAbs with the ability to discriminate between different states of integrin activation have revealed their usefulness in many studies on the regulation of activation of integrins belonging to the beta2 and beta3 subfamilies (Cabañas and Hogg, 1993; Dransfield et al., 1992a, 1992b; Du et al., 1991; Frelinger et al., 1991; O'Toole et al., 1990; van Kooyk et al., 1991, 1994). For the beta1 subgroup of integrins, however, availability of similar ``activation-reporter'' mAbs with the capacity to distinguish between different states of activation has been very limited.

In this report we describe a group of three mAbs, HUTS-4, HUTS-7, and HUTS-21, recognizing epitopes in the 355-425 region of the common beta1 subunit of VLA integrins, whose expression parallels the ligand binding activity of these adhesion receptors induced by various extracellular and intracellular stimuli.


MATERIALS AND METHODS

Cells and Cell Cultures

The colocarcinoma cell line COLO-320 (a generous gift from Dr. N. Hogg, Imperial Cancer Research Fund, London, United Kingdom), the erythroleukemic cell line K562, were grown in RPMI medium supplemented with 10% fetal calf serum (Biowhittaker, Fontenay Sous Bois, France), 50 IU/ml penicillin, 50 µg/ml streptomycin (ICN Biomedicals, Costa Mesa, CA). T lymphoblasts were obtained and cultured as described previously (Cabañas, 1993).

Antibodies

The control mAbs used in this study have all been previously described: TS2/16 (CD29) (Hemler et al., 1984), Lia1/2 (CD29) and X63 (negative control) (Campanero et al., 1992), TS2/7 (CD49a) (Hemler et al., 1984), 5E8D9 (CD49a) (Luque et al., 1994), Tea1/41 (CD49b) (Luque et al., 1994), P1B5 (CD49c) (Wayner and Carter, 1987), HP2/1 (CD49d) (Campanero et al., 1994; Arroyo et al., 1994), GoH3 (CD49f) (Sonnenberg et al., 1988), and HC1/1 (CD11c) (Cabañas et al., 1988).

Affinity Chromatography Purification of Activated beta1 Integrins

Purification of beta1 integrins in an activated form was carried out using an immunoaffinity chromatography column prepared by covalently coupling the stimulatory anti-beta1 mAb TS2/16 (at 2 mg/ml) to 3 ml of CNBr-activated CL-4B Sepharose (Pharmacia Fine Chemicals, Uppsala, Sweden) following the manufacturer's instructions. To preserve beta1 integrins in active conformation, divalent cation Mn at a concentration of 200 µM was always present throughout the immunoaffinity purification protocol.

beta1 Integrins were purified from Triton X-100 lysates of surgery samples of human lung, liver, and skeletal muscle tissues (obtained from the Department of Pathology, Hospital de la Princesa, Madrid). These tissues were diced, sieved, and lysed in 300 ml of lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 2 mM MgCl(2), 200 µM MnCl(2), 1% Triton X-100, 0.02% NaN(3), 1 mM PMSF, 0.2 units/ml aprotinin, and 5 mM iodoacetamide, pH 8.0) for 2 h at 4 °C. The cell lysate was centrifuged at 3,000 times g for 30 min at 4 °C and then ultracentrifuged at 100,000 times g for 1 h at 4 °C. The lysate was precleared by passing through a 2-ml pre-column of glycine-Sepharose CL-4B (pre-equilibrated in lysis buffer) and then loaded onto the 3-ml column of TS2/16-Sepharose CL-4B (pre-equilibrated in lysis buffer) at a flow rate of 0.5 ml/min. The column was then washed sequentially with 15 ml of lysis buffer and 15 ml of washing buffer (50 mM ethanolamine, 0.2% Triton X-100, 0.5 M NaCl, 2 mM MgCl(2), 200 µM MnCl(2), 1 mM PMSF, pH 10.0) and bound beta1 integrins were finally eluted with 50 mM ethanolamine, 0.5 M NaCl, 2 mM MgCl(2), 200 µM MnCl(2), 1% octyl glucoside, 1 mM PMSF, pH 12.0, at a flow rate of 0.5 ml/min. Fractions of 0.5 ml were collected and neutralized with 0.1 volume of 1 mM Tris, pH 6.7. Fractions containing beta1 integrins were identified by SDS-7% PAGE followed by silver staining. The yield of total beta1 integrins was 1.5 mg in 10 ml of neutralized elution buffer as estimated using the bicinchoninic-Protein Assay Reagent (Pierce Chemical Co).

