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J. Biol. Chem., Vol. 276, Issue 52, 48670-48678, December 28, 2001

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Real Time Analysis of the Affinity Regulation of ₄-Integrin

THE PHYSIOLOGICALLY ACTIVATED RECEPTOR IS INTERMEDIATE IN AFFINITY BETWEEN RESTING AND Mn²⁺ OR ANTIBODY ACTIVATION^*

Alexandre Chigaev, Ann Marie Blenc, Julie V. Braaten, Nateasa Kumaraswamy^§, Christopher L. Kepley, Ronald P. Andrews, Janet M. Oliver, Bruce S. Edwards, Eric R. Prossnitz^¶, Richard S. Larson, and Larry A. Sklar^**

From the  Department of Pathology and Cancer Center and the ^¶ Department of Cell Biology and Physiology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131, ^§ Commonwealth Biotechnologies, Inc., Richmond, Virginia 23235, and the  National Flow Cytometry Resource, Los Alamos National Laboratory, Los Alamos, New Mexico 87545

Received for publication, April 10, 2001, and in revised form, October 16, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

This work examines the affinity of ₄₁-integrin and whether affinity regulation by G protein-coupled receptor (GPCR) and chemokines receptors is compatible with cell adhesion mediated between ₄-integrin and vascular cell adhesion molecule-1. We used flow cytometry to examine the binding of a fluorescent derivative of an LDV peptide (Chen, L. L., Whitty, A., Lobb, R. R., Adams, S. P., and Pepinsky, R. B. (1999) J. Biol. Chem. 274, 13167-13175) to several cell lines and leukocytes with ₄-integrin ranging from about 2,000 to 100,000 sites/cell. The results support the idea that ₄-integrins exhibit multiple affinities and that affinity changes are regulated by the dissociation rate and conformation. The affinity varies by 3 orders of magnitude with the affinity induced by binding mAb TS2/16 plus Mn²⁺ > Mn²⁺ ` TS2/16 > activation because of occupancy of GPCR or chemokines receptor > resting receptors. A significant fraction of the receptors respond to the activating process. The change in ₄-integrin affinity and the corresponding change in off rates mediated by GPCR receptor activation are rapid and transient, and their duration depends on GPCR desensitization. The affinity changes mediated by IgE receptor or interleukin-5 receptor persist longer. It appears that the physiologically active state of the ₄-integrin, determined by inside-out signaling, has similar affinity in several cell types.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell-cell recognition in the vasculature is crucial in hemostasis, host defense, inflammation, and metastasis. The adhesion molecules that contribute to these interactions belong to the integrin family and their counterstructures (ICAMs and VCAMs),¹ and the selectin family and their counterstructures (cellular mucins such as PSGL-1). A set of "traffic signals" has been proposed as a means of targeting circulating leukocytes to specific vascular sites (1). These traffic signals include: 1) an up-regulation of the expression of selectins, ICAM-I and VCAM-I, on endothelial cells by cytokines; 2) the recognition of up-regulated selectins and VCAM-1 on endothelial cells for the initial capture of leukocytes from the circulation, followed by tethering and cell rolling; 3) the presence of chemokines and chemokine receptors for leukocyte stimulation and integrin activation; and 4) the utilization of up-regulated ICAM-1 and VCAM-1 on endothelial cells for leukocyte firm attachment and motility.

Adhesion between leukocytes and endothelial cells is regulated by expression levels of the adhesion molecules ("site density"), their affinity, and their "avidity" (2-4). Affinity regulation is related to the molecular binding recognition. In the case of selectins and mucins, recognition is believed to be constitutive and to depend simply upon the structure of the two molecules. In the case of ₁- and ₂-integrins, it depends additionally on conformational changes in the binding region of an integrin for its counterstructure on ICAM-1 or VCAM-1. The conformational changes are associated with the responses of cells to stimulation. Avidity regulation, on the other hand, represents the change in adhesive activity between cells with contributions from the number and the affinity of the integrin and its topography on the cell surface. Topographical regulation may include association with cytoskeleton, dimerization, or other forms of macromolecular assembly or clustering (5-7). VLA-4 (₄₁-integrin) is unique in its ability to act both as a low affinity receptor that mediates initial capture, tethering, or rolling and as a firm attachment receptor (8, 9). The avidity of VLA-4 has been shown to be sensitive to the presence of chemokines such as those binding to CCR3 (10). There are conflicting data, however, regarding whether changes in adhesion are due to the conformational state or the clustering of the receptor. One group of investigators suggests that clustering controls the initial tethering and rolling interactions of the resting receptor, whereas firm attachment is related to affinity changes (5, 11).

The affinity states of VLA-4 can be recognized by mAbs sensitive to its molecular conformation (8). Recently, it has also been shown that the conformations of VLA-4 can be evaluated with a peptide ligand derived from the LDV sequence of the VLA-4-binding region of fibrinogen. (12-14). Peptide binding affinity has been measured both by the K_d and the dissociation off rate of a radiolabeled analog using transfected cells expressing high levels of VLA-4. The K_d and off rate vary inversely. The specificity with respect to the ₄-integrin was characterized by blocking with the LDV sequence from fibrinogen and by blocking with the extracellular domain of VCAM-1.

