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Immobilized Stromal Cell-Derived Factor-1 Triggers Rapid VLA-4 Affinity Increases to Stabilize Lymphocyte Tethers on VCAM-1 and Subsequently Initiate Firm Adhesion¹

Jeffrey A. DiVietro^*, David C. Brown, Larry A. Sklar, Richard S. Larson and Michael B. Lawrence^2,*

^* Department of Biomedical Engineering, University of Virginia, Charlottesville, VA 22908; and Division of Hematology/Pathology, Cancer Center, University of New Mexico, Albuquerque, NM 87112

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The integrin VLA-4 (₄₁) mediates tethering and rolling events as well as firm adhesion of leukocytes to VCAM-1. Unlike selectins, VLA-4 integrin-mediated lymphocyte adhesiveness can be modulated by chemokines through intracellular signaling pathways. To investigate the effects of the chemokine stromal cell-derived factor-1 (SDF-1) on VLA-4-mediated lymphocyte adhesion, human PBL were flowed over VCAM-1 substrates in a parallel plate flow chamber with surface-immobilized SDF-1, a potent activator of firm adhesion. The initial tethering interactions had a median lifetime of 200 ms, consistent with the half-life of low-affinity VLA-4-VCAM-1 bonds. Immobilized SDF-1 acted within the lifetime of a primary tether to stabilize initial tethering interactions, increasing the likelihood a PBL would remain interacting with the surface. As expected, the immobilized SDF-1 also increased the ratio of PBL firm adhesion to rolling. An LDV peptide-based small molecule that preferentially binds high-affinity VLA-4 reduced PBL firm adhesion to VCAM-1 by 90%. The reduction in firm adhesion due to blockage of high-affinity VLA-4 was paralleled by a 4-fold increase in the fraction of rolling PBL. Chemokine activation of PBL firm adhesion on VCAM-1 depended on induction of high-affinity VLA-4 rather than recruitment of a pre-existing pool of high-affinity VLA-4 as previously thought.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Circulating leukocytes must interact and firmly adhere to the endothelium before transmigrating across the vasculature to sites of inflammation. The capture of a leukocyte from blood flow and subsequent rolling on the vascular endothelium can be mediated both by selectins interacting with their mucin-like ligands and by ₄₁ and ₄₇ integrin interactions with endothelial cell expressed ligands VCAM-1 and mucosal addressin cell adhesion molecule-1, respectively (1, 2, 3). Expression patterns of selectin and ₄ integrins contribute to the selectivity of leukocyte trafficking to sites of inflammation and homing to appropriate lymph nodes (4).

An additional level of control of leukocyte trafficking patterns is provided by localized presentation of inflammatory chemotactic peptides such as fMLP or chemokines such as stromal cell-derived factor-1 (SDF-1),³ which bind to specific G protein-coupled receptors on the leukocyte surface (5, 6). The subsequent induction of intracellular signals (inside-out signaling) triggers integrin-mediated arrest of the rolling leukocyte and eventual transmigration (7, 8, 9, 10, 11). Arrest requires an increase in ₂ or ₁ integrin avidity for endothelial ligands such as ICAM-1 and VCAM-1 to ensure more and perhaps stronger bonds to put a brake on the rolling leukocyte.

Increases in integrin binding avidity in leukocytes are also closely correlated with changes in cellular shape, membrane structure, and receptor number; with varied and frequently overlapping effects (12, 13, 14, 15). Integrin receptor affinity also increases in some cases following leukocyte exposure to inflammatory mediators (16, 17, 18), leading to situations when number, presentation on the plasma membrane, and ability to bind ligand are simultaneously modulated.

In the case of mononuclear leukocytes, the regulation of VLA-4 integrin avidity for VCAM-1 by chemokines does not depend on changes in receptor number. Rather, avidity modulation likely represents some combination of affinity and alterations in membrane clustering or cytoskeletal anchorage, although to what extent affinity changes may influence adhesion has been somewhat controversial. For instance, immobilized but not soluble chemokines have been reported to increase PBL tethering to VCAM-1 by rapid VLA-4 clustering (on the order of 0.1 s) rather than through alterations in affinity (19). Yet a number of chemokines, including soluble SDF-1, are capable of triggering transient VLA-4 affinity increases in cell lines with reconstituted G protein-coupled receptor activity (18, 20, 21). Furthermore, circulating PBL appear to have a pool of constitutively high-affinity VLA-4 integrins that can mediate arrest and firm adhesion independent of chemokine or inside-out signaling (22). It is unclear whether chemokine-triggered firm adhesion is therefore dependent primarily on the affinity or avidity state of VLA-4.