Generation and Selection of mAbs HUTS-4, HUTS-7, and HUTS-21

Balb/c mice were injected intraperitoneally with approximately 7.5 µg of purified beta1 integrins (in PBS containing 200 µM Mn) on days -48 and -33, and intravenously on day -3. Spleen cells were fused on day 0 with SP2 mouse myeloma cells at a ratio of 4:1 according to standard techniques and distributed in 96-well culture plates (Costar, Cambridge, MA). After 2 weeks, 576 hybridoma culture supernatants were harvested and their reactivity against human T lymphoblasts expressing beta1 integrins was estimated by flow cytometry under two different incubation conditions: (a) in the total absence of divalent cations (chelator EDTA was added to the hybridoma supernatants at a final concentration of 3 mM), and (b) in the presence of 500 µM divalent cation Mn. Hybridomas showing differential reactivity under these two divalent cation conditions were cloned by limiting dilution. Clones of hybridomas HUTS-4, HUTS-7, and HUTS-21 were selected because the reactivity of their secreted mAbs with T lymphoblasts was markedly higher in the presence of Mn. HUTS-7 (IgG1), HUTS-4 (IgG2b), and HUTS-21 (IgG2b) represent different mAbs produced by specific clones derived from three independent hybridomas.

Immunoprecipitation

T lymphoblasts (3 times 10^7 cells/ml) were iodinated with 0.5 mCi of sodium [I]iodine (ICN Biomedicals, Irvine, CA) using 1,3,4,6-tetrachloro-3alpha,6alpha-diphenylglycoluril (IODO-GEN, Sigma) and lysed in phosphate-buffered saline (PBS), pH 7.4, containing 1% Triton X-100, 1% BSA, 1 mM PMSF, for 20 min (unless otherwise indicated). The lysates were immediately clarified by centrifugation at 7000 times g for 30 min and pre-cleared with 30 µl of protein A-Sepharose (Sigma). For immunoprecipitation, equal amounts of pre-cleared radioactive cell lysates were incubated at 4 °C with 100 µl of corresponding hybridoma culture supernatants or 5 µg of purified mAb. After 2 h, 100 µl of culture supernatant containing the rat anti-mouse IgG mAb 187.1 were added and incubation proceeded for an additional period of 2 h at 4 °C. Finally, immunoprecipitates were removed by addition of 30 µl of protein A-Sepharose, incubation for 1 h with continuous stirring, and centrifugation at 200 times g followed by processing as described previously (Cabañas et al., 1988). Samples were analyzed in SDS-7% polyacrylamide gels followed by autoradiography with enhancing screens.

To demonstrate the effects of different divalent cation conditions, T lymphoblasts were lysed in 20 mM Hepes, 150 mM NaCl, 1% Triton X-100, 1% BSA, 1 mM PMSF, pH 7.4. After centrifugation and pre-clearing, appropriate amounts of divalent cations were added to different aliquots of the cell lysates to yield final concentrations of Ca (1 mM) and Mg (1 mM) or Mn (1 mM) prior to the 2-h incubation with the corresponding purified mAb (5 µg). Precipitates were subsequently removed with protein A-Sepharose as described above and the concentrations of divalent cations were preserved throughout the washing and processing procedures. Analysis of samples was carried out in SDS-7% PAGE and autoradiography. Immunoprecipitations of dissociated VLA integrin subunits after high pH treatment of cell lysates were done essentially as described (Bednarczyk et al. 1994). Basically, I-surface-labeled T lymphoblasts were lysed at 3 times 10^7 cells/ml in 10 mM Tris-HCl, pH 8.0, containing 1% Triton X-100, 150 mM NaCl, 1 mM Mn, 2% BSA, 1 mM PMSF. Aliquots of the cell lysates were mixed with a 10-fold (v/v) excess of 10 mM Tris-HCl, 500 mM NaCl, 0.2% Triton X-100, 1 mM Mn, pH 8.0; or 20 mM triethylamine, 500 mM NaCl, 0.2% Triton X-100, 1 mM Mn, pH 11.0. These mixtures were incubated at 37 °C for 30 min and then rapidly neutralized by the addition of 1/10 volume of 1.0 M Tris-HCl, pH 6.8. Immunoprecipitation of integrin polypeptides with the respective mAbs and processing and analysis of samples were carried out as described above.

Flow Cytometric Analysis

Cells were washed twice in RPMI medium or in Hepes/NaCl buffer (20 mM Hepes, 150 mM NaCl, 2 mg/ml D-glucose, pH 7.4) prior to their addition to the wells of a 96 round-bottom well plate. Approximately 5 times 10^5 cells were incubated in each well with 2.5 µg of the corresponding mAb for 20 min at 37 °C in 100 µl of RPMI medium or Hepes/NaCl buffer, containing the appropriate concentrations of divalent cations Ca, Mg, and Mn and stimuli. After this first incubation, cells were washed 3 times with 200 µl of RPMI and incubated for an additional period of 30 min at 4 °C with 75 µl of a 1:300 dilution of fluorescein isothiocyanate-conjugated sheep anti-mouse IgG secondary antibody (Sigma) in RPMI medium. Finally, after three washes with PBS, cells were fixed in 200 µl of 5% formaldehyde in PBS and their fluorescence measured using a FACScan flow cytometer (Becton Dickinson).