Very little is known about the characteristics of affinity regulation under physiological conditions. The missing details include the K_d and off rate of the physiological states of the receptors, the speed of the regulation, and the identity of the intracellular pathways that control affinity. In T cells, however, the pathway that controls affinity has been linked to p56^Lck (11). In this work, the affinity states of VLA-4 have been examined using a novel fluorescent peptide derived from the high affinity LDV sequence. We used flow cytometry to measure binding affinities and association and dissociation kinetics in real time and to examine affinity regulation in response to divalent cations and physiological mediators in both cell lines and blood leukocytes. The results suggest that affinity regulation as reflected in the dissociation rate is likely to play an important role in the adhesive characteristics of ₄-integrin on leukocytic cells.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials

The VLA-4-specific peptide (12, 13, 15), 4-((N'-2-methylphenyl)ureido)-phenylacetyl-L-leucyl-L--aspartyl-L-valyl-L-prolyl-L-alanyl-L-alanyl-L-lysine, and its FITC-conjugated analog were synthesized at Commonwealth Biotechnologies, Inc. (Richmond, VA). The purified peptide was dissolved in Me₂SO to 0.62 µM, based on absorbance measurements of the fluorescent peptide at 495 nm, and an extinction coefficient for fluorescein of 7.7 × 10⁴. FITC-conjugated monoclonal antibody, 44H6, against CD49d, was purchased from Serotec (Raleigh, NC). Phycoerythrin-labeled CD16 (CD16-PE) was purchased from Caltag (Burlingame, CA). Monoclonal antibody, HP2/1, against CD49d was purchased from Immunotech, Inc., (Westbrook, ME). Activating monoclonal antibodies directed against VLA-4 (TS2/16) were purified from hybridoma lines (ATCC, Manassas, VA), as previously described in detail (12, 13, 16). N-Formyl-Met-Leu-Phe-Phe was purchased from Sigma. Purified human eotaxin was purchased from Sigma. Recombinant human IL-5 was purchased from PharMingen (San Diego, CA). Leukotriene B4 (LTB4) was purchased from Sigma. RPMI 1640 and fetal bovine serum were from HyClone (Logan, UT). Human IL-8 and SDF-1 were purchased from PeproTech, Inc. (Rocky Hill, NJ). Recombinant human soluble VCAM-1 (rhVCAM) was purchased from R & D Systems Inc. (Minneapolis, MN). IL-5 (100 ng/µl stock) was used at 5 ng/µl. N-Formyl-Met-Leu-Phe-Phe (fMLFF) was prepared in 10 mM stocks in Me₂SO and used at a final concentration 0.1 µM. Eotaxin (stock 0.1 mM in sterile phosphate-buffered saline and 0.1% HSA) was used at 100 ng/ml. LTB4 (stock 100 µg/ml in ethanol) was used at a concentration of 3 µM. IL-8 was used at 20 nM. SDF-1 was used at 25 nM. TS2/16 was used at 5.6 µg/ml final concentration. HP2/1 was used at 10 µg/ml. RhVCAM was used at 0.4-1.0 µM.

Isolation of Human Leukocytes

Granulocytes-- Human blood was collected by venipuncture into sterile syringes containing 10 units of heparin/ml of blood in accordance with an approved human research protocol. Mixed granulocytes were typically isolated (15) with Mono-Poly resolving medium (ICN Biomedicals, Aurora, OH) plus 7.5% (v/v) sterile HEPES buffer (110 mM NaCl, 10 mM KCI, 10 mM glucose, 1 mM MgCl₂, and 30 mM HEPES, pH 7.4), by centrifugation at 400 × g for 35 min. Granulocytes were collected, washed in HEPES buffer, and then resuspended in HEPES buffer containing 1.5 mM CaCl₂ and 0.1% human serum albumin (Plasbumin-25; Bayer Pharmaceuticals, Elkhart, IN). The buffer had been depleted of lipopolysaccharide by affinity chromatography over polymyxin B-Sepharose (Detoxigel; Pierce). For some experiments, eosinophils were resolved by FACS analysis by staining the mixed granulocyte population with phycoerythrin-labeled CD16.

Isolation of Lymphocytes and Monocytes-- Mixed populations of lymphocytes and monocytes were isolated from Mono-Poly resolving medium as described for granulocytes, collecting cells from the layer located at the bottom of the plasma layer. The cells were washed in HEPES buffer and then resuspended in HEPES buffer/HSA/CaCl₂, as described above. Lymphocytes and monocytes were resolved from one another by FACS analysis of forward and side scatter intensity (15).

Isolation of Human Basophils-- Basophils were isolated from heparinized human blood by use of a Percoll gradient, as described previously (17). Purities from this initial step ranged from 15 to 66%. Basophil purity was routinely increased to >95% by negative selection using a negative selection cocktail (Stem Cell Technologies, Vancouver, Canada) and MidiMacs (Miltenyi Biotec, Auburn, CA) separation column. Basophils were resuspended in HEPES buffer with 1.5 mM CaCl₂ and stored on ice until used in assays.

Isolation of Human Eosinophils-- Mixed granulocytes were isolated from heparinized human blood by use of a Percoll gradient with a density of 1.088. The mixed granulocyte population was removed from the layer on top of the red cell layer, followed by red cell lysis with cold sterile water (two or three times). The resulting cells were resuspended in phosphate-buffered saline/fetal bovine serum with 50 µl of CD16-labeled and 10 µl of CD3-labeled magnetic beads and incubated for 30 min at 4 °C. This mixture was applied to a MACS (Miltenyi Biotec, Auburn, CA) separation column, and the CD16/CD3-negative flowthrough containing eosinophils was collected, washed, and resuspended in HEPES buffer/HSA/CaCl₂. These cells were labeled with CD16-PE to resolve any contaminating polymorphonuclear neutrophils by FACS analysis.