The use of peptide ligands tagged with either radiotracers or fluorophores has been a powerful approach to assess VLA-4 affinity in the context of a cell membrane and signaling apparatus. Both equilibrium binding assays and highly sensitive flow cytometry techniques suggest VLA-4 can exist in multiple affinity states on the cell surface and further indicate that there is temporal control of VLA-4 affinity in response to chemokines or chemotactic peptides (18, 23, 24). Although multiple VLA-4 affinity states and off rates have been observed, depending on the activating agent (25, 26), it has been difficult to isolate specific contributions of high-affinity VLA-4 to lymphoid cell tethering, rolling, or the transition of rolling to firm adhesion under controlled shear flow conditions.

In this study, we examined the role of VLA-4 affinity in SDF-1 triggered PBL adhesion to VCAM-1 under flow conditions. An LDV (4-(N-2-methylphenyl)ureido-LDVPAAK-OH) peptide based small molecule (compound BIO1211 with AAK sequence) that preferentially binds to high-affinity VLA-4 was used to examine the contribution of low- and high-affinity VLA-4 to lymphocyte interactions with VCAM-1 in vitro under the constraints created by hydrodynamic flow. Selective antagonism of high-affinity VLA-4 under dynamic flow conditions with a small peptide ligand allowed us to avoid a number of potential artifacts of mAbs, and rapid equilibrium was assured by the high diffusivity of the small molecule. By virtue of this approach, we were able to identify not only the changes in VLA-4 adhesive function, but also distinguish adhesive contributions of constitutively active VLA-4 vs chemokine regulated VLA-4. Our results suggested that SDF-1 modulates firm adhesion of PBL on VCAM-1 through extremely rapid (200 ms) VLA-4 affinity changes rather than exclusively through VLA-4 clustering.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Reagents

Recombinant human VCAM-1 and SDF-1 mAb were purchased from R&D Systems. SDF-1 was purchased from PeproTech. The VLA-4 specific peptide-based compound LDV was purchased from Commonwealth Biotechnologies and is derived from the compound BIO1211 with the addition of the AAK sequence to enable biotinylation (26). The mAb against VLA-4, HP2/1, was purchased from Immunotech. The anti-VCAM-1 mAb was purchased from BioSource International. The anti-CXCR4 PE-labeled mAb 12G5 was purchased from R&D Systems. The mAbs against L-selectin, CD3, CD20, CD45RA, and CD45RO were purchased from Ancell. Accurate One-Step Polymorphs solution was purchased from Accurate Chemical and Scientific.

PBL isolation

PBL were obtained from 50 ml of heparin (10 U/ml) anticoagulated whole blood. Blood was layered over Accurate One-Step Polymorphs solution and centrifuged at 500 x g for 50 min at 20°C. The mononuclear layer was incubated on tissue culture treated dishes for 1 h at 37°C. Nonadherent cells were centrifuged and resuspended in RPMI 1640 plus 10% FBS and incubated at 37°C for 15–18 h. The resulting PBL were 12% CD20⁺, 85% CD45RA⁺ (naive), and 40% CD45O⁺ (memory), and 93% CXCR4⁺. Overnight culture resulted in a 6-fold up-regulation of CXCR4 expression with no change in L-selectin or VLA-4 expression levels. Samples were washed and fluorescence was detected using a FACScan flow cytometer (BD Biosciences). PBL were resuspended in HBSS with 10 mM HEPES. For pertussis toxin (PTX) experiments, PBL were incubated in 250 ng/ml pertussis toxin for 3 h at 37°C. 2 mM CaCl₂ and 2 mM MgCl₂ were added immediately before use in the flow chamber. For every set of experiments, the same donors and isolations were used for each comparison, which explains the slight variation in control numbers between separate figures. Informed consent was obtained from all donors following Human Investigation Committee protocol 10671 (University of Virginia, Charlottesville, VA).

Preparation of adhesion substrates

Polystyrene slides were cut from bacteriological petri dishes (Falcon 1058; Fisher Scientific). VCAM-1 was adsorbed to the slide for 2 h at room temperature. For slides also containing SDF-1, the slides were rinsed with PBS before adsorbing the SDF-1 to the slides for 1 h room temperature. The slides were then blocked for nonspecific adhesion with 0.5% Tween 20 in PBS overnight at 4°C.

Site densities of adsorbed VCAM-1 and SDF-1 were determined by saturation binding radioimmunoassay using mAbs 1E5 for VCAM-1 and 79014 for SDF-1 as previously described (11).