Biotinylation of mAbs and Cross-competitive Binding Assays

Prior to biotin conjugation, purified mAbs at a concentration of 2 mg/ml, were dialyzed overnight against borate buffer (0.1 M boric acid, 0.025 M sodium tetraborate, 0.075 M sodium chloride, pH 9.4) at 4 °C. N-Hydroxysuccinimido-Biotin (Sigma) was freshly dissolved in dimethyl sulfoxide at 2 mg/ml and added to glass tubes containing the dialyzed mAbs, to yield a final concentration of 0.1 mg/ml. After continuous stirring of the mixture for 1 h at room temperature, unconjugated biotin was eliminated from the mAb solution by extensive dialysis against PBS containing 0.001% sodium azide.

In cross-competitive mAb binding assays, 5 times 10^5 T lymphoblasts were preincubated in round-bottomed wells with an excess of unconjugated mAbs (15-20 µg/ml) for 10 min at 37 °C in Hepes/NaCl buffer containing 0.5 mM Mn. Then, 2 µg of biotinylated mAbs were added to the wells and incubation proceeded for an additional period of 15 min at 37 °C. Unbound mAbs were then removed by washing the cells three times with 200 µl of RPMI, and 75 µl of a 1:300 dilution of fluorescein isothiocyanate-conjugated avidin (Sigma) in Hepes/NaCl buffer were added to the wells and incubated for 30 min at 4 °C. Finally, cells were washed three times in PBS, fixed in 200 µl of 5% formaldehyde in PBS, and their fluorescence determined by flow cytometry.

Western Blotting Analysis

Approximately 2 µg of affinity chromatography-purified beta1 integrins were electrophoresed on 7% SDS-polyacrylamide gels under nonreducing conditions and transferred to a 0.45-µm supported nitrocellulose membrane (Bio-Rad, Madrid, Spain) using a semi-dry transfer cell (Bio-Rad). After transfer, the membrane was saturated with PBS containing 5% (w/v) non-fat dry milk and 0.02% NaN(3) for 2 h at room temperature, and then incubated with the corresponding mAb in PBS, 2% BSA for 1 h at room temperature. After extensive washing in PBS, antibody binding to the membrane was detected with peroxidase-conjugated goat anti-mouse antibody (Sigma) followed by color development using 3-amino-9-ethylcarbazole (Sigma) as substrate.

Cell Attachment Assays

Cell adhesion assays were essentially performed as described previously (Arroyo et al., 1992; Luque et al., 1994). Briefly, 96-well flat-bottomed plates (Titertek, ICN Biomedicals) were coated with the beta1 integrin ligands laminin (a generous gift from Dr. M. A. Lizarbe, Facultad de Ciencias Químicas, Universidad Complutense, Madrid), collagen type I (ICN Biomedicals), or fibronectin (Sigma) at the indicated concentrations by overnight incubation at 4 °C. After saturation of free plastic sites of wells with 1% boiled BSA in PBS for 1 h at room temperature, plates were washed three times with PBS and once with 200 µl of Hepes/NaCl buffer. 50 µl of RPMI medium or Hepes/NaCl buffer containing 2.5 µg of purified stimulatory mAbs and/or the chlorides of divalent cations Mg, Ca, and Mn or chelators EGTA or EDTA were then added to the wells. 2.5 times 10^5 T lymphoblasts in 50 µl of RPMI medium or Hepes/NaCl buffer were added to each well and allowed to sediment onto the bottom of wells for 10 min at 4 °C. Plates were then transferred to a CO(2) incubator and incubated for 45 min at 37 °C. The percentage of cells that remained adhered after three gentle washes of wells with 200 µl of warm Hepes/NaCl buffer was calculated by measuring the absorbance of wells at 540 nm after fixation and staining with 0.5% crystal violet in 20% methanol.

Mapping of the beta1 Epitopes Recognized by mAbs HUTS-4, HUTS-7, and HUTS-21

Chinese hamster ovary transfectant cells (10^7) expressing human/mouse chimeric beta1 chains (Takada and Puzon, 1993) were lysed in 1 ml of 20 mM Tris, 150 mM NaCl, 1% Triton X-100, 0.05% Tween 20, pH 7.4. Wild type and chimeric beta1 integrins were immunopurified with anti-human beta1 mAb A1A5 immobilized to Sepharose. After washing the A1A5-Sepharose, the bound materials were recovered by boiling in SDS-PAGE buffer containing 1% SDS (w/v) for 5 min and separated by SDS-PAGE on 7% gels under nonreducing conditions. Proteins were transferred to Immobilon-P membrane (Millipore, Bedford, MA) and the membrane was blocked by incubating with 1% dry milk proteins for 1 h at room temperature. The membrane was used for Western blotting analysis with activation-dependent anti-beta1 mAbs HUTS-4, HUTS-7, and HUTS-21 or with the non-activation dependent mAb TS2/16 (positive control). Goat anti-mouse IgG conjugated with horseradish peroxidase (Bio-Rad) and ECL kit (Amersham, Buckinghamshire, UK) were used for detection of antibody binding.