LDV Peptide Binding to Peripheral Blood Mixed Leukocytes

100 µl of human peripheral blood were incubated with 15 µl of CD19-PE (SJ25C1) (Becton-Dickinson, San Jose, CA) and 15 µl of CD3-PerCP (Leu-4) (Becton-Dickinson) at room temperature for 15 min. An unstained negative control and an isotype control stained with mouse IgG1-FITC, mouse IgG1-PE, and mouse IgG1-PerCp (Becton-Dickinson) were also prepared. Following this incubation, 2 ml of FACS lysing solution (Becton-Dickinson) were added to each tube and incubated at room temperature for 10 min. The tubes were then centrifuged at 500 × g for 5 min. The supernatant was decanted, and the cells were washed with 1 ml of HEPES buffer containing 1.5 mM CaCl₂ and 0.1% human serum albumin, Immuno-U.S., Inc (Rochester, MI). The samples were centrifuged again at 500 × g for 5 min, and the supernatant was removed. FITC-labeled LDV peptide was added to cells in two concentrations: 0.6 and 3.0 nM. Unlabeled peptide, TS2/16, and MnCl₂ (3 mM) were added to appropriate tubes. The cells were then resuspended in HEPES buffer to a final volume of 200 µl/sample and incubated at 37 °C for 15 min. The samples were analyzed by flow cytometry, collecting 10,000 events. The gates were set on the light scatter characteristics of the lymphoid population, monocyte population, and granulocyte population. In addition B and T cell populations are separated on the basis of CD19 and CD3 positivity in the light scatter gate characteristic of the lymphoid cells. Eosinophils were identified by CD19+, CD3+, and CD49+ within the light scatter gate characteristic of granulocytes.

Cell Lines and Site-directed Mutants of Formyl Peptide Receptor in U937 Cells

JM-1 and SUP-T1 cell lines were purchased from ATCC (Manassas, VA). The cells were cultured at 37 °C in a humidified atmosphere of 5% CO₂ and 95% air. Site-directed mutants of formyl peptide receptor in U937 cells were prepared as described in Ref. 18. JM1, SUP-T1, and U937 cells (4 × 10⁶) grown in RPMI 1640 (supplemented with 2 mM L-glutamine, 100 units/ml penicillin, 100 g/ml streptomycin, 10 mM HEPES, pH 7.4, and 10% heat-inactivated fetal bovine serum) were harvested and resuspended in 1 ml of HEPES buffer containing 10 mM glucose and 0.1% HSA and stored on ice. Before the experiment the cells were diluted with HEPES buffer containing 10 mM glucose and 0.1% HSA to 1 × 10⁶ cells/ml and warmed up to 37 °C. Human VCAM-1-transfected Chinese hamster ovary (CHO) cells (CHO-VCAM cells) were kindly provided by Dr. D. Leavesley (Hanson Cancer Center, Adelaide, Australia).

Generation of CXCR2- and CXCR4-transfected U937 Cells

Human CXCR2 and CXCR4 cDNAs were obtained from a gt11 dibutyryl cAMP differentiated HL60 cDNA library by PCR using primers based on the published human sequences and containing terminal EcoRI restriction sites. The resulting 1.1-kb EcoRI-digested fragments were cloned into the mammalian expression vector pSFFV.neo and confirmed by dideoxy sequencing. U937 cells were transfected with the DNA constructs (1 µg) using Effectene (Qiagen Inc., Valencia, CA) as described by the manufacturer and selected with 1 mg/ml G418 (Invitrogen Corp., Carlsbad, CA) for 3-4 weeks. Receptor expression was confirmed by flow cytometry using monoclonal antibodies against CXCR2 and CXCR4.

Equilibrium Binding of LDV Peptide

Equilibrium and kinetic analyses of peptide binding followed approaches described previously (19). For equilibrium binding studies of the FITC-labeled LDV peptide, cell lines, eosinophils (either isolated or as mixed granulocytes), mixed lymphocytes and monocytes, and polymorphonuclear neutrophils were treated with various concentrations (typically 1-10 nM) of the fluorescent peptide in the presence or absence of 3 mM MnCl₂. Incubations were performed for short times at 37 °C and overnight on ice with qualitatively similar results. Because the equilibration time of high affinity small molecules is typically a function of their dissociation rate, we have found it experimentally convenient to perform the overnight incubations. Nonspecific binding was determined using 500-fold excess unlabeled peptide. Analysis was performed on a FACScan (Becton-Dickinson), and 10000 events were acquired. Because instrument sensitivity was varied between experiments, the mean channel fluorescence values are not comparable.

Kinetic Analysis of Peptide Binding and Dissociation by FACS Analysis

The cells (cell lines (1 × 10⁶ cells/ml)) or eosinophils (either 2-5 × 10⁵ cells/ml isolated cells or 5-10 × 10⁶ cells/ml of mixed granulocytes) were preincubated with the peptide for 10 min at 37 °C and 500 rpm stirring. Flow cytometric analysis was performed continuously for up to 1000 s. The samples were analyzed for 30-120 s to establish a base line, then the stimulus (Mn²⁺, eotaxin, LTB4, fMLFF, IL-5, SDF-1, or IL-8) was added, and FACS acquisition was immediately re-established, losing 5-10 s of the total time course. For dissociation kinetic measurements, cell samples preincubated with 6-15 nM of fluorescent peptide were treated with 500-fold excess unlabeled peptide, and the dissociation of the fluorescent peptide was followed. The resulting data were converted to mean channel fluorescence over time using Tru-Rate software developed by Seamer and Sklar (20) or using FACSQuery software developed by Bruce Edwards. Curve fits and statistics were performed using GraphPad Prism (San Diego, CA).