Laminar flow assays

The slides were incorporated into the lower wall of a parallel plate flow chamber. PBL at 1 x 10⁶ cells/ml were perfused into the flow chamber at a wall shear stress of 1 dyne/cm². In some experiments wall shear stress was varied between 0.5 to 2.0 dyne/cm². For experiments on the role of VLA-4 affinity on adhesion, PBL were incubated with 30 nM of the LDV compound (see below) for 5 min before flow. After 4 min of flow, seven fields of view were scanned, recording each field of view for 15 s. PBL that did not move in 15 s were counted as stationary (<1 µm), whereas PBL interacting with the surface that did move were counted as rolling. For tethering experiments, the field of view was focused at the leading edge of the adsorbed protein, which was determined by finding the most upstream area PBL would interact with the surface.

Quantitative antagonism of VLA-4 high affinity by LDV containing small molecules

Treatment of PBL with the LDV peptide ligand mimetic was 5 min before perfusion through the flow chamber to allow equilibrium to be achieved. The concentrations of the LDV peptide ligand mimetic were used as follows: The equation for fractional saturation is: Y = [L]/(K_d + [L]); where Y is fraction of ligand (L) bound (41). From Chigaev et al. (21), K_d values of LDV for different VLA-4 conformations are: K_d (resting VLA-4) = 36.1 nM (low affinity) and K_d (chemokine activated VLA-4) = 2.8 nM (high affinity). Most experiments were done with LDV at 30 nM such that Y (low affinity) = 30 nM/(36.1 + 30 nM) = 0.45 and Y (high affinity) = 30 nM/(2.8 + 30 nM) = 0.91. Therefore, at 30 nM, LDV binds to 45% of low-affinity VLA-4 and 91% of high-affinity VLA-4.

PBL tether data acquisition

To obtain tethering data, images were transferred from the VHS tape to a computer using a Scion LG-3 frame grabber in conjunction with Scion NIH Image 1.62. Movies were at standard video rate (30 frames/s). The number of tethers, or initial capture events, per minute was then determined by tracking any PBL that entered the field of view traveling with the bulk flow and then interacted with the surface, which was detected as a deceleration from bulk flow between any two consecutive frames. At 1 dyne/cm² wall shear stress, a tethering cell was quantitatively defined as one moving in the bulk flow (>250 µm/s) that decelerated greater than 35% (2600 µm/s²) within 1/30 s. Tethering at 0.5 dyne/cm² was similarly defined by decelerations of at least 35% within 1/30 s. PBL interactions were observed to initiate through cell surface contact or cell-adherent cell contact. Only the cell surface interactions (primary tethers) were included in the data. Transient tethers were defined as PBL that interacted with the surface and returned to bulk flow in <1 s. For PBL tethering experiments, a high-speed camera was used at 125 frames per second to quantify tethering events (FastCAM; Photron). Stable tethers were defined as PBL that captured from flow and remained interacting with the surface for greater than 1 s, either rolling or firmly adhered. PBL positions were obtained using a centroid tracking algorithm developed by Dr. W. Guilford (University of Virginia, Charlottesville, VA) and available as a plug-in to NIH Image J.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Under flow conditions VLA-4-mediated PBL tethering is extremely sensitive to flow and VCAM-1 site density

To characterize the relative frequency of rolling and firm adhesion of PBL on VCAM-1, PBL adhesive dynamics were quantified at flow rate and site densities titrated over 5- and 6-fold ranges, respectively (Fig. 1). VCAM-1 appeared relatively inefficient at supporting PBL tethering in flow compared with selectins, with virtually no interactions observed at 2.0 dyne/cm² wall shear stress even at site densities as high as 1200/µm². At a relatively low site density of VCAM-1 (250 sites/µm²), stable PBL tethering was optimal at 0.5 dyne/cm² wall shear stress and was significantly diminished at 1.0 dyne/cm² wall shear stress and above. Firm adhesion was favored over rolling at 0.5 dyne/cm² wall shear stress at the intermediate (600 sites/µm²) and highest VCAM-1 site densities (Fig. 1). For subsequent experiments, wall shear stress was 1 dyne/cm² for high VCAM-1 site density experiments (1200 sites/µm²) and 0.5 dyne/cm² for low VCAM-1 site density experiments (250 sites/µm²). Titrations were repeated with 1 mM Mn²⁺, which creates a high-affinity state of VLA-4 (26). When Mn²⁺ was present, only firm adhesion was observed at all wall shear stresses and VCAM-1 site densities tested. PBL did not roll when VLA-4 was in the Mn²⁺-induced high-affinity state.

View larger version (23K): [in this window] [in a new window]   FIGURE 1. Wall shear stress and VCAM-1 site density titration of stably interacting PBL. PBL in physiological assay medium (A), or with 1 mM Mn2+ (B) were perfused over VCAM-1 at site densities of 250, 640, and 1200 sites/µm2 at varying wall shear stresses. After 4 min of flow, at least seven fields of view were scanned for 15 s each. Those interacting lymphocytes that did not move in 15 s were defined as stationary, whereas lymphocytes that did move were defined as rolling.

