The chemokine SDF-1 stimulates integrin-mediated arrest of CD34+ cells on vascular endothelium under shear flow (original) (raw)
SDF-1 is displayed constitutively by human BM endothelium. The potential role of SDF-1 in human CD34+ progenitor adherence to BM endothelium has been studied in this work. We immunohistologically assayed the expression of SDF-1 by EC in biopsies of human BM. SDF-1 was expressed at high levels by BM venules. Anti–SDF-1 mAb reacted with EC lining the BM venules (Figure 1a, arrow), capillaries (Figure 1b, arrow), and sinusoids (data not shown), all of which have been recently reported to support stem cell rolling at sites of progenitor HPC emigration within the BM (22). These results suggest a key role for SDF-1 in the regulation of CD34+ progenitor recruitment to the human BM. Chemokines are implicated in the conversion of rolling interactions of circulating leukocytes into integrin-mediated arrest and emigration (6, 7). We therefore tested whether exposure of CD34+ cells to SDF-1 may drive rolling progenitors to develop firm adhesion to endothelial integrin ligands under physiological shear flow.
BM venules and capillaries reactive to anti–SDF-1 in human BM sections. SDF-1 immunostaining (arrows) of venous (a) and capillary (b) BM endothelium. ×100 (original magnification). No peroxidase staining of control mAb was observed on identical tissue sections.
Human CD34+ cells tether and roll on endothelial selectins and HA at physiological shear stresses. The potential for human CD34+ cells to roll on endothelial selectins expressed on BM vessel walls under physiological shear flow has not been studied before. We first characterized the efficiency by which CD34+ progenitors interact with purified selectins in vitro using parallel plate flow chamber assays designed to simulate the wall shear forces that exist in the BM microvasculature (4). Highly purified human CD34+ progenitors isolated from fresh cord blood were perfused over surfaces coated with physiological densities of endothelial selectins, either native or in the form of recombinant IgG fusion proteins. CD34+ cells express high and uniform levels of PSGL-1, the major P-selectin ligand, and highly heterogeneous levels of the cutaneous lymphocyte antigen (Figure 2a), a carbohydrate ligand whose expression on leukocytes closely correlates with their E-selectin binding activity (23–25). FACS® staining with an E-selectin–IgM chimera failed to detect high-affinity E-selectin ligands on human HPC, but revealed high-affinity ligands to a P-selectin–IgM chimera (26). Nevertheless, a similar fraction of CD34+ cells accumulated on both P-selectin and E-selectin substrates under different physiological shear stresses ranging between 0.5 dyn/cm2 and 2.5 dyn/cm2 (Figure 3a). All CD34+ cells tethered under flow to both selectins continued to roll on the selectins either immediately or when shear stresses were elevated (Figure 3b). Ca2+ chelation by EGTA abolished all adhesive interactions between CD34+ cells and P-selectin or E-selectin (Figure 4a and data not shown). P-selectin–mediated rolling of CD34+ cells was exclusively mediated by PSGL-1, the major P-selectin ligand on leukocytes (27, 28). This was confirmed by blocking CD34+ cell rolling on P-selectin with specific mAb to PSGL-1 (Figure 4a). When CD34+ cells accumulated on P-selectin or E-selectin were subjected to detachment by elevated shear forces, a higher fraction of CD34+ progenitors remained adherent to E-selectin than to P-selectin (Figure 3a). This was associated with a rolling velocity of CD34+ cells that was 2- to 2.5-fold lower on E-selectin than on P-selectin at every shear flow tested (Figure 3b). CD34+ cell rolling velocities on P-selectin increased by 50–80% when the site density of the selectin was reduced 2-fold (Figures 3b and 4b). However, neither P-selectin–mediated nor E-selectin–mediated rolling velocities varied with shear stress (Figures 3b and 4b). CD34+ cell rolling was comparable on both recombinant and native platelet-derived P-selectin (Figure 4b). The slower rolling and higher resistance to detachment of CD34+ cells adhered to E-selectin suggest that CD34+ cells express higher levels of adhesive ligands to E-selectin than adhesive ligands to P-selectin. CD34+ cells express moderate levels of L-selectin (29). Previously, L-selectin interactions with leukocyte ligands (including PSGL-1) were shown to prime homotypic interactions between myeloid cells and to augment secondary adhesion of circulating cells to adherent leukocytes and endothelium (30–32). In spite of the coexpression of functional PSGL-1 and L-selectin by CD34+ cells, we did not observe homotypic interactions between progenitors under any shear flow tested. These results and the previous findings by Mazo et al. (4) demonstrating the lack of endothelial ligands for L-selectin in the BM vasculature suggest that L-selectin plays little if any role in either primary or secondary adhesion of human CD34+ progenitors on the BM endothelium.
