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American Journal of Respiratory Cell and Molecular Biology. Vol. 28, pp. 607-615, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0105OC

Integrin Receptors Are Crucial for the Restimulation of Activated T Lymphocytes

Timur O. Yarovinsky, Martha M. Monick and Gary W. Hunninghake

Department of Internal Medicine, Division of Pulmonary, Critical Care, and Occupational Medicine, University of Iowa and Veteran Affairs Medical Center, Iowa City, Iowa

Address correspondence to: Dr. Timur O. Yarovinsky, University of Iowa, 100 EMRB, Iowa City, IA 52242. E-mail: timur-yarovinsky{at}uiowa.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Stimulation via the T-cell receptor results in proliferation of naive T cells and activation-induced death of activated T cells. The expression of Fas ligand and activation-induced cell death are major mechanisms by which immune responses are modulated in the lung. Although it is known that the binding of integrin receptors to extracellular matrix proteins provides co-stimulatory signals to naive T cells, it is not clear whether these signals are critical for activated T cells. The activation and differentiation of T cells is marked by significant changes in integrin expression and affinity. To determine the role of integrin signaling in restimulation of activated T cells, we blocked integrin receptors with RGD peptides. Using murine activated CD4+ T cells and the T-cell hybridoma DO11.10, we found that RGD peptides inhibit tyrosine phosphorylation of CD3 {epsilon}-chain and ZAP-70, clustering of T-cell receptors, extracellular signal-regulated kinase mitogen-activated protein-kinase activation, and Fas ligand expression and prevent activation-induced cell death. We demonstrate that activated T cells are sensitive to integrin co-stimulation and that integrin receptors are required for the successful restimulation of activated T cells. This indicates that matrix proteins may play a major role in regulating T-cell–mediated immune responses in the lung.

Abbreviations: activation-induced cell death, AICD • calcein acetoxymethyl ester, calcein AM • antigen-presenting cell, APC • bovine serum albumin, BSA • extracellular signal-regulated kinase, ERK • ethidium homodimer, EthD-1 • focal adhesion kinase, FAK • Fas ligand, FasL • fetal calf serum, FCS • fluorescein isothiocyanate, FITC • interleukin 2, IL-2 • mitogen-activated protein kinase, MAP-kinase • phosphate-buffered saline, PBS • relative fluorescence intensity, RFI • Arg-Gly-Asp, RGD • Arg-Gly-Asp-Ser, RGDS • Arg-Gly-Glu-Ser, RGES • staphylococcal enterotoxin B, SEB • T-cell receptor, TCR • Tris-buffered saline with 0.1% Tween 20, TTBS


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The activation of T lymphocytes is a critical event in the pathogenesis of a number of lung diseases (1). The engagement of T-cell receptors (TCR) of naive T lymphocytes results in the proliferation and production of proinflammatory cytokines, whereas the restimulation of activated T lymphocytes leads to activation-induced cell death (AICD) (2). The deletion of activated T lymphocytes is a major mechanism that regulates immune responses, including those that occur in the lung (3). Failure to delete activated T cells results in excessive and profound immune responses and, in some instances, autoimmune diseases (2). Excessive production of Fas ligand (FasL) is also known to induce epithelial cell apoptosis in the lungs of patients with the acute respiratory distress syndrome (4).

The activation and differentiation of T lymphocytes are accompanied by marked changes in the expression and affinity of various integrin receptors (5). The co-stimulatory role of integrins in the response of naive T cells to TCR engagement has been well documented (6–9). Integrin receptors have been shown to decrease the threshold for activation of naive T cells by stabilizing their adhesion to antigen presenting cells (APC) (10). However, studies attempting to define the role of integrin receptors in AICD triggered by restimulation of activated T cells have produced conflicting data. Some studies show augmentation of AICD by integrins (11–13), whereas others suggest that integrins may inhibit AICD (14, 15). Numerous reports, showing that immobilized anti-CD3 antibody alone triggers AICD of T-cell hybridomas and activated T cells, suggest that co-stimulation is not necessary for FasL expression and AICD if T cells are restimulated with a strong signal (16–19). Although a number of studies have shown that integrin receptors may have some regulatory effect on FasL and interleukin 2 (IL-2) expression in activated T cells (9, 12, 14, 15, 20, 21), none of the studies addressed whether integrin receptors are necessary for proximal TCR signaling during the restimulation of activated T cells.