RESULTS

Characterization of mAbs Specific for beta1 Epitopes Selectively Expressed by Activated States of VLA Integrins

The mAbs produced by the hybridomas HUTS-4, HUTS-7, and HUTS-21 were initially selected because their reactivity with T lymphoblasts was markedly higher when the incubation was done in normal RPMI medium to which 1 mM Mn had been added, compared with RPMI medium where the divalent cations had been removed by the addition of 3 mM EDTA (Fig. 1A). In contrast, the expression of the epitopes recognized by two control anti-beta1 mAbs, TS2/16 and Lia1/2, remained unchanged in these two different incubation conditions. These results indicate that mAbs HUTS-4, HUTS-7, and HUTS-21 might be recognizing epitopes which are specifically expressed by Mn-induced activated conformations of VLA integrins. T lymphoblasts were used in this mAb selection protocol because activation of the VLA integrins expressed on these cells can be rapidly induced either with the stimulatory anti-CD29 mAb TS2/16 and/or by the presence of extracellular Mn (Fig. 1B).


Figure 1: Expression of the epitopes recognized by mAbs HUTS-4, HUTS-7, and HUTS-21 on T lymphoblasts is induced by Mn and correlates with beta1 integrin-mediated cell adhesion. A, flow cytometry analysis of the differential expression of epitopes HUTS-4, HUTS-7, and HUTS-21 on T lymphoblasts depending on whether incubation is done in the absence of extracellular divalent cations by removal with 3 mM EDTA (left panel) or in normal RPMI medium to which 1 mM Mn has been added (right panel). B, T lymphoblast adhesion to beta1 integrin ligands type I collagen (20 µg/ml), laminin (10 µg/ml), and fibronectin (10 µg/ml) is stimulated by the addition of Mn (1 mM) to the extracellular medium and by the presence of anti-beta1 mAb TS2/16 (5 µg/ml) and completely blocked in the absence of extracellular divalent cations by removal with 3 mM EDTA. Percentages of adherent cells represent means of triplicates ± standard deviation and one representative experiment out of six independent ones is shown.



The nature of the molecules recognized by the three HUTS mAbs was investigated by immunoprecipitation and SDS-PAGE analysis from I surface-labeled lysates of human T lymphoblasts. Fig. 2shows that the precipitates obtained with these three mAbs from cells solubilized in a standard lysis buffer (1% Triton X-100, 1% BSA in PBS, pH 7.4, without Ca or Mg) consisted of one major band with an electrophoretic mobility identical to the beta1 integrin subunit, both under reducing (130 kDa) and nonreducing conditions (110 kDa). In order to confirm the beta1 specificity of the epitopes recognized by the HUTS mAbs and also the dependence of their expression upon activation of solubilized integrins, we compared the precipitates obtained from T lymphoblasts lysed under different divalent cation conditions. Fig. 3shows that when the lysis and the subsequent immunoprecipitation protocol were performed under conditions where VLA integrins are inactive, i.e. in the total absence of divalent cations obtained by removal with 3 mM EDTA, only one major band corresponding to the beta1 subunit could be detected in the precipitates obtained with the three HUTS mAbs. Interestingly, when lysis and immunoprecipitation were carried out in the presence of both Ca (1 mM) and Mg (1 mM) (which in terms of divalent cations represents a situation equivalent to the physiological extracellular conditions), again only the same single band corresponding to the beta1 subunit could be observed in the HUTS precipitates. In contrast, the presence of Mn (2 mM) in the lysis buffer and throughout the immunoprecipitation protocol induced important qualitative and quantitative changes in the immunoprecipitates of these mAbs. Thus, Mn induced the appearance in the precipitates of additional bands corresponding to alpha1, alpha2, and the 80 kDa fragment of alpha4, associated with the common beta1 subunit. Moreover, the presence of Mn also induced an increase in the intensity of the band corresponding to the beta1 subunit. Definitive demonstration of the beta1 specificity of the epitopes detected by the HUTS mAbs was obtained from lysates of T lymphoblasts containing activated VLA integrin heterodimers (in the presence of 2 mM Mn) that had been previously dissociated in their alpha and beta constituent subunits by a short high pH pretreatment (pH 11.0, 30 min at 37 °C) followed by rapid reneutralization to pH 8. After this high pH pretreatment of the cell lysates only the beta1 subunit was observed in the HUTS precipitates (not shown).


Figure 2: MAbs HUTS-4, HUTS-7, and HUTS-21 immunoprecipitate a single polypeptide with identical electrophoretic mobility to the beta1 integrin subunit. I-Labeled T lymphoblasts were lysed in PBS, 1% Triton X-100 buffer without Ca and Mg and immunoprecipitation carried out as described under ``Materials and Methods'' with the following mAbs: TS2/16 (beta1), lanes A and K; TS2/7 (VLA-1), lanes B and L; Tea 1/41 (VLA-2), lanes C and M; P1B5 (VLA-3), lanes D and N, HP2/1 (VLA-4), lanes E and O; Lia 1/2 (beta1), lanes F and P; GoH3 (VLA-6), lanes G and Q; HUTS-4, lanes H and R; HUTS-7, lanes I and S; and HUTS-21, lanes J and T.