Immunofluorescence flow cytometry of human CD34+ progenitors stained with antibodies to major vascular adhesion receptors and to the SDF-1 receptor CXCR4. (a) Cutaneous lymphocyte antigen (CLA) expression (detected by the HECA-452 mAb PSGL-1) and CD44 expression are shown. (b) Expression levels of the LFA-1 integrin CXCR4 and the integrin VLA-4. Negative control staining with nonbinding preimmune mouse IgG is shown in the filled histograms.
CD34+ cells roll on endothelial selectins and CD44 ligand under physiological shear flow. (a) Accumulation of CD34+ cells continuously perfused on substrates coated with E-selectin–IgG or P-selectin–IgG at stepwise incremented shear stresses; resistance of accumulated cells to detachment by elevated shear stresses. Cells were perfused for 45 seconds at 1 dyn/cm2, and then the flow was increased by stepwise increments every 5 seconds. The number of cells bound at the end of each interval of incremented shear stress was determined as described in Methods. Data points are presented as mean ± SD of 4 fields of view. Note that cells continued to attach to the substrate to a maximum shear stress of 3 dyn/cm2. Selectin-IgG fusion proteins were each coated at 4 μg/mL on substrate-immobilized protein A, yielding 130 sites/μm2 of fusion protein, corresponding to 260 selectin sites/μm2. (b) Velocities of CD34+ cells rolling at representative shear stresses on the P-selectin–IgG or E-selectin–IgG substrate as described in a. Each mean value represents a minimum of 15 cells ± SEM, determined in 2 fields of view. (c) Effect of shear stress on the CD44-mediated rolling of CD34+ cells tethered to immobilized hyaluronan. CD34+ cells were allowed to settle for 30 seconds on HA (coated at 1 mg/mL) and then subjected to a shear stress of 0.5 dyn/cm2 for 5 seconds, followed by 3 increments each of 0.5 dyn/cm2, 1 dyn/cm2, 2 dyn/cm2, and 3 dyn/cm2, with each increment lasting 5 seconds. The number of cells remaining adherent at the end of the indicated shear interval was determined in 4 representative fields and was expressed relative to the number of cells adhering to the HA-coated substrate at a shear stress of 2.5 dyn/cm2. The percentage of rolling cells within the adherent cells at low and high shear stresses is shown above the data points. The mean velocity of 20 CD34+ cells rolling on HA at representative shear stresses is shown in black squares below the graph. SEM of mean velocities determined at 6.5 dyn/cm2 and 9.5 dyn/cm2 were 2.8 μm/s and 2.7 μm/s, respectively. The data shown in a–c are representative of 3 independent assays using CD34+ cells from different donors.
CD34+ cells and adult PBL express comparable levels of functional P-selectin ligand. (a) Accumulation of CD34+ cells and PBL perfused in identical numbers over immobilized P-selectin–IgG coated at 2 μg/mL. In control experiments, cells were briefly pretreated with the PSGL-1–blocking mAb KPL-1(αPSGL-1), or were perfused in a cation-free binding medium in the presence of EGTA. (b) Velocities of CD34+ cells and PBL at representative shear stresses on P-selectin–IgG or native (platelet-purified) P-selectin coated on substrates at densities supporting a comparable strength of adhesion of each cell type. Each mean value represents a minimum of 15 cells ± SEM, determined in 2 fields of view. *P < 0.002. **P < 0.016.