The capture of a leukocyte from flow, defined in this study as tethering, was quantified as any window of 1/30 s in which the leukocyte decelerated rapidly (>2600 µm/s²) due to bonds forming with the substrate. Following the initial tethering on VCAM-1, PBL displayed one of three characteristic adhesive phenotypes. In some cases, PBL would detach immediately and return to flow, characteristic of a transient tether event (outcome 1). Alternatively, PBL remained on the VCAM-1 substrate by either rolling (outcome 2) or stopping to form a firm adhesion (outcome 3). The combination of rolling and firm adhesion characterized our definition of stable tethers.

Fig. 2 shows representative instantaneous velocity tracings of three PBL that tethered in the microscope field of view yet displayed different adhesive phenotypes. In the first case (Fig. 2A), a PBL decelerated and interacted with the VCAM-1 surface for 0.18 s before returning to the flow field. The tether event did not lead to any further surface interactions detectable at 125 frames/s (8 ms/frame) video capture rates. The second case (Fig. 2B) shows a tethering event that lead to a prolonged surface interaction as the PBL rolled on the VCAM-1 surface. Before the rolling interaction was initiated there were two very short-lived transient tethers that failed to convert to rolling. In the third case (Fig. 2C) a PBL tethered on VCAM-1 and arrested within a cell diameter, forming a firm adhesion. The combination of instantaneous velocity analysis and cumulative distance-time plots allowed quantitative distinctions to be drawn between populations of PBL displaying transient (Fig. 2A) and stable tethers that last longer than 1 s (Fig. 2, B and C). The last two illustrated cases (Fig. 2, B and C) were classified as stable tethers and involved rolling and firm adhesion, respectively (see Materials and Methods). All interactions were abolished by mAb HP2/1 (anti-₄) (data not shown).

View larger version (26K): [in this window] [in a new window]   FIGURE 2. Tracking of tethering lymphocytes. Initial capture events were separated into transient (those that interacted with the surface for <1 s), and stable (those that interacted for >1 s). Instantaneous velocity profiles (left) and the cumulative distance moved per time (right) of three tethering lymphocytes are shown. A lymphocyte that transiently interacted with the surface for 0.18 s (A), and two lymphocytes that stably interacted with the surface, one that rolled (B) and one that firmly adhered after tethering (C) are shown. Lymphocytes were perfused over a VCAM-1 (250 sites/µm2) surface with (C) or without (A and B) 1100 sites/µm2 SDF-1 at 0.5 dyne/cm2 wall shear stress. Images were captured at 125 frames per second.

Influence of immobilized SDF-1 on PBL adhesive dynamics on VCAM-1

To quantify immobilized SDF-1 chemokine effects on VLA-4-mediated PBL adhesive dynamics, tethering interactions were categorized as either transient (<1 s) or stable (>1 s) (see Fig. 2). To justify the use of 1 s as a discriminator between transient and stable tethering events, histograms of tether durations were plotted for unstimulated PBL that interacted with low site density VCAM-1 (250 sites/µm²) with and without SDF-1 (Fig. 3, A and B). The distribution of PBL tether lifetimes was distinctly bimodal, with >80% of the adhesive interactions lasted either <1 s or longer than 20 s at 0.5 dyne/cm² wall shear stress. Within the population of transient tethers, >90% lasted <0.6 s (Fig. 3, A and B, insets). The observed bimodal distribution of tether lifetimes was in sharp contrast to the exponentially distributed tether lifetimes observed with leukocytes tethering on selectins (27, 28).

View larger version (15K): [in this window] [in a new window]   FIGURE 3. Results of tether durations. Lymphocytes were perfused at 0.5 dyne/cm2 wall shear stress over a VCAM-1 (250 sites/µm2) substrate (A), a VCAM-1 (250 sites/µm2) plus SDF-1 (1100 sites/µm2) substrate (B), or a VCAM-1 (250 sites/µm2) substrate (C) with 1 mM Mn2+ added to the cell suspension. At least 50 lymphocytes were tracked for each condition for up to 20 s and the amount of time each lymphocyte interacted with the surface was obtained. Insets in A and B represent data of only those lymphocytes that interacted for 1 s or less.