Figure 3: The addition of Mn to lysates of T lymphoblasts induces the appearance of the beta1-associated integrin alpha subunits in the immunoprecipitates with mAbs HUTS-4, HUTS-7, and HUTS-21. Cell surface labeled T lymphoblasts were lysed in 20 mM Hepes, 1% Triton X-100 without divalent cations (-cation) and then Mn or Ca + Mg were added to aliquots of lysates to give a 1 mM final concentration of corresponding divalent cation. Immunoprecipitation was performed as described under ``Materials and Methods.'' alpha4-F corresponds to the 80-kDa proteolytic fragment of the alpha4 integrin subunit. The stimulatory anti-beta1 mAb TS2/16 was used as an invariant control in immunoprecipitations. Identical results were obtained when the blocking anti-beta1 mAb Lia 1/2 was used as an invariant control.



Activation of VLA Integrins Is Accompanied by Increased Expression of the HUTS Epitopes on the beta1 Subunit

The important changes induced by Mn in the level of expression of the HUTS epitopes on the beta1 subunit indicated that these mAbs recognize reporter epitopes of functional activation of VLA molecules. We therefore examined whether the induction of beta1 integrin activation by several well characterized stimuli other than Mn is also accompanied by a similar induction of the expression of these epitopes. We induced activation of beta1 integrins on T lymphoblasts with the CD29 mAb TS2/16, phorbol 12,13-dibutyrate, or the calcium ionophore A23187. Fig. 4shows that preincubation of T lymphoblasts with these stimuli, either alone or combined, in medium containing Ca and Mg results in increased levels of expression of the HUTS-21 epitope. Identical results were obtained with epitopes HUTS-4 and HUTS-7 (not shown).


Figure 4: Expression of the epitope recognized by mAb HUTS-21 is enhanced by cell preincubation with stimulatory anti-beta1 mAb TS2/16, phorbol 12,13-dibutyrate, and calcium ionophore A23187. T lymphoblasts were preincubated with 5 µg/ml TS2/16, 100 nM phorbol 12,13-dibutyrate (PDBU), or 2 µM calcium ionophore A23187 in Hepes/NaCl buffer containing 1 mM Mg and 1 mM Ca for 30 min at 37 °C. Biotin-conjugated mAb HUTS-21 was then added and binding subsequently detected with avidin-fluorescein isothiocyanate. Flow cytometric analysis was performed as described under ``Materials and Methods.'' Numbers represent percentage of positive cells.



To further characterize the parallelism observed between the process of functional activation of VLA integrins and augmented levels of expression of these beta1 epitopes, the effects of temperature and divalent cation conditions on the T lymphoblast surface expression of HUTS epitopes were analyzed. Fig. 5A shows that Mg alone (in the presence of the Ca chelator EGTA) was able to induce increased expression of the epitope recognized by mAb HUTS-21 on T lymphoblasts (compared to background levels observed in the absence of divalent cations) only when the incubation was carried out under physiological temperature conditions (37 °C). Ca, however, did not induce detectable expression of this epitope either under 37 °C nor under 4 °C temperature conditions. Interestingly, when both extracellular Mg and Ca were present, the level of expression of the HUTS-21 epitope was only marginally above background. Mn induced an important increase in epitope expression at 37 °C but, in contrast, this induction was minimal at 4 °C. Similar results were obtained for epitopes HUTS-4 and HUTS-7 under all conditions (data not shown). We also studied in parallel the regulation exerted by these different divalent cation conditions on functional activation of beta1 integrins expressed on T lymphoblasts, under physiological temperature conditions, by measuring the percentage of these cells that effectively adhere to collagen, laminin, and fibronectin (Fig. 5B). A close correlation between functional activity of beta1 integrins and expression of HUTS epitopes was observed under all the divalent cation conditions tested.


Figure 5: Regulation of HUTS-21 epitope expression and beta1 integrin-mediated T cell adhesion by extracellular divalent cations. A, expression of the beta1 epitope HUTS-21 is induced at 37 °C by Mg or Mn and is inhibited by the presence of Ca or low temperature. The anti-beta1 mAb TS2/16 is included as an invariant control. B, T lymphoblast adhesion to beta1 integrin-specific ligands type I collagen, laminin, and fibronectin at 37 °C under different divalent cation conditions. Divalent cations and Ca chelator EGTA were used at the following concentrations: 1 mM Ca, 1 mM Mg, 0.5 mM Mn, 2 mM EGTA.



To rule out the possibility that the observed increase in the expression of the HUTS epitopes during the process of beta1 integrin activation on T lymphoblasts was a phenomenon restricted to this cell system or a consequence of their in vitro culturing and stimulation with phytohemagglutinin/interleukin-2, we analyzed the expression of HUTS epitopes on freshly isolated peripheral blood lymphocytes. We also extended our studies to other beta1-positive cell types including the B lymphoblastoid cell lines RAMOS (which only expresses VLA-4 among beta1 integrins), the erythroleukemic cell line K562 (which only expresses the VLA-5 member), and the colocarcinoma cell line COLO-320 (which expresses VLA-1, VLA-2, VLA-3, VLA-5, and VLA-6). Table 1shows that, similarly to the results already described with T lymphoblasts, Mn induced an important increase in the expression of the HUTS-21 epitope (above the level observed in medium containing Ca + Mg) on peripheral blood lymphocytes and on the RAMOS, K562, and COLO-320 cell lines only when physiological temperature conditions were used. In contrast, Mn did not induce the expression of this epitope in the JY B lymphoblastoid cells, which express hardly detectable amounts of the beta1 integrin subunit on their surface. Similar results were obtained with epitopes HUTS-4 and HUTS-7 (data not shown).