In the present study, we blocked integrin signaling with RGD peptides to demonstrate that co-stimulatory signals mediated by integrin receptors are crucial for the restimulation of activated T cells. We demonstrate that RGD peptides inhibit cell death of activated CD4+ T cells restimulated with staphylococcal enterotoxin B (SEB) in the presence of APC. In addition, the blockade of integrin signaling with RGD peptides results in the inhibition of AICD triggered by immobilized anti-CD3 antibody in the absence of APC. The inhibition occurs at the level of proximal TCR signaling because RGD peptides decreased the clustering of TCR and tyrosine phosphorylation of the CD3 {epsilon}-chain and ZAP-70 tyrosine kinase. Our observations provide strong evidence that the restimulation of activated T cells is dependent on integrin co-stimulation. These observations suggest that matrix proteins may modulate immune responses in the lung.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Arg-Gly-Asp-Ser (RGDS) and Arg-Gly-Glu-Ser RGES peptides were obtained from BACHEM Bioscience (King of Prussia, PA), Peninsula Laboratories (San Carlos, CA), and Sigma (St. Louis, MO). SEB was purchased from Toxin Technology (Sarasota, FL). Polystyrene 6-µm microbeads were acquired from Polysciences (Warrington, PA). Tissue culture plates and flasks were from Corning Costar Inc. (Corning, NY). All other reagents were purchased from Sigma unless otherwise noted.

Antibodies
Low-endotoxin and azide-free antibodies, purchased from BD Pharmingen (San Diego, CA), were used for cell stimulation (anti-CD3 {epsilon}-chain [145-2C11] and anti-CD28 [37.51]) and function blocking assays (anti-CD18 [GAME-46], anti-CD29 [Ha2/5 and HMß1-1], and anti-CD61 [2C9.G2]). Fluorescently labeled antibodies against CD4 (L3T4), FasL (MFL3), and TCR ß-chain (H57-597); isotype control antibodies; and fluorescein isothiocyanate (FITC)-labeled annexin V were purchased from BD Pharmingen. The following antibodies were used for immunoblotting and immunoprecipitation: rabbit polyclonal antibodies against phosphorylated extracellular signal-related kinase (ERK)-1 and ERK-2 MAP-kinases (Cell Signaling Technology, Beverly, MA) and against ERK-2 (C-14), focal adhesion kinase (FAK, C-20), ZAP-70 (LR), and normal rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA). Mouse monoclonal anti-FAK (clone 77) and anti-ZAP-70 (clone 29) were purchased from BD Transduction Laboratories (Lexington, KY). Phosphotyrosine-specific mouse monoclonal antibody 4G10 was purchased from Upstate Biotechnology (Lake Placid, NY). Secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA).

Cell Culture
All media and reagents for cell culture were obtained from Life Technologies (Rockville, MD) unless otherwise noted. The cells were maintained in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS), non-essential amino acids, 2 mM L-glutamine, 5 x 10-5 M ß-mercaptoethanol (Sigma), and 50 µg/ml gentamycin (Mediatech, Herndon, VA) (referred to herein as complete medium). Activated CD4+ T cells were cultured in the presence of 20 U/ml IL-2 to avoid cytokine deprivation apoptosis. Stimulation of the cells for immunoprecipitation/Western blotting analysis and conjugation with the microbeads was performed in Leibovitz's L-15 medium supplemented with 10% FCS.

Splenocytes were isolated from spleens of exsanguinated 8- to 12-wk-old female C57BL/6 or BALB/c mice (Harlan, Indianapolis, IN). The cells were stimulated with 10 µg/ml SEB or immobilized anti-CD3 plus anti-CD28 antibodies for 48 h and expanded for additional 24–96 h in the presence of IL-2. CD4+ T cells and T-cell–depleted splenocytes were isolated using an immunomagnetic negative selection kit (StemCell Technologies, Vancouver, BC, Canada) or CD4 and CD8 Dynabeads (Dynal, Lake Success, NY). T-cell hybridoma DO11.10 was provided by Dr. Barbara Osborne (University of Massachusetts) and Dr. Philippa Marrack (National Jewish Medical and Research Center, University of Colorado) and was used for studying biochemical events after TCR activation. This cell line is one of the best characterized cell lines for studying AICD and TCR signaling (18, 22).

Assessment of AICD
Purified CD4+ T cells (106/ml) that received primary stimulation with SEB and expanded in the presence of IL-2 were restimulated with SEB (10 µg/ml) in 24-well tissue culture plates for 18–20 h in the presence of antigen-presenting cells (T-cell depleted splenocytes, 2.5 x 105/ml). Immobilized anti-CD3 antibody was used for the restimulation of CD4+ T cells that received primary stimulation with immobilized anti-CD3 plus anti-CD28 antibodies. The death of CD4+ cells was measured by staining with FITC-labeled annexin V and R-phycoerythrin (PE)-labeled anti-CD4. A total of 20,000 cells that satisfied a gate on forward and side scatter to eliminate aggregates and debris were evaluated using a FACScan flow cytometer (Becton Dickinson, Mountain View, CA) at the University of Iowa Flow Cytometry Facility. Data analysis was performed using WinMDI 2.8 software (Scripps Institute, La Jolla, CA). Cell death was expressed as the percentage of annexin V stained cells among CD4+ cells.