Calibration of Surface Markers

Expression of adhesion molecules was measured with fluorescent mAbs and quantified by comparison with a standard curve generated with Quantum Simply Cellular microspheres (Flow Cytometry Standards, San Juan, Puerto Rico) stained in parallel with the same mAb. This produces an estimate of the total mAb-binding sites/cell (21).

Calibration of Peptide Binding

Quantum^TM 24 Premix microbeads (Flow Cytometry Standards Corp.) were used to quantitate the fluorescence intensity of cells bearing VLA-4 specific peptide. One or two drops of beads were added to 0.5 ml of isotonic phosphate-buffered saline (pH 7.2), and the mean channel numbers for all calibrated microbeads were collected. The number of molecules of equivalent soluble fluorochromes was plotted versus the mean channel numbers for the four fluorescent microbeads. The graph was used to estimate the total number of molecules of equivalent soluble fluorochromes/cell.

Cell Suspension Adhesion Assays

Cell suspension adhesion assays were described previously (21). Briefly, U937 cells were labeled with green fluorescent fluo 4 (7.5 µM, 15 min, 37 °C), and CHO-VCAM transfectants or CHO parental cells (nontransfected) were labeled with red fluorescent hydroethidine (250 µM, 15 min, 37 °C). Labeled cells were washed, resuspended in HEPES buffer (110 mM NaCl, 10 mM KCl, 1 mM MgCl₂, 1.5 mM CaCl₂, 30 mM HEPES, 10 mM glucose, rendered nonpyrogenic by affinity chromatography over Polymixin B, pH 7.4), and stored on ice until used in assays. U937 cells were preincubated for 5 min at 37 °C in the presence of no addition, LDV peptide (1 µM), anti-₄-integrins mAb HP2/1. Then cells were mixed, and cell suspensions containing 2 × 10⁶ U937 plus 2 × 10⁶ CHO-VCAM or CHO parental cells in 1.0 ml of HEPES buffer were stirred in a 12 × 75-mm polystyrene tube with a 2 × 5-mm magnetic stir bar (400 rpm, 37 °C). After 2 min of continuous stirring, the cells were sampled every 30 s and analyzed in the flow cytometer to determine the numbers of nonadherent singlet U937 (green fluorescent), nonadherent singlet CHO (red fluorescent), and cell conjugates containing U937 adherent to CHO (red and green cofluorescent). Every experiment included samples with Mn²⁺ (3 mM) added after 2 min of initial stirring. The percentage of U937s forming conjugates was derived by dividing the number of conjugates (dual color events) by the number of conjugates plus nonadherent singlet U937.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Design of the ₄-Integrin-specific Fluorescent Peptide-- We have taken advantage of an ₄-integrin-specific peptide developed at Biogen (12-14) derived from the Ile-Leu-Asp-Val (ILDV) binding sequence of the alternatively spliced connecting segment-1 of fibronectin. This sequence is homologous and isosteric with the Gln-Ile-Asp-Ser (QIDS) peptide found in the VCAM-1-binding site (22). Based on the published structure of a tight binding inhibitor of _41-integrin, compound 13 in Ref. 14, containing the sequence Leu-Asp-Val-Pro-Ser-Thr (LDVPST), and prior experience in peptide-receptor interactions, we replaced the C-terminal serine and threonine with Ala-Ala-Lys-FITC (Fig. 1A). The structure-activity relationships suggested that this change would not interfere with LDV peptide binding (14).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Specificity of the LDV peptide binding. A, line drawing of the VLA-4-specific peptide. The VLA-4-specific peptide, 4-((N'-2-methylphenyl)ureido)-phenylacetyl-L-leucyl-L--aspartyl-L-valyl-L-prolyl-L-alanyl-L-alanyl-L-lysine, and its FITC-conjugated analog were synthesized at Commonwealth Biotechnologies, Inc. The purified peptide was dissolved in Me2SO and used as described under "Experimental Procedures." B, aggregation between CHO and U937 cells regulated by LDV peptide and anti-4-integrin mAb HP2/1 was performed as described under "Experimental Procedures." The data are plotted as the mean percentages of CHO forming conjugates at steady state phase of aggregate formation (between 4 and 7 min after start). The data are representative of three experiments. C, binding of fluorescent LDV peptide blocked by recombinant soluble human VCAM-1. U937 cells were preincubated with recombinant soluble human VCAM-1 (1 µM) or nonfluorescent LDV peptide (1 µM) in the presence of Mn2+ for 1 h. Then fluorescent LDV peptide was added, and fluorescence was measured. The data represent the mean channel fluorescence. The autofluorescence of U937 cells was subtracted.

Specificity of the LDV Peptide Binding-- To investigate the capability of the peptide to specifically block the VCAM-1/VLA-4 interaction, we set up cell suspension adhesion assays using CHO cells transfected with human VCAM-1, and U937 cells, expressing VLA-4 (Fig. 1B). The aggregation experiment showed a low basal level of aggregation between nontransfected CHO cells and U937, which did not increase following integrin activation by Mn²⁺. VCAM-1 expression in CHO cells dramatically increased cell aggregation, and Mn²⁺ in this case stimulated the aggregation. The difference between basal aggregation and aggregation of VCAM-transfected cells thus represents VCAM-dependent aggregation. VCAM-dependent aggregation was blocked using LDV peptide or anti-₄-integrin mAb HP2/1. Thus, the peptide is acting through the ₄-integrin.

To obtain direct evidence of binding specificity, we used rhVCAM or nonfluorescent peptide in competition with the fluorescent LDV peptide (Fig. 1C). Preincubation of U937 cells with an excess of rhVCAM or the unlabeled peptide completely blocked binding of fluorescent LDV peptide. A more detailed analysis of binding is described below.