Blocking of the vascular receptor CD44 on HPC has been shown to interfere with their engraftment in mouse BM (1). CD44 can support rolling interactions of tumor cells and subsets of activated lymphocytes on its carbohydrate ligand, hyaluronate, as well as on activated vascular endothelium (33–35). Although high and uniform CD44 levels were detected on human CD34+ cells (Figure 2a), only a fraction of cells adhered to purified HA; yet all adherent cells could roll on the CD44 ligand when subjected to elevated shear stresses (Figure 3c). CD34+ cell rolling on HA was entirely CD44 dependent, as verified by mAb blocking, and did not require divalent cations as did selectin-mediated rolling (data not shown). A fraction of cells arrested on HA without rolling even at elevated shear stresses, but more than 70% of the cells that remained adherent to HA in high physiological shear flow rolled on the CD44 ligand (Figure 3c). Adherent cells rolling on high-density HA were as resistant to detachment by physiological shear stresses as those with E-selectin–mediated rolling were (Figure 3, a and c); rolling velocities were comparable for E-selectin and CD44 (Figure 3, b and c). Neither CD34+ progenitors nor CD44-expressing lymphocytes adhered to other glycosaminoglycans such as heparin and chondroitin sulfate, which were identically immobilized on the substrate (data not shown). This is consistent with the notion that the bond between CD44 and HA is specialized in mediating rolling adhesions under shear flow. Nevertheless, CD44 failed to promote initial CD34+ progenitor tethering to HA at shear stresses higher than 1 dyn/cm2, in contrast to tethering to E-selectin and P-selectin (Figure 3a and data not shown). This suggests that CD44 interactions with HA are unlikely to take place in the absence of endothelial selectins, which are obligatory for initial cell tethering to BM endothelium.
CD34+ cells express moderate levels of the P-selectin ligand PSGL-1 (Figure 2a). PBL express about 10-fold higher levels of PSGL-1, as determined by FACS® staining (data not shown). However, only a portion of the protein is properly glycosylated and can bind P-selectin (36). It was therefore necessary to compare the level of adhesiveness to P-selectin of CD34+ cells and fresh PBL under physiological shear flow. Although similar fractions of CD34+ cells and PBL accumulated at low physiological flow on P-selectin (Figure 4a), CD34+ cells rolled on the selectin at significantly reduced velocities compared with PBL (Figure 4b). CD34+ cells also resisted detachment from P-selectin by elevated shear stresses more effectively than did PBL (data not shown). Because slower rolling and higher resistance to detachment correspond to a higher number of adhesive bonds (37), these data collectively suggest that CD34+ cells express more functional P-selectin ligands than do freshly isolated adult T lymphocytes.
SDF-1 triggers LFA-1–mediated firm adhesion to ICAM-1 of CD34+ cells and PBL rolling on P-selectin. All CD34+ cells express moderate levels of the integrin LFA-1 (Figure 2b). In stasis, strong LFA-1–dependent adhesion of CD34+ cells to the major LFA-1 ligand ICAM-1 could be induced with short pretreatment with agonists such as phorbol esters and the LFA-1–activating mAb TS2.1 (data not shown). In contrast to the efficient attachment with which CD34+ cells tethered to endothelial selectins, CD34+ cells failed to adhere to substrates coated with ICAM-1 under physiological shear flow (Figure 5a) or at stasis (data not shown). However, when ICAM-1 was coimmobilized with P-selectin (P-selectin/ICAM-1), it supported efficient CD34+ cell tethering under physiological shear flow (Figure 5a). Nevertheless, the vast majority of tethered and rolling cells failed to spontaneously arrest on the substrate. The