Death of DO11.10 cells or CD4+ cells induced by restimulation with immobilized anti-CD3 antibody was measured in 96-well plates. Tissue culture plates were coated with 0.125–2.5 µg/ml of anti-CD3 antibody in phosphate-buffered saline (PBS) for 1 h at 37°C or overnight at 4°C. T cells were stimulated at 105 cells/well for 20–24 h, and the dead cells were stained for 15 min with 8 µM ethidium homodimer (EthD-1) (Molecular Probes, Eugene, OR). Fluorescence intensity in the wells was measured using a 540/10-nm excitation filter and a 620/10-nm emission filter on a Victor2 (EG&G Wallac, Gaithersburg, MD) microplate reader. In some experiments, live cells were counterstained with 1 µM calcein acetoxymethyl ester (calcein AM) (Molecular Probes) for 30 min, and the percentage of dead cells was estimated by manual counting using a fluorescent microscope. These experiments established a linear correlation between the number of dead cells and the fluorescence intensity of EthD-1.

Flow Cytometry Analysis for Surface Expression of FasL
DO11.10 cells (106/well of 24-well tissue culture plates) were stimulated with immobilized anti-CD3 or isotype control antibodies, washed, and stained with PE-labeled anti-FasL or isotype control antibodies. Samples stained with isotype-matched control antibodies were used as negative controls to determine the percentage of FasL-positive cells.

Cell Adhesion Assay
T cells (105 cells/well of 96-well tissue culture plates) were stimulated with immobilized anti-CD3 or isotype control antibody in complete medium for 30 min at 37°C in a humidified atmosphere with 5% CO2. In some experiments, the plates were blocked with 1% bovine serum albumin (BSA), human serum albumin, or ovalbumin in PBS. Where indicated, T cells were pretreated with 10 µg/ml of antibodies against integrin receptors for 30 min on ice. After the incubation period, the wells were washed three times with pre-warmed PBS containing calcium and magnesium using wide-bore pipet tips, and the plates were tapped on blotting paper between each wash. T cells resuspended in PBS at known concentration were added to wells (104–105 cells/well) to make a standard curve in each plate. The cells were stained with calcein AM (2 µM) at 37°C for 30 min. The fluorescence was measured using a 485/8-nm excitation filter and a 535/10-nm emission filter on the fluorescence microplate reader. The percentage of adherent cells was determined by comparing the fluorescence intensity in sample wells to the standard curve.

Immunoprecipitation and Western Blotting
The cells (8 x 106/ml) were treated with 2 mM RGDS peptides or PBS as a control for 10 min at 37°C. Stimulation with immobilized antibodies was performed by incubating 0.5 ml of the cell suspension in triplicate wells of 6-well tissue culture plates pre-coated with 2.5 µg/ml anti-CD3 or isotype control antibody, each containing 0.5 ml of pre-warmed medium. Activation was stopped by placing the plates on ice and adding 0.5 ml ice-cold PBS containing 1 mM sodium orthovanadate. Adherent and nonadherent cells were lysed with ice-cold lysis buffer (0.05 M Tris, pH 7.4; 0.15 M NaCl; 1% NP-40) supplemented with 1x complete protease inhibitors (Roche Molecular Biochemicals, Indianapolis, IN), 1x phosphatase inhibitors (Calbiochem, La Jolla, CA), and 1 mM sodium pervanadate. In some experiments, the cells were stimulated with 10 µg/ml soluble anti-CD3 or isotype control antibody. Immunoprecipitation was performed by incubation of precleared lysates, equivalent to 107 cells, with 4 µg of control or anti-CD3, anti-FAK, or anti-ZAP-70 antibodies. Overnight incubation at 4°C with rotation was followed by the addition of 20 µl/sample Protein A-agarose (Santa Cruz Biotechnology) for 1 h. The immunoprecipitates were washed four times with lysis buffer containing 1 mM sodium orthovanadate and released from agarose by boiling in 2x sample buffer. Western blot analysis was performed by separating 30 µg of cell lysate/lane (for phospho-ERK) or immunoprecipitated proteins in 10% SDS-PAGE gel and semi-dry transfer onto nitrocellulose membrane (ECL; Amersham, Arlington Heights, IL). The membranes were blocked with 3% BSA for phosphotyrosine immunoblotting or 5% milk in Tris-buffered saline with 0.1% Tween 20 (TTBS) for 1 h and incubated with the primary antibody for 1 h at room temperature or overnight at 4°C. The blots were washed four times with TTBS and incubated for 1 h with horseradish-peroxidase conjugated secondary antibodies. Immunoreactive bands were developed using a chemiluminescent substrate (ECL Plus; Amersham). Autoradiographs were obtained by exposing Kodak Biomax MR films (Eastman Kodak, Rochester, NY) to the membranes for 10 sec to 3 min. The loading of the proteins of interest was evaluated by stripping the blots and re-probing with rabbit anti-ERK-2 or mouse anti-FAK or anti-ZAP-70 antibodies. The intensity of immunoblotting signal was measured using the Fluor-S MultiImager system and Quantity One software (Bio-Rad, Hercules, CA). The signal from phosphorylated proteins was normalized to the signal from loaded total proteins derived from re-probed blots and expressed relative to the normalized signal from nonstimulated samples.