Equilibrium Binding of Fluorescent LDV Peptide to Cell Lines-- Initial studies were conducted to determine whether the peptide would bind to several cell lines including those representative of monocytes (Fig. 2A, U937), T cells (Fig. 2B, SUP-T1), and B cells (Fig. 2C, JM1). With this range of concentrations (to 25 nM), considerably higher levels of binding could be detected so that it was possible to measure peptide binding both in the presence and absence of Mn²⁺. The K_d values (0.3-1 nM) were all similar in the presence of Mn²⁺. In absence of Mn²⁺, the K_d was ~12 nM in U937 cells (Fig. 2A). Following earlier observations (12-14), we examined equilibrium binding in the presence of Mn²⁺, TS2/16, and their combination (Figs. 2D and 3). As indicated previously, states of varying affinity were detected, with dissociation rates (Fig. 3B) varying inversely with the K_d (Fig. 3A). These results are also consistent with the idea that peptide binding association rate constants do not vary appreciably between these receptor states (Table I).

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Equilibrium binding of the fluorescent peptide to cell lines. Equilibrium binding experiments were conducted with LDV fluorescent peptide on U937 (A), SUP-T1 (B), and JM-1 (C) cell lines as described under "Experimental Procedures." D, equilibrium binding of the fluorescent peptide to U937 in the presence of TS2/16 activating mAb. The data are plotted as mean channel fluorescence versus peptide concentration. The data are representative of three experiments. Dissociation constants (Kd) were calculated using a one-site (hyperbolic) binding equation. MCF, mean channel fluorescence.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Regulation of affinity and dissociation in 4-integrin conformations. A, equilibrium binding of LDV fluorescent peptide to U937 cells in the presence or absence of Mn2+ and the combination of activating antibody TS2/16 and Mn2+. Dissociation constants (Kd) were calculated using a one-site binding equation. B, dissociation kinetics of LDV fluorescent peptide from U937 cells induced by 500-fold excess of unlabeled peptide. Binding is shown as mean channel fluorescence versus time. To obtain dissociation rate constants (koff), the data were fitted to a one-phase exponential decay equation. The values for the rate constants are summarized in Table I. MCF, mean channel fluorescence.

Signaling between FPR, CXCR2 and CXCR4 Receptors, and ₄-Integrin-- Next we took advantage of the transfectants of the U937 cell line expressing the FPR, CXCR2, and CXCR4 to evaluate the relationship of signaling between receptors and the ₄-integrin. Experiments (Fig. 4) showed that the binding of the LDV peptide to ₄-integrin responded to the formyl peptide when the FPR was present in native form (wild type FPR) but not in an inactive mutant form (D71A). These experiments also showed that the duration and magnitude of binding to ₄-integrin was increased in the presence of a nondesensitizing FPR mutant, ST, in which the tail phosphorylation sites were removed (Fig. 4B) (23, 24). U937 cells transfected with CXCR2 and CXCR4 also rapidly responded to IL-8 and SDF-1, respectively, but the responses were transient (Fig. 4, C and D).

View larger version (32K): [in this window] [in a new window]   Fig. 4.   Response kinetics of LDV peptide binding to stimulation of CXCR4, CXCR2, wild type FPR, and FPR mutants in U937 transfectants. Cell suspensions were incubated with 3 nM fluorescent VLA-4-specific peptide and stimulated with fMLFF (A and B), SDF-1 (C), and IL-8 (D). A, nontransfected U937, stimulated with Mn2+, nondesensitizing ST, and nonactivating D71A ST. B, expanded scale for wild type FPR, ST, and D71A ST. C, rapid and transient response of CXCR4-transfected U937 cells to SDF-1. D, response of CXCR2 receptor-transfected U937 cells to IL-8. Binding is shown as mean channel fluorescence versus time. MCF, mean channel fluorescence.

Integrin Affinity States and Cell Activation-- We can begin to evaluate the relationship of receptor states to cell activation by examining the association and dissociation rate constants. Because the association rate constant does not appear to vary greatly among receptor conformations (Table I), we expect that when the peptide is in excess as compared with ₄-integrin, the initial rate of peptide binding will be proportional to the number of receptors available. In these experiments, there is a gap in data collection between the time of peptide addition and the beginning of the binding analysis (Fig. 4). From these results, we can only conclude that active conformations of ₄-integrin begin to appear within seconds of the time the cells are stimulated with chemoattractant or Mn²⁺.

However, it is also apparent that the binding initiated by Mn²⁺ addition is of greater magnitude compared with the chemoattractant (Fig. 4), which could in principle be due to a difference in site number or affinity. Because the change in binding initiated by chemoattractant is rapid and reversible, it is not an easy matter to determine the number of activated sites. However, it was possible to address the dissociation rate as shown in Fig. 5 by pre-equilibrating the U937 cells with a near saturating VLA-4 binding peptide concentration. Fig. 5A shows the dissociation characteristics of the peptide ligand as a function of time following chemoattractant addition. Dissociation is initiated at different times by the addition of the nonfluorescent LDV peptide. Qualitatively it is observed that the dissociation rate varies with time following chemoattractant. The dissociation rate at 10-30 s after chemoattractant binding is about an order of magnitude greater than that observed with Mn²⁺.