The Three HUTS mAbs Induce Functional Activation of beta1 Integrins

Next, we addressed the question of whether the HUTS mAbs were able to exert functional effects on the VLA integrin-mediated cell adhesion. Fig. 6shows that the presence of mAb HUTS-21 (similar results were obtained with mAbs HUTS-4 and HUTS-7, data not shown) in RPMI medium induces significant stimulation of the adhesion of T lymphoblasts to VLA ligands fibronectin, type I collagen, and laminin. This stimulatory effect of mAb HUTS-21 was similar to that observed with the stimulatory CD29 mAb TS2/16 under all the conditions tested. These results demonstrate that under ``permissive'' conditions for integrin-mediated adhesion the HUTS mAbs are able to induce the functional activation of beta1 integrins.


Figure 6: mAb HUTS-21 (circle) stimulates the adhesion of T lymphoblasts to beta1-specific ligands fibronectin, type I collagen, and laminin. Adhesion assays were performed in RPMI medium as described under ``Materials and Methods.'' mAbs were used at a final concentration of 5 µg/ml. Adhesion stimulatory anti-beta1 mAb TS2/16 (), and the irrelevant anti-CD11c mAb HC1/1 (box) were used as controls. The total dependence of this cell adhesion system on VLA integrin function was demonstrated by the complete blockade with the inhibitory anti-beta1 mAb Lia 1/2 (not shown). Similar results were obtained with mAbs HUTS-4 and HUTS-7.



mAbs HUTS-4, HUTS-7, and HUTS-21 Recognize Overlapping Epitopes Located in the 355-425 Region of the beta1 Subunit

To answer the question of whether the epitopes recognized by each of the three HUTS mAbs are physically distant or localized on overlapping regions of the beta1 integrin subunit, cross-competitive cell binding experiments were carried out using biotinylated antibodies. We found that the three HUTS mAbs recognize the same or overlapping epitopes on the beta1 chain as cell preincubation with one of them (HUTS-21) cross-blocked the binding of the other two (Fig. 7A). Reciprocal blockade of binding was observed when cells were preincubated with mAb HUTS-7 or mAb HUTS-4. Using a panel of purified human/mouse chimeric beta1 molecules and Western blotting techniques, we have been able to unequivocally map these activation-reporter epitopes to the region located between residues 355-425 of the beta1 polypeptide. As shown in Fig. 7B, mAb HUTS-21 detects a chimeric beta1 polypeptide containing human residues 1-425, but does not recognize chimeric beta1 molecules that only contain the human residues 1-354. Similar results were obtained with mAbs HUTS-4 and HUTS-21 (not shown).


Figure 7: A, mAbs HUTS-4, HUT-7, and HUTS-21 cross-compete for binding to the beta1 polypeptide. In each sample, T lymphoblasts were first incubated with the mAb shown at the left side of the arrow and then with the biotinylated mAb shown at the right side of the arrow, as described under ``Materials and Methods.'' Anti-VLA-1 (CD49a) mAb 5E8D9 was used as a control. B, the epitope recognized by mAb HUTS-21 maps within the region 355-425 of the human beta1 polypeptide. Wild type and human/mouse chimeric beta1 molecules h587/m, h425/m, and h354/m were immunopurified as described under ``Materials and Methods.'' Purified materials were separated by SDS-PAGE under nonreducing conditions, transferred onto Immobilon-P membrane, and blotted with mAbs HUTS-21 or TS2/16. Identical results were obtained with mAbs HUTS-4 and HUTS-7.




DISCUSSION

Unlike other cell surface receptor molecules which are constitutively activated, integrins can only bind ligand and mediate cellular adhesion after their activation is induced by specific stimuli. Reversible functional transitions between inactive and activated states of integrins represent therefore a key mechanism by which cells control their adhesive properties.

In this report, we describe a group of three mAbs, named HUTS-4, HUTS-7, and HUTS-21, that recognize specific epitopes on a novel regulatory region of the common beta1 subunit of VLA integrins whose expression parallels functional activation and ligand binding by these adhesion receptors. These mAbs were obtained after immunization of mice with a purified preparation of activated human beta1 integrins. The protocol for purification of VLA integrins was designed to yield these molecules in active conformation with high ligand binding affinity. The initial selection of the HUTS hybridomas was based on the marked increase in the binding of these mAbs induced by the presence of Mn, suggestive of their specificity for epitopes on VLA integrins whose expression accompanies functional activation of these molecules.