Analysis of TCR Clustering
DO11.10 cells were treated with RGDS peptides or PBS as a control for 10 min at 37°C and mixed with the equal numbers of 6-µm beads that were pre-coated with anti-CD3 or isotype control antibodies and blocked with 1% BSA. To minimize the effects of RGDS peptides on adhesion and to facilitate contacts between the cells and the beads, they were centrifuged at 200 x g for 3 min. The pellets were incubated at 37°C for 10 min, and the reactions were stopped by adding sodium azide to the final concentration (0.1%) and washing with ice-cold staining buffer. FITC-labeled anti-TCR ß-chain antibody was used at 1:25 dilution in ice-cold staining buffer. After the staining, cells were washed several times with ice-cold PBS containing calcium and magnesium plus 0.1% sodium azide, fixed with 10% paraformaldehyde and mounted onto poly-L-lysine slides. Slides were coverslipped using Vectashield (Vector Laboratories, Burlingame, CA) and analyzed by using a Bio-Rad MRC-1024 confocal microscope at the University of Iowa Central Microscopy Research Facility. Ten random fields (512 x 512 pixels each) were collected for each experimental group, and all cell–bead conjugates within those fields were examined. Quantitative image analysis was applied to determine the degree of TCR clustering at the cell–bead conjugates using ImagePro Plus software (Media Cybernetics, Silver Spring, MD) and the following protocol. Two circles,each 20 pixels in diameter, were placed at the center of the cell–bead interface and free membrane of the same cell. The mean fluorescence intensity within both circles was recorded. For each interface, the clustering index was determined as the ratio of intensity at the interface to the intensity at the free membrane. If the clustering index exceeded a value of 2, the conjugates were scored as TCR clustered (positive); otherwise, the conjugates were scored as unclustered (negative).

Statistical Analysis
The effects on AICD were analyzed using the ANOVA test for repeated measures. Post hoc Dunett and Bonferroni tests were applied if a significant difference was found (P < 0.05). The difference in the degree of TCR clustering between the cells treated with RGDS peptides and control cells was analyzed by comparing the mean clustering index for the experimental groups using two-tailed Student's t test. The difference in the number of cell-bead conjugates with clustered TCR was analyzed using the Chi-square goodness-of-fit test. All calculations were performed with GraphPad Prism software version 3.00 (GraphPad Software, San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
RGDS Peptides Block AICD
As a model of activated T cells, we used splenocytes from BALB/c mice that received primary stimulation with SEB for 48 h and were purified to more than 95% homogeneity, using the expression of the CD4 marker. They were expanded in the presence of IL-2 for an additional 48–96 h (23). Primary stimulation with SEB resulted in the selective expansion of SEB-responsive CD4+ T cells as demonstrated by an increase of Vß8+ T cells among CD4+ cells (from 29% in unstimulated population to 83% in activated population, data not shown). To determine whether activated T cells were dependent on co-stimulatory signals provided by integrins, we blocked integrin receptors with RGDS peptides during restimulation of these cells with SEB in the presence of APC (Figure 1). To determine the percentage of CD4+ T cells undergoing AICD, flow cytometry was used after staining with annexin V as a marker for apoptotic cells. The presence of RGDS peptides at a concentration of 1 mM inhibited AICD by more than 2-fold, whereas control RGES peptides had no effect. Thus, integrin signaling is required to induce cell death after the restimulation of activated T cells.



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  		Figure 1. Blockade of integrin receptors with RGDS peptides prevents AICD of CD4+ T cells after restimulation with SEB. Activated CD4+ T cells were isolated by negative selection from the 48-h culture of BALB/c splenocytes stimulated with 10 µg/ml SEB, expanded in the presence of IL-2 and restimulated with 10 µg/ml SEB in the presence of added APC (freshly purified splenocytes depleted of T cells) and 1 mM of RGDS or RGES peptides for 18 h. The cells were stained for CD4 expression with PE-labeled antibody and for phosphatidylserine expression with FITC-labeled annexin V. The percentage of apoptotic (annexin V+) cells among CD4+ cells was determined by gating for CD4+ cells with FACScan flow cytometer. Fluorescence of gated CD4+ cells at the FL1 channel (annexin V-FITC) is shown; the representative data from one of three independent experiments are shown.