presence of ICAM-1 did not increase the percentage of CD34+ cells tethering to the substrate, which was comparable to that seen on P-selectin alone (data not shown). Short pretreatment of CD34+ cells with soluble SDF-1 for 1 minute did not increase the rate of cell tethering to the P-selectin/ICAM-1 substrate at 1 dyn/cm2, but caused about 30% of the cells accumulating on the adhesive substrate to arrest on the P-selectin/ICAM-1 substrate (Figure 5a). However, these arrested CD34+ cells did not develop firm adhesion to the substrate — at elevated shear stresses they exhibited poor resistance to detachment and resumed rolling, as did CD34+ cells accumulated and rolling on identical P-selectin/ICAM-1 substrates in the absence of SDF-1 (Figure 5b). Prolonged pretreatment of CD34+ cells with saturating levels of SDF-1 (1 μg/mL for up to 5 minutes) caused cell aggregation or deformation and reduced CD34+ progenitor accumulation on identical adhesive substrates (data not shown). In contrast to cells treated with soluble SDF-1, cells tethered to P-selectin/ICAM-1 substrate containing immobilized SDF-1 developed high adhesiveness to the substrate. About 60% of the cells accumulating on the P-selectin/ICAM-1/SDF-1 substrate came to full arrest either immediately or after a short period of rolling on the substrate (Figure 5a), and remained firmly adhered to the substrate even when the shear stress was elevated to the supraphysiological level of 15 dyn/cm2 (Figure 5b). The resistance to detachment by elevated shear stresses of cells tethered to P-selectin/ICAM-1 substrate containing immobilized SDF-1 was far greater than that of cells accumulated in flow on P-selectin/ICAM-1 alone or after treatment with soluble SDF-1 (Figure 5b). At all shear stresses tested, the vast majority of adherent cells remained arrested on the adhesive substrate that contained immobilized SDF-1 (Figure 5b and data not shown), but none of the cells adhering to the P-selectin/ICAM-1 substrate remained arrested in the absence of SDF-1 (Figure 5b). CD34+ cells accumulating on P-selectin/ICAM-1 substrate containing immobilized SDF-1 also readily spread on the substrate, whereas little or no spreading was detected of unstimulated CD34+ cells or of cells stimulated with soluble SDF-1 adhering to the P-selectin/ICAM-1 substrate (data not shown). Immobilized SDF-1 alone, on the other hand, failed to tether CD34+ cells under shear flow, although it slightly enhanced the attachment of CD34+ cells to ICAM-1 substrates (Figure 5a). In contrast to its dramatic effect on the conversion of rolling adhesions to firm arrests on P-selectin/ICAM-1, immobilized SDF-1 elevated only marginally the number of cells that initially accumulated on P-selectin/ICAM-1 (Figure 5b). Similar results were obtained with PBL: under identical experimental conditions, the vast majority of PBL rolling on P-selectin came to full arrest either immediately or after a short rolling period on the P-selectin/ICAM-1 substrate only when in the presence of coimmobilized SDF-1 (Figure 5a). Immobilized SDF-1 also stimulated firm, shear-resistant adhesion of PBL to the P-selectin/ICAM-1 substrate (Figure 5b); this adhesion was blocked by treatment with LFA-1–specific mAbs (data not shown). These experiments demonstrate a considerable synergy among P-selectin, ICAM-1, and immobilized SDF-1 with respect to the ability of CD34+ cells and of fresh lymphocytes to develop firm LFA-1–mediated arrest subsequent to rolling on P-selectin. Because soluble SDF-1 failed to stimulate LFA-1 adhesiveness with comparable efficiency to immobilized SDF-1, regardless of the time course of treatment, presentation of the chemokine on the adhesive surface appears to be crucial for its activity in stimulating firm integrin adhesion under physiological shear flow.