Stable PBL tethering was more frequent following of SDF-1 stimulation

The presence of SDF-1 coimmobilized with VCAM-1 (250 sites/µm²) caused a highly significant increase (p < 0.001) in the number of stable tethers and a reduction in the relative number of transient tethers (Fig. 3B). No differences were observed in the duration of the transient tethers on either the VCAM-1 or the VCAM-1 plus SDF-1 surfaces, with median lifetimes of VLA-4 tethers lasting only 200 and 170 ms, respectively. The differences were not statistically different (p > 0.45). Almost all (>95%) tethers observed with 1 mM Mn²⁺ were stable, and no rolling was observed (Fig. 3C).

Immobilized SDF-1 increases the probability of stable tether formation on VCAM-1 but fails to enhance PBL tethering frequency

Previous investigations have reported that immobilization of SDF-1 amplified PBL tethering frequency on VCAM-1 under flow conditions. However, the PBL tethering interaction has never been analyzed using computer-assisted image processing techniques and high-speed video microscopy, which can detect highly transient interactions that take place between flowing leukocytes and adhesive surfaces that are otherwise invisible at video frame rates (28, 29, 30).

Fig. 4A shows that 90% of PBL tethering to the high site density VCAM-1 (1200 sites/µm²) remained stably interacting with the surface, whereas only 49% of PBL tethering to the low VCAM-1 site density (250 sites/µm²) formed stable interactions (51% formed transient tethers). The low VCAM-1 site density was therefore used to examine possible effects of immobilized SDF-1 on whether PBL tethers were stable or transient. To quantify the impact of immobilized SDF-1 on PBL tethering frequency, PBL interactions with VCAM-1 were analyzed with high-speed video and the microscope objective focused on the edge of the VCAM-1 coating so that all PBL interactions would represent first encounters with the VCAM-1, thereby removing a potential source of systematic error created by upstream adhesive interactions. With PBL flux thus controlled, we observed that total PBL tethering frequency on VCAM-1 was unaffected (p = 0.5) by coimmobilized SDF-1 (Fig. 4B). Total tethering frequency of PBL on the high site density VCAM-1 was also unchanged with SDF-1 coimmobilized to the surface (data not shown). Although the PBL tethering frequency was unchanged by the presence of coimmobilized SDF-1, the percentage of tethers that were stable increased significantly from 49 to 76% (Fig. 4B). The loss of transient tethering events was almost exactly balanced by an increase (p < 0.02) in the number of stable tethers (Fig. 4B). Interestingly, SDF-1 effects appeared to be extremely rapid with the loss of transient tethers, suggesting that during the 200 ms median lifetime of a VLA-4-VCAM-1 transient tether, the PBL had a high probability of converting to a stable interaction.

View larger version (22K): [in this window] [in a new window]   FIGURE 4. SDF-1 stabilizes lymphocyte adhesions immediately following tethering. PBL were perfused over VCAM-1 surfaces at a site density of 250 sites/µm2 at a wall shear stress of 0.5 dyne/cm2 with and without immobilized SDF-1 (1100 sites/µm2), or perfused over VCAM-1 at 1200 sites/µm2 and 1 dyne/cm2. A, The percentage of tethers that were stable for both site densities is shown. B, Shown is the mean total tethering frequency (percentage of PBL close to the surface that tether), stable tethering frequency, and transient tethering frequency for the low site density VCAM-1. Total tethering frequencies were not significantly different. Both the transient tethering rates and stable tethering rates were significantly different comparing PBL on VCAM-1 with PBL on VCAM-1 plus SDF-1 (p < 0.05).

Immobilized SDF-1 alters PBL adhesion characteristics to VCAM-1

Consistent with previous reports (18, 31), immobilized SDF-1 increased the number of firmly adhered PBL (Fig. 5). Blocking the ₄ subunit of VLA-4 with the mAb HP2/1 abolished all PBL interactions with the surface, confirming that the tethering, rolling, and firm adhesive events were all ₄ integrin-mediated. The ratio of firmly adhered to rolling PBL was increased for both high and low VCAM-1 site densities. When VCAM-1 was used at the lower site density (250 sites/µm²) to permit transient tethers (50% of the tethers were transient without immobilized SDF-1, see Fig. 4), immobilized SDF-1 increased the total number of PBL on the surface as well as the ratio of firmly adherent to rolling PBL (Fig. 5B). This observation was consistent with our previous tethering data showing that at the low VCAM-1 density immobilized SDF-1 acts rapidly to stabilize tethering interactions and decrease transient interactions (Fig. 4), thereby increasing PBL accumulation (rolling or firm adhesion) with the surface.