 
Because restimulation with SEB requires the presence of APC, it is not clear whether the inhibitory effects of RGD peptides are directed toward integrin receptors expressed by T cells or APC. To determine the direct effects of RGD peptides on T cells, we restimulated activated CD4+ T cells with immobilized anti-CD3 antibody. This allowed us to avoid the confounding effects of the peptides on APC in the subsequent experiments. As an additional model for activated T cells, we used CD4+ T cells that received primary stimulation via immobilized anti-CD3 and CD28 antibodies and the T cell hybridoma DO11.10 (18, 22). Pilot experiments established that stimulation of T cells was under saturating conditions when anti-CD3 antibody was immobilized at 2.5 µg/ml or less (data not shown). More than 90% of activated CD4+ T cells and DO11.10 cells undergo AICD when stimulated in this manner (Figure 2). The presence of 1 mM RGDS peptides, but not the control RGES peptides, prevented AICD of DO11.10 and CD4+ T cells stimulated with immobilized anti-CD3 antibody. The inhibitory effect of RGDS peptides was dose dependent and significant at a concentration of 0.125 mM (data not shown). Pretreatment of DO11.10 cells with RGDS peptides for 30 min at 37°C followed by washing before stimulation did not inhibit AICD (data not shown). Thus, RGDS peptides must be present extracellularly during the activation of T cells with immobilized anti-CD3 antibody to inhibit AICD. These data suggest that the blockade of integrin receptors on activated T cells with RGDS peptides prevents AICD induced by restimulation with SEB in the presence of APC or by restimulation with immobilized anti-CD3 antibody in the absence of APC.



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  		Figure 2. RGDS peptides prevent AICD of T cells after restimulation with immobilized anti-CD3 antibody. (A) T-cell hybridoma DO11.10 was stimulated with immobilized anti-CD3 antibody in the presence or absence of 1 mM RGDS or RGES peptides for 24 h and stained for dead (EthD-1, red) or viable (calcein-AM, green) cells and analyzed by fluorescent microscopy. Yellow scale bar indicates 100 µm. (B) AICD of purified activated CD4+ T cells (left panel) or DO11.10 cells (right panel) was measured as the relative fluorescence intensity (RFI) of EthD-1 using microplate reader. The values are means ± SEM of one representative experiment performed in triplicate (CD4+ cells) or three independent experiments (DO11.10 cells). Asterisk indicates significant difference in the level of AICD of DO11.10 cells stimulated in the absence or presence of RGDS peptides (P < 0.001).

 
The restimulation of activated T cells leads to the expression of FasL, which is necessary for AICD, but may also induce lung epithelial cell apoptosis and acute lung injury (4, 16). More than 30% of DO11.10 cells stimulated with immobilized anti-CD3 antibody expressed FasL at 6 h (Figure 3). The presence of RGDS peptides during cell stimulation effectively blocked FasL expression, whereas control RGES peptides had no effect. RT-PCR analysis showed that RGDS peptides inhibited the expression of FasL at the RNA level (data not shown). These data suggest that the induction of FasL expression by immobilized anti-CD3 antibody is dependent on co-stimulatory signals provided by integrin receptors. RGDS peptides added at 4 h post-stimulation had no effect on AICD (data not shown), suggesting that RGDS peptides have no effect of Fas-mediated apoptosis. Thus, RGDS peptides inhibit AICD via the downregulation of FasL.



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  		Figure 3. RGDS peptides inhibit expression of FasL in DO11.10 cells stimulated with immobilized anti-CD3 antibody. DO11.10 cells were stimulated with immobilized anti-CD3 antibody in the presence or absence of 1 mM RGDS or RGES peptides. Surface expression of FasL was analyzed by flow cytometry after staining with PE-labeled antibody against FasL at 6 h post-stimulation. Representative data from one of three independent experiments are shown.

 
RGDS Peptides Downregulate ERK Phosphorylation Induced by Immobilized, but Not Soluble, Anti-CD3 Antibody
The ERK MAP-kinase is involved in signaling downstream from the TCR. Its activation is necessary for FasL gene expression in DO11.10 cells stimulated by anti-CD3 antibody (22). Stimulation of DO11.10 cells with immobilized anti-CD3 antibody resulted in sustained ERK phosphorylation (Figure 4A). The level of ERK phosphorylation reached maximum by 30 min and remained at high levels beyond 30 min (data not shown). The presence of RGDS peptides significantly decreased the level of ERK phosphorylation, whereas control RGES peptides had no effect (Figure 4A). A different pattern of ERK phosphorylation was observed in DO11.10 cells stimulated with soluble anti-CD3 antibody (Figure 4B). The level of ERK phosphorylation reached a maximum by 5 min post-stimulation but returned to the basal levels by 30 min. RGDS peptides had no effect on the level of ERK phosphorylation in DO11.10 cells stimulated with soluble anti-CD3 antibody. Thus, soluble anti-CD3 antibody induces transient phosphorylation of ERK MAP-kinase, whereas immobilized anti-CD3 antibody induces sustained activation of ERK. Restimulation of T cells with soluble anti-CD3 antibody does not induce AICD (24). ERK activation in the presence of RGDS peptides and immobilized anti-CD3 antibody resembles the pattern of ERK activation found in the cells stimulated with soluble anti-CD3 antibody. These findings indicate that sustained activation of ERK during the stimulation of activated T cells with immobilized anti-CD3 antibody requires co-stimulatory signals mediated by integrin receptors.