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Dissociation rates after VLA-4 activation in U937 cells. A, wild type FPR cells were incubated with 12 nM fluorescent LDV peptide. The cells were stimulated with 1 µM formyl peptide, and then blocking peptide was added after 0, 10, and 60 s after formyl peptide. B, exponential fits of data in A. The dissociation kinetics were fitted to a two component exponential decay equation. Dissociation rate constants (k1 and k2) are shown on the figure. The proportion of the active receptor was 0% at time 0, 72% at 10 s, 88% at 30 s, and 29% at 60 s. The binding is shown as relative fluorescence versus time (data were normalized to values between 0 and 1 of relative mean channel fluorescence).

Fig. 5B shows an analysis of the dissociation characteristics over time. The data have been fit with single (not shown) and double exponentials. The single exponential analyses yielded a systematic error in the fit to the data. When we fit the dissociation data to two components, one consisting of the rate for resting VLA-4 (0.06/s), the second component was consistently 0.01/s, a rate 10 times faster than in the presence of Mn²⁺. Notably, as the time after stimulus increased, the dissociation characteristics appeared to alter from receptors that were in the 0.01/s dissociation intermediate form to receptors that increasingly returned to a low 0.06/s dissociation form. These data are summarized in the legend to Fig. 5. When the same experiments were conducted with ST cells, after stimulation a dissociation rate of 0.01/s was maintained for at least 10 min.

Number of ₄-Integrin Molecules Detected by LDV Peptide Binding and anti-₄-Integrin mAb-- The initial studies of cell lines were extended to blood cells. First, we compared the site numbers detected by peptide binding to the numbers of ₄-integrin molecules detected by commercial anti-₄-integrin mAb using independent calibration methods for the peptide and antibody binding assays (Fig. 6A). We found a strong correlation between the number of mAb-binding sites and the number of molecules of the fluorescent LDV peptide. The values determined for leukocytes ranged from about 2,000 peptides bound for eosinophils (not shown) and 3,000 each for T and B cells to 6,000 for monocytes. The antibody determinations were consistent ranging from under 2,000 to 4,000. We expected the number of bound antibodies to be less than the number of peptides because of the possibility of bivalent binding.

View larger version (24K): [in this window] [in a new window]   Fig. 6.   Binding of the LDV fluorescent peptide to blood cells. A, correlation between number of 4-integrin molecules detected on peripheral blood leukocytes using anti-4-integrin mAb (44H6) and number of bound fluorescent peptide molecules. The traces of binding to granulocytes represent lower levels of detection of 4-integrin. The labeling procedure was described under "Experimental Procedures." B-D, equilibrium binding of fluorescent LDV peptide to mixed lymphocytes (B), eosinophils (C), and basophils (D). Experiments were conducted as described under "Experimental Procedures." The data are plotted as total, nonspecific, and specific binding as mean channel fluorescence versus peptide concentration. Nonspecific binding was determined in the presence of 500-fold excess of nonfluorescent LDV peptide. Specific binding was calculated as difference between total binding and nonspecific. The data are representative of three experiments. MCF, mean channel fluorescence.

The number of ₄-integrin molecules detected on peripheral blood cells was significantly lower than on the cell lines representing incomplete differentiation (data not shown). In particular, for SUP-T1, we detected about 20,000 peptide sites and 14,000 antibody sites. For JM1, we estimated about 6,000 peptide sites and 13,000 antibody sites. For U937 cells, we estimated about 40,000 peptide sites and 100,000 antibody sites. The under-reporting of peptide-binding sites for JM1 and U937 based on the semi-quantitative estimates was not further explored.

Equilibrium Binding of Fluorescent LDV Peptide to Blood Cells-- Equilibrium binding studies showed low levels of binding above the levels of cell autofluorescence. The apparent K_d in the presence of Mn²⁺ was ~0.4 nM, a value similar to that detected on cell lines and reported previously (12-14). The low site number was inadequate to detect significant binding in the absence of Mn²⁺ (Fig. 6, B and C) or anti-₄-activating mAb 8A2 (Fig. 6D). Although the total binding was low, the low level of nonspecific binding suggested that it might also be possible to examine the response of leukocytes to physiological stimuli.

Response Kinetics of LDV Peptide Binding to Leukocyte Stimulation-- Responses to IgE receptor cross-linking on purified basophils (Fig. 7A), IL-5 on basophils (Fig. 7B) and eosinophils (Fig. 7D), LTB4 on eosinophils (not shown), and eotaxin on eosinophils (not shown) have all been detected. In particular, the responses to IL-5 and IgE receptor cross-linking suggest that changes in ₄-integrin conformation occur within the detection limits (seconds) of these experiments. The responses to both eotaxin and LTB4 were similar to SDF-1 and IL-8 stimulation in U937 cells (Fig. 4, C and D). They were rapid and transient, whereas the responses to IL-5 and IgE receptor cross-linking were longer, and the fluorescent LDV peptide remained bound to cell surface for at least 400-600 s. This allows us to evaluate receptor states by examining dissociation rate constants.

View larger version (31K): [in this window] [in a new window]   Fig. 7.   Response kinetics of LDV peptide binding to leukocyte stimulation. A and B, basophils were purified as described under "Experimental Procedures" and then preincubated with 3 nM fluorescent peptide. The samples were analyzed for 30-120 s to establish a base line, and then 22E7, anti-FcRI, and IgE receptor cross-linking mAb or IL-5 was added, and FACS acquisition was immediately re-established, losing 5-10 s of the total time course. Nonspecific binding was determined in the presence of 500-fold excess of nonfluorescent LDV peptide. C and D, for dissociation kinetic measurements, eosinophils preincubated with fluorescent peptide were treated with IL-5 or vehicle for 10 min at 37 °C, and then 500-fold excess unlabeled peptide was added, and the dissociation of the fluorescent peptide was followed. Binding is shown as mean channel fluorescence versus time. A summary of dissociation rate constants obtained in dissociation experiments with basophils, eosinophils, and U937 cells transfected with FPR is shown in Table II. MCF, mean channel fluorescence.