View larger version (16K): [in this window] [in a new window]   FIGURE 5. SDF-1 exposure, by either immobilized or soluble means, increases firm adhesion of PBL to VCAM-1. A, PBL were perfused over a VCAM-1 substrate (1200 sites/µm2), a VCAM-1 plus SDF-1 (1100 sites/µm2) substrate, or exposed to 2.5 nM SDF-1 immediately before perfusion over the VCAM-1 substrate. The numbers of rolling and stationary cells are shown at the 4 min mark. Also shown are PBL incubated with the VLA-4 blocking Ab, HP2/1. The number of firmly adhered PBL was significantly different from control for both immobilized SDF-1 and soluble SDF-1 (*, p < 0.005). B, PBL were perfused over a VCAM-1 substrate (250 sites/µm2) with or without immobilized SDF-1 (1100 sites/µm2). There were statistically different increases in both total PBL (**, p < 0.005) and firmly adhered PBL (**, p < 0.05) with immobilized SDF-1.

SDF-1 mediates PBL adhesion increases through G protein-coupled receptor signaling

To verify that the SDF-1 effects were a result of "inside-out" G protein signaling, G_i subunits were inactivated using PTX. Unstimulated PBL display both rolling and firm adhesion phenotypes and were unaffected by PTX treatment. In contrast, PTX blocked any increase in the number of firmly adherent PBL in response to SDF-1 (Fig. 6), confirming that SDF-1 effects were G protein-sensitive.

View larger version (15K): [in this window] [in a new window]   FIGURE 6. SDF-1-mediated firm adhesion is G protein-sensitive. Lymphocytes were incubated with PTX to block Gi signaling before perfusion over VCAM-1 (1200 sites/µm2) surfaces at 1 dyne/cm2. Means of firmly adhered and rolling lymphocytes are shown for PTX treated lymphocytes with and without SDF-1 coimmobilized on the VCAM-1 surface. PTX significantly reduced firm adhesion (*, p < 0.005) in immobilized SDF-1 assays, but does not alter adhesion of nonchemokine-stimulated PBL.

SDF-1 modulation of VLA-4 affinity and PBL adhesive dynamics on VCAM-1 surface

It is currently unclear whether and to what extent affinity increases or avidity modulation involving VLA-4 are responsible for the enhancement of firm adhesion following PBL exposure to SDF-1 (19, 22, 26). It is possible that SDF-1 may not even modulate VLA-4 affinity at all according to some reports (19). To investigate the effect of VLA-4 affinity modulation on PBL adhesion to VCAM-1 in shear, the LDV-based small molecule was used to selectively block the high-affinity VLA-4 (32). To examine the putative contribution of high-affinity VLA-4 on PBL rolling and firm adhesion without the presence of transient tethering interactions, we used the high site density of VCAM-1 (1200 sites/µm²) to ensure that most (90%) of PBL tethers formed long-lived, stable interactions predictive of rolling and firm adhesion.

Although the LDV small molecule preferentially depletes the population of high-affinity VLA-4, there is some binding to low-affinity VLA-4 (see Materials and Methods). Using the published K_d values of 36.1 nM for the LDV small molecule-VLA-4 interaction on resting U937 cells and 2.3 nM for a state analogous to chemokine activation (18), 45% of low-affinity VLA-4 and 91% of high-affinity VLA-4 are blocked at 30 nM. Therefore a strategy was devised to establish that the LDV small molecule effects were due to blocking high-affinity VLA-4, rather than an effect of simply fewer VLA-4 molecules available to bind VCAM-1. To accomplish this effect, we used the VLA-4 blocking mAb HP2/1 at subsaturation levels to decrease the total number of VLA-4 molecules available without preferentially blocking the high-affinity conformation. HP2/1 mAb does not selectively target the high-affinity VLA-4.

Fig. 7 compares the effect of a titration of mAb HP2/1 vs the LDV small molecule on unstimulated PBL rolling and firm adhesion on VCAM-1 at 1200 sites/µm². Both blockage agents reduced the overall number of stable tethers, but with significantly different effects on the distribution between rolling and firm adhesion. Random blockage of VLA-4 with mAb HP2/1 slightly favored a proportional increase in firm adhesion relative to rolling (Fig. 7B). In contrast to the effects of mAb HP2/1, the preferential blockage of high-affinity VLA-4 with the LDV small molecule significantly increased the relative populations of rolling vs firmly adherent PBL (Fig. 7A; p < 0.01). As with HP2/1, increasing concentrations of the LDV small molecule reduced overall adhesion. HP2/1 mAb did not decrease the likelihood of PBL firm adhesion for any concentration tested.