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  		Figure 4. RGDS peptides inhibit phosphorylation of ERK induced by immobilized, but not soluble, anti-CD3 antibody. DO11.10 cells were stimulated with 2.5 µg/ml plate-immobilized (A) or with 10 µg/ml soluble anti-CD3 antibody (B) in the presence or absence of RGDS peptide (1 mM) for the indicated time points. ERK phosphorylation was analyzed by immunoblotting using phosphospecific antibody. The same blots were stripped and re-probed with antibody against ERK-2. Representative blots from one of three independent experiments are shown.

 
RGDS Peptides Block T-Cell Adhesion and FAK Phosphorylation Induced by Anti-CD3 Antibody
Activation by anti-CD3 antibody has been demonstrated to induce the adhesion and spreading of T cells on various substrates (25, 26). We observed that immobilized anti-CD3 antibody effectively induced the adhesion of DO11.10 cells to tissue culture plates, whereas immobilized isotype control antibody had no effect. RGDS peptides effectively blocked adhesion of DO11.10 cells induced by various amounts of immobilized anti-CD3 antibody, whereas anti-CD3-induced T-cell adhesion remained high in the presence of RGES control peptide (Figure 5A). Similarly, RGDS peptides, but not the control RGES peptides, blocked adhesion of activated CD4+ cells restimulated with immobilized anti-CD3 antibody (data not shown).



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  		Figure 5. Stimulation with immobilized anti-CD3 antibody results in integrin-mediated cell adhesion and FAK phosphorylation. (A) Adhesion of DO11.10 cells stimulated with immobilized anti-CD3 antibody for 30 min in the presence of RGDS or RGES peptides (1 mM) was measured by calcein AM assay. Representative data from one of three independent experiments are shown (mean ± SEM). (B) Tyrosine phosphorylation of FAK in DO11.10 cells stimulated with immobilized anti-CD3 antibody for 15 min in the presence or absence of RGDS peptides was estimated by immunoprecipitation and phosphotyrosine immunoblotting. Representative blots from one of three independent experiments are shown. IP, immunoprecipitation; WB, western blot. (C) Pretreatment of DO11.10 cells with the antibody against ß3, but not ß1 or ß2, integrin receptors blocks anti-CD3–induced T-cell adhesion. The data of one from three representative experiments, each performed in triplicate, are shown (mean ± SEM).

 
The activation of FAK upon the engagement of integrin receptors is a major event in signal transduction by integrins. FAK is phosphorylated upon cross-linking of various ß1 integrins (27), {alpha}Lß2 integrin LFA-1 (28), or the vitronectin receptor {alpha}vß3 (29). We observed that treatment of DO11.10 cells with immobilized anti-CD3 antibody increased the level of FAK tyrosine phosphorylation (Figure 5B). Treatment of DO11.10 cells with RGDS peptides prevented the increase of FAK tyrosine phosphorylation. These findings suggest that restimulation of activated T cells with immobilized anti-CD3 antibody is accompanied by adhesion and tyrosine phosphorylation of FAK.

We next defined the integrin receptors that contribute to the restimulation of activated CD4+ T cells. Antibodies against the most abundantly expressed ß chains of integrin receptors were tested for their ability to inhibit anti-CD3–induced adhesion. Pretreatment of DO11.10 cells with the antibody against ß3 chain (CD61) of the integrin receptor for vitronectin prevented anti-CD3–induced adhesion, whereas antibodies against the common ß1 chain (CD29) of receptors for various extracellular matrix proteins and ß2 (CD18) chain of LFA-1 had no effect (Figure 5C). Similar results were observed when activated CD4+ T cells were pretreated with the antibodies against ß chains of integrin receptors (data not shown). The vitronectin receptor {alpha}vß3 has been shown to be expressed by activated T lymphocytes and to mediate their adhesion to vitronectin and fibronectin (29). These observations suggest that vitronectin receptor may also be responsible for the adhesion of activated CD4+ cells after the stimulation with immobilized anti-CD3 antibody.