Integrin Affinity States and Leukocyte Activation-- To determine dissociation rate constants basophils, eosinophils, and U937 cells transfected with ST FPR were treated with 22E7 anti-FcRI-IgE receptor cross-linking mAb, IL-5, or fMLFF, respectively, and then 500-fold excess of nonlabeled LDV peptide was added to induce dissociation. An example of those experiments is shown in Fig. 7 (C and D). A summary of dissociation rate constants is presented in Table II. In all experiments we were able to detect the difference between the resting state and the activated state of the ₄-integrin. Moreover, dissociation rate constants were similar for all cells and all cell treatments (Table II), but dissociation rate constants in activated cells were at least 10 times greater than for Mn²⁺- or TS2/16-treated cells (Table I). These results lead us to the idea that the physiologically active state of the ₄-integrin, initiated by inside-out signaling, is similar in several cell types and largely independent of the activating receptor pathway. This state is also different from one that can be induced by Mn²⁺- or anti-₄-integrin-activating mAb.

View this table: [in this window] [in a new window]   Table II Summary of dissociation rate constants for U937 cells transfected with FPR, basophils, and eosinophils, obtained in dissociation experiments (similar to the one shown on Fig. 7C and D) It was not possible to perform a dissociation experiment after treatment of cells with SDF-1, IL-8, LTB4, and eotaxin because responses were too short in duration (for example see Fig. 4, B and C).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Both receptor clustering and receptor conformation (affinity) have been considered as playing roles in the avidity of integrins. However, for the _4-integrin VLA-4, clustering has been attributed as the initial factor in avidity regulation (5). Because it has been recognized previously that ₄-integrin affinity can be regulated in cell lines by mechanisms associated with conformational changes (9-14), our goal was to determine whether affinity regulation could occur on a time frame and magnitude to be physiologically relevant.

We took advantage of the low molecular weight peptide ligand previously characterized as a radioligand (12, 13) and modified it to be suitable for fluorescence detection by flow cytometry, a technique previously known for its sensitivity and its applicability to real time analysis of ligand binding and dissociation (25). A particular advantage of using low molecular weight peptides is the possibility that, unlike large molecules such as antibodies, diffusion-limited rates of binding will allow binding of the peptide on a time scale relevant to transient cell responses and transient ₄-integrin affinity changes. The association rate constants for the parent peptide suggested that it bound rapidly to the receptor (12). The parent peptide contained an LDVPST sequence (Fig. 1). Because the carboxyl PST sequence appeared not to interfere with the activity of the peptide (13), we hypothesized that the C terminus could be modified without consequence for activity and selected the sequence LDVPAAK-FITC. We used criteria similar to those established previously to determine specificity with respect to ₄-integrin (Fig. 1). We showed that the peptide blocked ₄-integrin function and that its own binding was inhibited by ligands representative of fibronection and VCAM-1. The IC₅₀ for rhVCAM for 0.3 nM fluorescent peptide was ~50 nM in presence of Mn²⁺ (not shown), indicating that the K_d of VCAM is in the range of tens of nanomolar (13).

We showed that the peptide we selected behaved with properties similar to the originally described peptide. It bound to cell lines at high levels and to leukocytes at low levels consistent with the number of ₄-integrin molecules expressed on the cell surface (Figs. 2 and 6). The measurements can be equally well made on cell mixtures and potentially even in diluted blood.

The K_d of the peptide was similar in all cells tested in the presence of Mn²⁺, 0.3-1.0 nM with a K_d of ~12 nM in the resting state. We showed that the K_d varied inversely with the dissociation rate constant, suggesting that the on rate constant was essentially independent of receptor conformation (Ref. 12 and Fig. 3). Semi-quantitative estimates of site density provide correspondence within a half-log based on antibody and peptide binding. However, it is worth noting that such systematic comparisons by flow cytometry do not yet exist in the literature.

We have also shown that in both leukocytic cell lines and leukocytes the affinity of the binding can be modulated rapidly by divalent cations as well as soluble ligands for different signaling receptors (Figs. 3-5 and 7 and Table II). The equilibrium binding data are consistent with multiple receptor states in which contributions of individual conformations are additive (13). The changes in affinity occur within seconds and are as rapid as can be measured by manual flow cytometric approaches. The changes are reversible over minutes in response to chemokines and chemoattractants (Fig. 5).

Because the earlier results of the Biogen group indicate that there is an inverse relationship between the K_d and the dissociation rate for the LDV radiopeptide, it is worth commenting on the comparable affinity measurements in flow cytometry with the fluorescent peptide. As in all homogeneous binding measurements, the reliability of the specific binding analysis depends in part upon the level of nonspecific binding. It is our experience that nonspecific binding becomes appreciable when the total ligand concentration is in the range of several tens of nanomolar, although this value is influenced by the site density on cells. As a practical matter, measurements of K_d for tens of thousands of receptors/cell is most accurate below 100 nM total ligand concentration. However, the unique strength of the flow cytometric measurement is the ability to make a homogeneous measurement of the dissociation rate as reflected as in Fig. 7 even when the ₄ site densities of several thousand/leukocyte are likely to preclude detection even by a radioligand. Thus, we can obtain off rates that provide an independent measurement of the affinity of the binding interaction. It is reasonable to suggest therefore that the duration of the molecular adhesive interactions will be found to contribute in an important way to duration of cell-cell contacts during adhesion. The duration of cell-cell adhesion under conditions where cell-cell contacts are interrupted by the presence of agents that block those molecular contacts is likely to represent one aspect of cell avidity.