View larger version (22K): [in this window] [in a new window]   FIGURE 7. LDV changes PBL adhesive dynamics. PBL were incubated with LDV (A) or HP2/1 (B) at varying concentrations before perfusion over VCAM-1 (1200 sites/µm2) substrates at 1 dyne/cm2. The mean number of firmly adhered and rolling PBL per field of view is shown for each condition. C, PBL blocked with a subsaturating concentration of HP2/1 (50 ng/ml) or LDV (30 nM) were perfused over VCAM-1 (1200 sites/µm2) substrates with or without coimmobilized SDF-1 (1100 sites/µm2) at 1 dyne/cm2. Both HP2/1 and LDV significantly decreased total adhesion and firm adhesion for all conditions tested (*, p < 0.0005). D, The percentage of PBL that are firmly adhered is shown. LDV significantly decreased the percentage of firmly adhered PBL for both untreated and immobilized SDF-1 cases. **, p < 0.0005.

In contrast to the effects of mAb HP2/1, PBL treated with the LDV small molecule had a higher probability of rolling compared with firm adhesion even when interacting with immobilized SDF-1. The relative effects of mAb HP2/1 and the LDV small molecule on PBL firm adhesion on VCAM-1 coimmobilized with SDF-1 were dramatic (Fig. 7D), with 91% of mAb HP2/1-treated PBL having formed firm adhesions and only 54% of the LDV small molecule-treated PBL having formed firm adhesions. The LDV small molecule increased the likelihood PBL would roll on both VCAM-1 and VCAM-1 plus SDF-1 while reducing the likelihood of firm adhesion. The HP2/1 mAb did not significantly change the ratio of rolling to stationary PBL.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The VLA-4 integrin can mediate capture, rolling, and firm adhesion steps of lymphocytes to vascular endothelium through interactions with cytokine-induced VCAM-1 (33, 34). In contrast to selectins, the VLA-4 integrin is also responsive to stimulating agents such as Mn²⁺, phorbol esters, mAbs, and chemokines; all of which can increase the adhesiveness of VLA-4-expressing leukocytes for VCAM-1 (35, 36, 37). The dual adhesive modalities of VLA-4 allow a PBL in particular to react to endothelial cell expressed chemokines such as SDF-1 in ways the selectins or ₂ integrins are unable to duplicate functionally. In this report, we observed that the chemokine SDF-1 increased VLA-4 affinity, and that the induction of high affinity was critical for arrest and firm adhesion on VCAM-1. Furthermore, immobilized SDF-1 did not appear to alter PBL tethering frequency, yet triggered conversion of VLA-4 to high affinity within the lifetime of a tether bond (200 ms). Although both low- and high-affinity VLA-4 supported PBL tethering, low-affinity VLA-4 favored rolling over firm adhesion, consistent with a model in which chemokine activation of VLA-4 affinity increases take place subsequent to PBL tethering.

To address the contributions of VLA-4 affinity to lymphocyte adhesion to VCAM-1 under flow conditions, independent of receptor clustering, we developed an affinity-selective small molecule blocker of VLA-4-VCAM-1 binding. The small molecule has several technical advantages over mAbs and large soluble proteins; primarily being that its size is less likely to perturb cell function and secondarily, that its affinity for ligand is much more easily measurable than is the case with the large molecules. Indeed, Scatchard analysis has shown the small molecule blocker to have monospecific and saturable binding to VLA-4, with distinct affinities for resting and activated VLA-4 (18, 20). In contrast to peptides that are unable to discriminate between high- and low-affinity VLA-4, the LDV-based small molecule has a 40-fold higher affinity for the Mn²⁺-induced state of VLA-4 than for resting VLA-4, thereby allowing functional discrimination between activated and nonactivated VLA-4.

When PBL were exposed to the LDV-based small molecule at concentrations sufficient to block >90% of high-affinity VLA-4, the probability of rolling was dramatically enhanced and firm adhesion was virtually abolished. Most strikingly, it appeared that immobilized SDF-1 acted rapidly, on the order of 200 ms, to convert transient tethering interactions to rolling followed by firm adhesion. The blocking effects of the small molecule as well as the parallel effect of G protein-coupled receptor inhibition strongly implicated SDF-1 induction of high-affinity VLA-4 as critical for the arrest of PBL, consistent with observations of fMLP-stimulated monocytes under flow conditions (38). SDF-1 effects, although rapid, were secondary to the initial VLA-4 tethering event as both low- and high-affinity states of VLA-4 equivalently supported tethering.

SDF-1 has also been shown to amplify PBL tethering rather than firm adhesion, in contrast to what has been observed with the chemokine IL-8 stimulation of neutrophil arrest on ICAM-1 (11, 19). Despite the use of high spatial-temporal resolution video microscopy, we were unable to detect any increase in PBL capture events under flow when SDF-1 was immobilized on VCAM-1. Unbiased computer tracking (C. Paschall and M. B. Lawrence, manuscript in preparation), however, revealed the presence of a significant population of brief PBL tethering events with a lifetime of 180 ms that may have been previously undetected. It appeared that in our parallel plate flow system, the frequency of PBL interactions with VCAM-1 was independent of the presence of chemokines, as the PBL interaction frequency with VCAM-1 was the same whether SDF-1 was present or not.