RGDS Peptides Block Proximal TCR Signaling
The most proximal event in TCR/CD3 signaling is the phosphorylation of immunoreceptor tyrosine-based activation motifs in the CD3 signaling complex (30). We tested whether the blockade of integrin receptors with RGD peptides had any effect on proximal TCR signaling. RGDS peptides downregulated tyrosine phosphorylation of the CD3 {epsilon}-chain induced by immobilized anti-CD3 antibody (Figure 6A). Phosphorylation of the CD3 signaling complex attracts and activates ZAP-70 tyrosine kinase (30). We observed that the activation of DO11.10 cells with immobilized anti-CD3 antibody induced the detectable tyrosine phosphorylation of ZAP-70 at 5 min post-stimulation, that peaked at 15 min (Figure 6B). Treatment of DO11.10 cells with RGDS peptides reduced the level of ZAP-70 tyrosine phosphorylation at all time points, whereas RGES peptides had no effect. These results demonstrate that RGDS peptides are capable of inhibiting the most proximal signaling events induced by immobilized anti-CD3 antibody.



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  		Figure 6. RGDS peptides inhibit proximal TCR signaling. (A) Tyrosine phosphorylation of CD3-{epsilon} chain was analyzed by immunoprecipitation and phosphotyrosine immunoblotting after stimulation of DO11.10 cells with immobilized anti-CD3 antibody for 15 min in the presence or absence of RGDS peptide. Representative blot from one of three independent experiments is shown. (B) Tyrosine phosphorylation of ZAP-70 was analyzed at different time points after stimulation with immobilized anti-CD3 antibody. Representative blots from one of two independent experiments are shown. IP, immunoprecipitation; WB, western blot.

 
RGDS Peptides Prevent Clustering of the TCR Induced by Immobilized Anti-CD3 Antibody
Clustering of the TCR after stimulation is necessary for signaling. Reorganization of the TCR complex and other molecules at the interface between APC and T cells has been characterized as the "immunological synapse" (31, 32). Latex microbeads coated with anti-CD3 antibody have been widely used as artificial APC and induce TCR/CD3 aggregation at the interface between cells and beads (33). Because RGDS peptides inhibit the adhesion of T cells induced by immobilized anti-CD3 antibody, this may also negatively influence the interaction of T cells with anti-CD3–coated beads. To reduce this effect, we forced the contacts between the beads and DO11.10 cells by centrifugation. Using these conditions, anti-CD3 antibody immobilized on the microbeads induced clustering of the TCR ß-chain (Figure 7A, top panel). No clustering of the TCR was induced by isotype control antibody (data not shown). Increased intensity of TCR ß-chain staining at the cell–bead interface was observed in more than 40% of cell–bead conjugates (Figure 7B). Treatment of DO11.10 cells with RGDS peptides significantly decreased the number of cell–bead conjugates with clustered TCR ß-chains (Figure 7A, bottom panel, and Figure 7B). In addition, the mean clustering index for all cells conjugated with beads was significantly lower when the cells were treated with RGDS peptides (data not shown). As a control, we studied the clustering of the TCR/CD3 complex in T cells activated by soluble anti-CD3 antibody cross-linked with a secondary Cy2-labeled antibody. No significant difference was observed in the aggregation of the TCR/CD3 complex induced by cross-linking in the presence or absence of RGDS peptides (data not shown). Thus, TCR clustering is impaired by RGDS peptides when the cells are stimulated with immobilized anti-CD3 antibody. This suggests that aggregation of the TCR/CD3 signaling complex during stimulation with immobilized anti-CD3 antibody depends on the participation of integrin receptors. Therefore, we can conclude that integrin receptors provide co-stimulatory signals during stimulation of activated T cells in part by regulating the assembly of the TCR/CD3 signaling complex.



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  		Figure 7. RGDS peptides decrease clustering of TCR induced by anti-CD3 antibody immobilized on microbeads. (A) DO11.10 cells were incubated with RGDS peptides, and contacts between the cells and 6-µm beads coated with anti-CD3 antibody were facilitated by centrifugation. After a 10-min incubation, the cells were stained with FITC-labeled antibody against TCR ß-chain and analyzed by confocal fluorescent microscopy. Asterisks indicate microbeads. (B) Clustering of TCR at the cell–bead interface was analyzed quantitatively as described in MATERIALS AND METHODS to determine the number of conjugates with clustered TCR. Data from two independent experiments with 10 random fields for each group are shown (mean ± SEM). Asterisk indicates significant difference in the percentage of conjugates with clustered TCR between control and RGDS-treated groups (P < 0.01).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A number of lung diseases, including sarcoidosis, hypersensitivity pneumonitis, and pneumonia, are characterized by the infiltration of lung tissues with activated T lymphocytes (1). Restimulation of activated T cells leads to expression of FasL, which is implicated in increased lung injury via Fas-mediated apoptosis of lung epithelial cells (4). However, the expression of FasL is also a critical step in AICD, which may eliminate excess activated T cells (16, 17). In this study, we provide evidence that restimulation of activated T cells depends on integrin-mediated co-stimulation. The blockade of integrin receptors with RGD peptides prevented AICD of CD4+ T cells restimulated with SEB in the presence of APC or with immobilized anti-CD3 antibody alone. RGD peptides blocked the proximal events associated with TCR signaling. These observations suggest that lung matrix proteins may modulate T-cell–mediated immune responses in the lung.