Tethering and rolling on P-selectin are prerequisites for SDF-1–triggered firm arrest of CD34+ cells on ICAM-1–containing surfaces. (a) CD34+ cell and T lymphocyte tethering to various adhesive substrates containing P-selectin, SDF-1, and ICAM-1 at a shear stress of 1 dyn/cm2. Motions of individual cells tethered to the different substrates were monitored over a 45-second period, and were divided into 3 categories as described in Methods. The fraction of each category within each experimental group (i.e., rolling, rolling-associated arrests, and immediate arrests) is presented in the stacked bars. The categories were analyzed only for cells that remained bound to the substrate at a shear stress of 2.5 dyn/cm2. (b) Resistance to detachment by incremented shear stresses of CD34+ cells accumulated at 1 dyn/cm2 on P-selectin/ICAM-1. The absolute numbers of cells accumulated during 1 minute at 1 dyn/cm2 and the number of cells remaining bound at the end of a 5-second interval of each shear increment are depicted. The percentage of stationary (arrested) cells within the cells remaining bound at a median shear stress (7.5 dyn/cm2) is shown in parentheses near the data points for each experimental group. ICAM-1 and SDF-1 were coated at 0.4 μg/mL and 10 μg/mL, respectively. P-selectin/ICAM-1 spots were prepared by mixing P-selectin and ICAM-1 in PBS/1% octyl glucoside and diluting the mixture in coating medium to final concentrations of 1 μg/mL and 0.5 μg/mL, respectively. Substrates were washed and then coated with SDF-1 as described in Methods. To assess the effect of soluble SDF-1, cells were preincubated in binding medium containing 1 μg/mL SDF-1 for 1 minute and perfused unwashed over the P-selectin/ICAM-1 substrate. Results shown in a and b are presented as mean of 2 determinations ± range. Sol., soluble; Imm., immobilized.
VLA-4 supports stable tethering of CD34+ cells to VCAM-1 under flow that is augmented by SDF-1. VLA-4 is a key CD34+ stem cell integrin (Figure 2b) that has recently been shown to work in parallel with selectins in promoting primary rolling adhesions of murine HPC on BM endothelium (4). We therefore asked whether VLA-4 on human CD34+ cells could support rolling adhesions on isolated VCAM-1, and whether SDF-1 could modulate VLA-4 adhesiveness to VCAM-1 under shear flow. Efficient tethering to VCAM-1 occurred at 0.75 dyn/cm2. This is a lower shear stress than that seen to allow CD34+ cell tethering to endothelial selectins, yet tethering efficiency of CD34+ cells was comparable to that of PBL under the same conditions (Figure 6a). Initial tethering on VCAM-1 was completely inhibited by treatment with mAb against the α4 integrin subunit as well as by EDTA chelation of divalent cations (data not shown). Tethering and accumulation of CD34+ cells varied strongly with the concentration of VCAM-1 coating on the substrate (Figure 6a and data not shown). Tethering was diminished on VCAM-1 coated at 0.5 μg/mL and lower concentrations, suggesting that VLA-4–mediated cell adhesion to VCAM-1 is supported by multivalent VLA-4:VCAM-1 bonds. After tethering to high-density VCAM-1, CD34+ cells spontaneously arrested on the VLA-4 ligand without rolling, even at elevated shear stresses. In contrast, PBL established both rolling adhesions and spontaneous arrests on various VCAM-1 substrates (Figure 6a; ref. 38).
Immobilized SDF-1 stimulates CD34+ cell adhesion to VCAM-1. (a) Accumulation of CD34+ cells or PBL on sVCAM-1–coated substrates at 0.75 dyn/cm2, and resistance to detachment of accumulated cells by incremented shear stresses. (b) Effect of soluble (1 μg/mL) or immobilized SDF-1 or MIP-1α (co-coated at 2 μg/mL with sVCAM-1) on the accumulation of CD34+ cells at 0.75 dyn/cm2 and on the resistance of accumulated cells to detachment by elevated shear forces. (c) Inhibition by pertussis toxin (PTX) of SDF-1–triggered firm adhesion of CD34+ cells to sVCAM-1 alone or coated together with 2 μg/mL of SDF-1. In b and c, sVCAM-1 was coated at 2 μg/mL and 5 μg/mL, respectively.