Interestingly, immunoprecipitation analysis revealed that, when divalent cations were not present in the lysis buffer, the HUTS mAbs only precipitated the beta1 subunit of VLA integrins. However, the presence of Mn (but not of Ca and Mg) in the lysis buffer resulted in the appearance in the precipitates of the associated alpha subunits. A possible explanation for these results is that upon T cell solubilization a certain proportion of VLA integrin heterodimers are dissociated in their beta1 and alpha constituent subunits. In the absence of divalent cations or presence of both Ca and Mg in the lysis buffer, the epitopes recognized by these mAbs are only expressed on the population of dissociated beta1 polypeptides. The implication is that, under these two divalent cation conditions, i.e. complete absence of divalent cation and presence of both Ca and Mg, these beta1 epitopes are masked on the inactive integrin alpha:beta heterodimers. On the contrary, in the presence of Mn these epitopes are expressed not only by the fraction of dissociated beta1 chains but also by the beta1 polypeptides (in a specific Mn-induced conformation) which are forming part of activated intact integrin alpha:beta1 heterodimers. These activated conformations of beta1 integrins can result from direct binding of Mn to the cation binding site present in the N-terminal region of the beta1 chain. Alternatively, the allosteric changes in beta1 polypeptide detected by the HUTS mAbs can be transmitted by the alpha subunits upon Mn binding to one or more of their cation binding sites.

Our data show that expression of the HUTS epitopes correlates with functional activation of VLA integrins. We have demonstrated that this parallelism not only applies when activation of beta1 integrins is induced from outside the cells with the stimulatory CD29 mAb TS2/16 or Mn, but also when integrin activation is induced by intracellular signals (inside-out signaling) generated upon cell treatment with phorbol esters or calcium ionophores.

Several recent reports have shown that Ca generally exerts inhibitory effects on the functional activity of integrins belonging to the beta1, beta2, and beta3 subfamilies (Dransfield et al., 1992a; Grzesiak et al., 1992; Hemler et al., 1990; Masumoto and Hemler, 1993; Staatz et al., 1989; Weitzman et al., 1993). In contrast, in the absence of extracellular Ca (i.e. in the presence of the Ca chelator EGTA), Mg supports effective integrin-mediated cell adhesion in a dose-dependent manner. We have characterized in detail the divalent cation regulation of beta1 integrin functional activity on T lymphoblasts by measuring their adhesion to three different ligands specific for VLA integrins: type I collagen, laminin, and fibronectin. As previously reported in other cellular systems (Masumoto and Hemler, 1993; Luque et al., 1994; Weitzman et al., 1993), we find that Mg and Mn induce activation of beta1 integrins and support cell adhesion. In contrast, Ca exerts inhibitory effects on beta1 integrin function as no adhesion to ligands collagen and laminin (and only weak adhesion to fibronectin) occurred when Ca was the only divalent cation present in the extracellular medium. Furthermore, the presence of extracellular Ca partially inhibited the level of cell adhesion supported by Mg. The level of expression of the epitopes recognized by the HUTS mAbs correlated with functional activity of beta1 integrins under all the divalent cation conditions.

Physiological temperature has been characterized as a requirement for integrin activation and subsequent adhesion in a number of cell systems, reflecting the need for an intact cell metabolism (Campanero et al., 1990; Dransfield et al., 1990; Marlin and Springer, 1987; Rothlein and Springer, 1986; van de Wiel-van Kemenade et al., 1992). The use of the HUTS activation-reporter mAbs has also allowed us to confirm that an intact cell metabolism is an absolute requirement for beta1 integrin activation, as only minimal levels of expression of these epitopes on T lymphoblasts could be detected at 4 °C, regardless of the stimulus used.

Our findings on the temperature and divalent cation regulation of beta1 integrin function and correlated expression of the HUTS epitopes have been confirmed for different VLA heterodimers using other cellular systems, including the B lymphoblastoid cell line RAMOS (VLA-4), the erythroleukemic cell line K562 (VLA-5), and the colocarcinoma cell line COLO 320 (which expresses several members of the VLA integrin subfamily). The fact that a similar regulation by temperature and divalent cations was also observed in freshly isolated lymphocytes rules out possible in vitro culturing effects and indicates that the HUTS epitopes indeed represent physiologic epitopes.

Competitive cell binding studies indicate that mAbs HUTS-4, HUTS-7, and HUTS-21 recognize common or overlapping epitopes on the beta1 polypeptide. A small region of the human beta1 subunit (residues 207-218) with important regulatory properties on the function of VLA integrins has been recently identified using a panel of beta1-specific mAbs (Takada and Puzon, 1993). All these mAbs recognize overlapping epitopes in the 207-218 region but they have completely different effects on VLA integrin function: while some of these antibodies (including TS2/16) activate beta1 integrins and induce cell adhesion, others exert a blocking effect on beta1 integrin-mediated adhesion. The epitopes recognized by the three HUTS mAbs have been mapped to a distant segment (residues 355-425) which is in the vicinity to the cysteine-rich domains in the primary sequence of the beta1 polypeptide (Argraves et al., 1987). The HUTS mAbs, therefore, define this sequence as a novel regulatory region in the beta1 subunit that is exposed upon activation of integrin heterodimers. Interestingly, a mAb (D3GP3) which recognizes a conformational epitope of the beta3 integrin subunit and induces fibrinogen binding to a limited population of integrin alphaIIb/beta3 molecules has also been mapped to a similar region close to or within the cysteine-rich core of the beta3 subunit (422-692) (Kouns et al., 1990). Therefore, common structural and functional features which can be evidenced by the expression of activation epitopes seem to be conserved among the beta subunits of the integrin family.