It is well established that integrin receptors provide co-stimulatory signals to naive T cells by stabilizing contacts between TCR and MHC-petide complexes and by decreasing the threshold for activation (6–9). On the other hand, activated T cells have a lower threshold for activation and are considered less dependent on co-stimulatory signals (9, 21). Numerous reports, showing that immobilized anti-CD3 antibody alone triggers AICD of T-cell hybridoma and activated T cells, suggest that co-stimulation is not necessary for FasL expression if T cells are restimulated with a strong signal (16–19). Soluble RGD peptides and their nonpeptidic mimetics can dampen immune responses, presumably by disrupting cognate interactions between APC and T cells and by blocking T-cell migration (34, 35). Consistent with the strong role of integrins in na-ve T-cell activation, previous studies showed that RGD peptides block integrin-mediated co-stimulation of naive T cells (6, 7). However, the blocking effects of RGD peptides on restimulation of activated T cells have not been characterized. We found that RGD peptides not only blocked the expression of FasL but also inhibited activation signals induced by immobilized anti-CD3 antibody. It seems from previous studies (36) and our study that immobilized anti-CD3 antibody leads to a sustained activation of ERK MAP-kinase, which is required for the expression of FasL (22). It is well established that soluble anti-CD3 antibody provides only a partial activating signal to T cells, and immobilization of anti-CD3 antibody is required for full T-cell activation (37–39). Although a large fraction of, if not all, CD3 molecules on the cell surface could be engaged by soluble anti-CD3 antibody, the signal achieved by this stimulation is transient, probably due to fast internalization of the engaged molecules. In contrast, immobilized anti-CD3 antibody engages a relatively small fraction of CD3 molecules, but the effect is prolonged. Although RGDS peptides had no effect on ERK phosphorylation induced by soluble antibody, they abrogated ERK phosphorylation induced by immobilized anti-CD3 antibody and rendered it more similar to the weak, transient ERK activation by soluble antibody. These findings suggest that integrin receptors are necessary to amplify TCR signaling.

We found that RGDS peptides prevented the adhesion of T cells and the activation of FAK induced by immobilized anti-CD3 antibody. We also observed that an antibody against the ß3 chain of the vitronectin receptor (CD61) blocked the adhesion of T cells induced by immobilized anti-CD3 antibody with the same efficiency as RGDS peptides. These findings correlate with a recent report demonstrating that vitronectin and fibronectin present in FCS may facilitate AICD and suggest that adhesion mediated by ß3-integrins may provide co-stimulatory signals facilitating AICD (40). Another study demonstrated that a surface-associated isoform of fibronectin may be produced by activated T cells and enhance T-cell proliferation via an integrin-dependent mechanism (41). Either of these sources of matrix proteins may have provided the integrin-mediated effects that we observed in this study.

To determine whether integrin receptors are essential for the early events in the re-stimulation of activated T cells, we analyzed the formation of clusters of TCR/CD3 complexes upon stimulation with anti-CD3 antibody immobilized on microbeads. We used the model of T-cell activation with anti-CD3 antibody immobilized on microbeads because mobilization of various molecules to the cell–bead interface could be visualized and analyzed by microscopy (33). In addition, we previously established that anti-CD3 antibody immobilized on microbeads efficiently triggers AICD of DO11.10 cells (42). We found that TCR clustering at the cell–bead interface was significantly reduced in the presence of RGDS peptides. It is reasonable to suggest that a similar reduction of TCR/CD3 clustering occurs in the case of T-cell activation by plate-bound anti-CD3 antibody in the presence of RGDS peptides. Apparently, this is also accompanied by the inhibition of proximal TCR signaling because tyrosine phosphorylation of CD3 {epsilon}-chain and ZAP-70 tyrosine kinase induced by immobilized anti-CD3 antibody was greatly reduced in the presence of RGDS peptides. Thus, RGDS peptides disrupt clustering of the TCR/CD3 signaling complex and inhibit the most proximal events triggered by immobilized anti-CD3 antibody. These novel observations suggest that integrin receptors are necessary for the structural organization and signaling function of the TCR/CD3 complex after the restimulation of activated T cells. They also suggest that engagement of integrin receptors of T cells may be an important mechanism for modulating immune responses in the lung.


   Acknowledgments
 
The authors thank Dr. Barbara Osborne (University of Massachusetts) and Dr. Philippa Marrack (National Jewish Medical and Research Center, University of Colorado) for the kind gifts of DO11.10 cells. They also thank Noah S. Butler for helpful discussions and suggestions and Dr. Mary E. Wilson and Dr. Gail Bishop (University of Iowa) for their critical reading of the manuscript and helpful comments. This work was supported by grants from National Institutes of Health (NIH-HL-37121 and HL-03860) and VA Merit Review (G.W.H.).

Received in original form July 7, 2002

Received in final form October 29, 2002


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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