One of the goals of the current study was to determine whether VLA-4 on human PBL converts to a functional high-affinity state after a physiological SDF-1 stimulation and whether high-affinity VLA-4 is critical for firm adhesion to VCAM-1. Several laboratories have shown that VLA-4 can exist in multiple affinity states (26, 32, 35, 36), but the correlation of affinity and adhesion has frequently been clouded by the use of soluble ligands as reporters. Chan et al. (38) have shown that chemokines, including SDF-1, increase monocyte VLA-4 affinity for VCAM-1 leading to arrest and firm adhesion. However, significant differences in adhesiveness due to chemokine activation of VLA-4 have been reported for T lymphocytes and PBL. For instance, it has been reported that SDF-1 exposure does not increase the lifetime of VLA-4-VCAM-1 tethers or soluble VCAM-1 binding of PBL as might be supposed if VLA-4 were in a higher affinity state (25). Additionally, a number of PBL specific chemokines appear to trigger arrest on VCAM-1 through VLA-4 avidity modulation rather than affinity modulation.

Part of the complexity of interpreting VLA-4 function comes from the mixture of transient binding, rolling, and firm adhesion phenotypes typically observed for PBL. High-speed video microscopy allowed us to determine that PBL interactions were strikingly segregated into two statistically distinguishable populations, with >90% of the interactions quantified grouped into either transient (<1 s) or stable (>20 s) categories. Only 10% of the PBL adhesive interactions failed to be grouped in either category. If a PBL tethering event lasted over a second, then the likelihood of slow rolling or firm adhesion became very high (+90%) and the interaction was classified as stable. The relative lifetimes of transient tethers correlated surprisingly well with estimates of the lifetime of resting VLA-4 bonds as assessed by quantitative flow cytometry (18). In the absence of Mn²⁺ stimulation, a significant proportion of VLA-4-VCAM-1-mediated PBL tethers survived no more than a fraction of a second, consistent with solution phase estimations of resting VLA-4 k_off for recombinant human VCAM-1 being greater than 5 s^–1 (18). When Mn²⁺ was used to induce high-affinity VLA-4 almost no transient interactions were observed. The PBL immediately firmly adhered to the surface after the initial interaction, suggesting the high-affinity VLA-4 has a much lower k_off than VLA-4 on resting PBL.

A number of recent observations suggest that lymphocyte populations are shifted toward different adhesive phenotypes by flow, VCAM-1 site density, VLA-4 affinity modulation, and chemokines (21, 24, 38, 39). A schematic illustrating the multiple adhesive phenotypes observed in this study of lymphocytes interacting with VCAM-1 surfaces is shown in Fig. 8. After an initial VLA-4–VCAM-1-mediated interaction (stage 1), a lymphocyte can either immediately detach (stage 2) or remain interacting with the surface (stage 3). Lymphocytes that remain interacting with the surface could be either rolling (stage 5) or stationary (stage 4). Rolling lymphocytes may eventually stop and firmly adhere to the surface (stage 6). Exposure of lymphocytes to Mn²⁺ removed any possibility of vectoral or adhesion linked signaling and proved to be the most potent initiator of PBL firm adhesion. Nevertheless, immobilized SDF-1 when copresented with VCAM-1 was a highly effective inducer of firm adhesion, but required a brief rolling interaction before arrest. Likely, some time was required for the transduction of intracellular signal from the CXCR4 receptor to the VLA-4 and for the VLA-4 receptor to find a VCAM-1 molecule. In vivo it is possible that lymphocytes might at various times be exposed to either soluble or immobilized SDF-1 during the course of an immune system challenge.

View larger version (35K): [in this window] [in a new window]   FIGURE 8. Schematic of possible pathways for a tethering PBL. After an initial capture-tether interaction (stage 1), a PBL can immediately detach (stage 2) or remain interacting on the surface (stage 3). Those PBL that remain stably interacting with the surface were observed to either roll (stage 5) or be firmly adhered (stage 4). Rolling PBL can stop and transition to firmly adhered PBL (stage 6).

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by National Institutes of Health Grant HL54614.

² Address correspondence and reprint requests to Dr. Michael B. Lawrence, Department of Biomedical Engineering, MR5 Building Room 2111, University of Virginia, P.O. Box 800759, Charlottesville, VA 22908. E-mail address: mbl2a{at}virginia.edu

³ Abbreviations used in this paper: SDF, stromal cell-derived factor; PTX, pertussis toxin.

Received for publication July 3, 2006. Accepted for publication December 27, 2006.

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Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

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