Consistent with previous cell suspension adhesion assays using basophils (26), the active state induced physiologically by fMLFF, IL-5, or IgE is one that is intermediate in dissociation rate between the resting state and the one induced by Mn²⁺- or anti-₄-integrin-activating mAb (Figs. 5 and 7 and Table II). The process of turning the affinity of the ₄-integrin on and off is tied to the activity of the chemoattractant receptor and appears to be accounted for by a single step conversion between the two states, nominally "off" and "on" (Fig. 5). This result was verified in the U937 cells with the nondesensitizing formyl peptide receptor. Even at times longer than 1 min of stimulation, a dissociation rate of 0.01/s characteristic of active ₄-integrin was detected (data not shown). Taken together, these observations are consistent with the possibility that ₄-integrin affinity regulation contributes to the overall avidity of cell adhesion mediated by ₄-integrin. To our knowledge, these measurements represent the first time that changes in the affinity or conformation of ₄-integrin have been identified or measured in real time on leukocytes at natural receptor abundance. Moreover, the regulation of ₄-integrin on different cell lines appears to be similar, with comparable affinities and off rates in response to different stimuli. Different extracellular stimuli lead to activation of different pathways that end at the same integrin effector molecules. In this case, the physiologically active state of integrins appears to be the same (at least in terms of affinity state) and independent from the activating receptor. Moreover, the differences in kinetics of receptor desensitization appear to be reflected by different kinetics of the affinity changes. They are of short duration in the case of CXCR2, CXCR4, and CCR3 and of longer duration in the case of IgE and IL-5 receptors. Other data suggest (not shown) that the activated states of ₄-integrin with varying dissociation rates and varying affinities also lead to varying avidities and varying cell-cell adhesion times. Because the level of ₇-integrin was typically or less of the ₁-integrin on the cells we tested (not shown), our results refer specifically to the ₄₁-integrin, VLA-4.

However, the relative contribution of ₄₁-integrin clustering as compared with ₄₁-integrin affinity to regulate avidity remains under investigation. It has been suggested that clustering measured by microscopy can alter adhesion in a subsecond time frame (5), and it remains to be seen whether the affinity changes induced by ₄₁-integrin arise in a similar time frame. It should be possible using rapid mix flow cytometry (27) to achieve resolution of peptide binding in the subsecond time frame to determine whether ₄₁-integrin affinity changes occur that rapidly. The clustering and conformational changes may very well cooperate to play different roles at different times, i.e. clustering or high affinity, so that cells can attach, roll, and firmly adhere.

The role of receptor density in modifying adhesive interactions on leukocytes underscores the importance of the relatively low number of ₄₁-integrin receptors on blood leukocytes. ₄₁-Integrin can in principle serve as its own rolling receptor for VCAM-1, presumably using the resting or clustered states of ₄₁-integrin for rolling and the activation pathway mediated by chemokines receptors for firm attachment via the activated state. It seems possible, given the low number of ₄₁-integrin on leukocytes and the variable number of VCAM-1 on endothelial cells, that another receptor pair contributes to rolling adhesion. For example, cell adhesion via ₄₁-integrin can activate LFA-1 (28). Moreover, PSGL-1 on leukocytes and P-selectin on endothelial cells may be used in combination with ₄₁-integrin and VCAM-1 to initiate rolling interactions. Because P-selectin/PSGL-1 tethering has been shown to induce ₂-integrin activation in some cases (29), it will be important to assess whether PSGL-1 engagement also leads to ₄₁-integrin activation, and the binding of the fluorescent peptide may prove to be a useful tool in this regard or to determine conditions under which the engagement of low affinity ₄₁-integrin leads to a ₄₁-integrin conformational change. The distinct time courses of ₄₁-integrin activation in response to stimulation is an area worthy of further investigation.

	ACKNOWLEDGEMENTS

We thank Dr. Richard Freer of Commonwealth Biotechnologies, Inc. for the helpful discussion regarding the peptide synthesis and Hisashi Tsuji for skillful technical support. Help from the University of New Mexico Clinical Research Center in recruiting of blood donors is gratefully acknowledged.

	FOOTNOTES

^* The work was supported by National Institutes of Health Grants RR14175 and RR01315 (to L. A. S.), HL56384 (to J. M. O.), and CA88339, American Cancer Society Grant RPG09601LBC (to R. S. L.), and the New Mexico Cigarette Tax, which supports the Flow Cytometry Facility of the University of New Mexico Cancer Research and Treatment Center.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence should be addressed: Dept. of Pathology and Cancer Center, CRF 219, University of New Mexico Health Sciences Center, Albuquerque, NM 87131. Tel.: 505-272-4249; Fax: 505-272-6995; E-mail: lsklar@salud.unm.edu.

Published, JBC Papers in Press, October 18, 2001, DOI 10.1074/jbc.M103194200

	ABBREVIATIONS

The abbreviations used are: ICAM, intercellular cell adhesion molecule; VCAM, vascular cell adhesion molecule; FPR, formyl peptide receptor; VLA, very late antigen; IL, interleukin; mAb, monoclonal antibody; FITC, fluorescein isothiocyanate; rhVCAM, recombinant  human soluble VCAM-1; fMLFF, N-formyl-Met-Leu-Phe-Phe; HSA, human serum albumin; FACS, fluorescence-activated cell sorter; CHO, Chinese hamster ovary.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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