To study the effect of SDF-1 on VLA-4–mediated accumulation and adhesion strengthening on VCAM-1 under shear flow, CD34+ cells were perfused at 0.75 dyn/cm2 on a substrate coated with low-density VCAM-1, which alone was incapable of supporting CD34+ cell accumulation under shear flow (Figure 6b). Brief preactivation of CD34+ cells with soluble SDF-1 failed to enhance the attachment of CD34+ cells or PBL to the VCAM-1 substrate. Reminiscent of the lack of effect of soluble SDF-1 on LFA-1 adhesiveness shown in Figure 6, prolonged pretreatment of CD34+ cells with soluble SDF-1 deformed the cells and reduced their VLA-4–dependent tethering to a level below that of intact cells (data not shown). In sharp contrast, immobilized SDF-1 induced a high fraction of the CD34+ cells tethered to VCAM-1 at 0.75 dyn/cm2 to immediately arrest on the VLA-4 ligand. All arrested cells developed high resistance to detachment by elevated shear forces (Figure 6b). A major portion of cells also readily spread on the VCAM-1/SDF-1 substrate (data not shown). The strong proadhesive effect of immobilized SDF-1 was G-protein sensitive, as shown by the fact that pertussis toxin treatment suppressed almost all SDF-1–stimulated adhesion to VCAM-1 (Figure 6c). In contrast, MIP-1α , a stimulant of β1 integrin–mediated CD34+ cell adhesion to fibronectin (39), failed to enhance adhesion of cells to VCAM-1 under shear flow (Figure 6b), although it stimulated VLA-4 adhesiveness of T cells to VCAM-1 under similar experimental conditions (data not shown). Platelet activating factor, a lipid stimulant of HPC differentiation (40), similarly failed to enhance CD34+ cell adhesion when coimmobilized with VCAM-1 (data not shown). This may reflect the lower expression levels of MIP-1α and platelet activating factor receptors on CD34+ cells relative to the levels of the SDF-1 receptor CXCR4. Indeed, chemokine triggering of integrin adhesiveness under shear flow requires a high level of functional chemoattractant receptors (41).
SDF-1 adsorbed onto TNF-activated HUVEC triggers firm adhesion of CD34+ cells under shear flow. To elucidate the mechanism by which SDF-1 affects stem cell adhesiveness to endothelium under shear flow, we studied the interactions of cord blood–derived CD34+ progenitors with primary cytokine–activated HUVEC, a well-established vascular EC system. Upon cytokine activation, this endothelium expresses high levels of E-selectin, ICAM-1, and VCAM-1 (42, 43), and supports rolling and firm adhesion of leukocytes under physiological shear flow (44–46). Because the availability of human BM-derived EC is limited, we used this primary model as a surrogate for BM vascular endothelium. CD34+ cells accumulated efficiently on activated HUVEC under shear flow of 1 dyn/cm2, but failed to accumulate on resting HUVEC. The majority of CD34+ cells that accumulated on activated HUVEC at physiological shear flow started to roll on the endothelial monolayer when subjected to elevated shear stresses. CD34+ cell rolling on TNF-stimulated HUVEC was slow, with mean velocities similar to those observed in parallel experiments on purified E-selectin (data not shown). The majority of primary rolling adhesions to activated HUVEC were mediated by E-selectin (Figure 7a). The remaining rolling interactions of CD34+ cells with TNF-activated HUVEC pretreated with an E-selectin–blocking mAb were eliminated by blocking VLA-4 function on CD34+ cells (Figure 7a). Because VCAM-1 is the exclusive vascular ligand for VLA-4, these data suggest that endothelial VCAM-1 accounts for a small portion of CD34+ cell tethering and rolling in this vascular endothelium model, but together, VCAM-1 and E-selectin support all primary adhesions of CD34+ cells to TNF-activated HUVEC under physiological shear flow. Pretreatment of the activated endothelium with anti–P-selectin mAb did not interfere with CD34+ cell accumulation or rolling on the activated HUVEC, consistent with a lack of P-selectin, as verified by flow cytometry (data not shown). L-selectin ligands were also absent from the HUVEC cells tested in this study (data not shown), and appear to be absent from BM. Cytokine-activated endothelium expresses glycosaminoglycan ligands for CD44 that support rolling adhesions of lymphoid cells in a divalent cation–independent manner (35, 47). However, pretreatment of CD34+ cells with a CD44-blocking mAb did not affect tethering or rolling of CD34+ cells on TNF-stimulated HUVEC, indicating that this EC model does not express CD44 ligands that are functional under shear flow. Rolling of CD34+ cells on TNF-activated HUVEC was also eliminated by chelation of Ca2+ and Mg2+, consistent with exclusive roles for E-selectin and VLA-4, but not for CD44, in promoting rolling of CD34+ cells in this EC model. This is probably due to the absence of hyaluronan-like CD44 ligand on the lumenal surface of stimulated HUVEC under the experimental conditions.