Binding of the HUTS mAbs to specific epitopes on the beta1 integrin chain results in enhanced cell adhesion to VLA integrin ligands fibronectin, laminin, and type I collagen. This stimulation by HUTS mAbs of beta1 integrin-mediated adhesion is similar to that induced with mAb TS2/16. Although the overall effect of these mAbs on cell adhesion is similar, the mechanism by which the HUTS and TS2/16 mAbs induce enhanced adhesion may be somehow different. In this regard, the epitope recognized by mAb TS2/16 in the 207-218 region of the beta1 subunit is constitutively expressed, regardless of the state of integrin activation. This is evidenced by the high level of expression of this epitope even in the presence of EDTA. It has been recently shown that upon binding of mAb TS2/16 to the beta1 polypeptide an important increase in the ligand binding affinity of VLA integrins occurs, presumably mediated by a change in conformation which favors their interaction with ligands (Arroyo et al., 1992, 1993; van de Wiel-van de Kemenade et al., 1992). In contrast, the expression of the epitope(s) recognized by the HUTS mAbs is(are) not constitutive but depends on the state of integrin activation. Therefore, it is tempting to speculate that upon binding of HUTS mAbs to the beta1 subunit of partially preactivated VLA heterodimers already showing weak interaction with ligand, the conformation of these integrin molecules is ``locked'' in a state where ligand is bound with high affinity and their reversion to an inactive state is thus prevented. In fact, some preliminary data indicate that under some conditions, the HUTS epitopes behave as ligand-induced binding sites since their expression on beta1 integrins also depends on the interaction with ligands. A similar mechanism has been proposed to explain the functional effects exerted by other activation-reporter mAbs specific for the beta2 or beta3 integrins. For instance, binding of activation-reporter mAb 24 to the alpha subunits of leukocyte (beta2) integrins inhibits T cell proliferative responses, lymphokine-activated killer activity of T lymphoblasts, and f-Met-Leu-Phe-induced neutrophil chemotaxis. All these functional effects of mAb 24 could be explained by assuming that it prevents ``de-adhesion'' of integrin/ligand pairs, possibly by freezing integrins in an active conformation with ligand firmly bound (Dransfield et al., 1992b). Furthermore, the epitope recognized by mAb 24 has been shown to be of the ligand-induced binding site type since its expression also depends on the interaction of integrin LFA-1 with ligand intercellular adhesion molecule-1 (Cabañas and Hogg, 1993).

Two recent reports describe the use of a mAb (15/7), which detects an activation dependent conformational epitope on the beta1 molecules, in studies of the T cell responses in secondary lymphoid tissue (Picker et al., 1993) and of the expression of activated forms of beta1 integrins in chronic inflammatory diseases (Arroyo et al., 1995). However, full characterization of the specificity and properties of this mAb has not been published. Two additional mAbs specific for activation dependent epitopes of the beta1 molecule have been recently characterized. One of these mAbs, named 9EG7, detects an epitope on the mouse beta1 subunit which is specifically expressed in the presence of Mn (Lenter et al., 1993). In contrast to the adhesion-enhancing effect of the HUTS mAbs, mAb 9EG7 inhibits integrin-ligand interactions. A second mAb, named SG/7, detects an epitope on the human beta1 molecule whose expression depends on the presence of either Mn or Ca and cannot be induced on the cell surface after phorbol ester treatment (Miyake et al., 1994). The control of the expression of the epitope detected by mAb SG/7 by Ca is therefore clearly different from that of HUTS epitopes. Since none of the mentioned studies have characterized the regions of beta1 integrin subunit containing these activation epitopes it would be interesting to carry out comparative studies between these and the HUTS mAbs.


FOOTNOTES

*
This work was supported by grants from the Comunidad Autónoma de Madrid CAM 230/92 and Fondo de Investigaciones Sanitarias FIS 93/0157 (to C. C.), FIS 95/0212 and PB 92/0318 (to F. S. M.), and GM 47157 and GM 49899 (to Y. T.). 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: Departamento Bioquímica y Biología Molecular, Facultad de Medicina, Universidad Complutense, 28040 Madrid, Spain. Tel.: 34-1-3941382; Fax: 34-1-3941691.

(^1)
The abbreviations used are: VLA, very late activation antigen(s); mAb, monoclonal antibody; PMSF, phenylmethylsulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin; PBS, phosphate-buffered saline.


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