CD34+ cell accumulation and development of firm adhesion on TNF-activated HUVEC under physiological shear flow. (a) Accumulation of CD34+ cells on intact and TNF-activated HUVEC; effect of EC-associated SDF-1 and blocking mAb’s against E-selectin or VLA-4. Cells were perfused at 1 dyn/cm2 for 45 seconds and then subjected to incremented shear stresses as described in Methods. The number of cells accumulated on the different HUVEC monolayers under the indicated experimental conditions was determined in 4 representative fields of view. Accumulated cells were divided into 2 categories: firmly arrested cells were cells that came to full arrest during accumulation at 1 dyn/cm2 and remained bound and stationary throughout the detachment assay (i.e., at a shear stress of 15 dyn/cm2). Rolling cells were defined as cells that continued to roll on the HUVEC monolayer immediately after tethering at 1 dyn/cm2, or cells that began to roll on the EC at elevated shear stresses and either remained adherent or moved from the field of view. The fractions of arrested or rolling cells among the cells initially accumulated in the field of view are shown in the stacked bars. (b) Resistance to detachment by shear flow of CD34+ cells accumulated at 1 dyn/cm2 on TNF-activated HUVEC. The absolute number of cells accumulated during 1 minute at 1 dyn/cm2 and the number of cells remaining bound at the end of each shear increment (each lasting 5 seconds) are depicted. Values are given as mean ± range of determinations in 4 fields of view. One of 4 independent experiments.
We next asked whether SDF-1 made available to flowing CD34+ cells on the lumenal surface of activated HUVEC can induce firm adhesion of CD34+ cells under shear flow. Because TNF-activated HUVEC do not produce endogenous SDF-1, soluble SDF-1 was overlaid on the cytokine-activated HUVEC monolayer for several minutes, and free chemokine was removed by extensive washing with binding medium. The presence of SDF-1 did not enhance CD34+ cell accumulation on stimulated EC, but did increase the percentage of CD34+ cells that, upon tethering and rolling on HUVEC, became firmly adherent and remained arrested even at high shear stresses (Figure 7a). In the absence of SDF-1, 80% of the cells originally tethered to the TNF-activated EC continued to roll on it without coming to arrest (Figure 7a). SDF-1 also increased the resistance of cells to detachment or to clearance from the site of initial accumulation by accelerated rolling at elevated shear stresses (Figure 7b). The proadhesive effects of SDF-1 on CD34+ cell adhesion to activated HUVEC were totally inhibited by pretreating the cells with pertussis toxin (Figure 7a). TNF-stimulated HUVEC are known to produce various chemokines and lipid mediators, such as GRO and platelet activating factor (48–50). However, pertussis toxin treatment of CD34+ progenitors had no effect on the low levels of spontaneous progenitor arrest on TNF-activated HUVEC that were observed in the absence of SDF-1 (Figure 7a and data not shown). Taken together, these results suggest that neither chemokines nor other Gi-protein stimulants produced by TNF-activated HUVEC trigger firm integrin adhesion of CD34+ cells to the endothelium under shear flow. Notably, firm adhesion of CD34+ cells triggered by endothelium-associated SDF-1 was fully integrin dependent — it could be blocked by a mixture of anti–LFA-1 and anti–VLA-4 mAbs (data not shown). These results indicate that SDF-1 on activated HUVEC can stimulate CD34+ cell integrins under physiological shear flow. This stimulation is essential to translate selectin-mediated rolling into firm shear-resistant arrest of CD34+ cells on vascular endothelium expressing the integrin ligands ICAM-1 and VCAM-1.