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. 2000 Aug 21;192(4):495–506. doi: 10.1084/jem.192.4.495

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© 2000 The Rockefeller University Press

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).

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graphic file with name JEM000195.f5ab.jpg — Immobilized chemokine induces rapid clustering of VLA-4 at adhesive contact zones. (A) Effect of VCAM-1 density on frequency of PBTL tethering, measured at a shear stress of 0.5 dyn/cm². All tethers measured on the indicated VCAM-1 densities were transient and diminished below a threshold VCAM-1 density (180 sites/μm²). (B) Confocal microscopy analysis of immunofluorescence staining of VLA-4 on PBTLs briefly incubated with control (HSA-coated) or SDF-1–coated beads (control and SDF-1, respectively), washed, fixed, and stained with the nonblocking VLA-4–specific mAb, B5G10. For PMA stimulation, cells were incubated with PMA for 2 min before VLA-4 staining. (C) Microclustering of VLA-4 and CXCR4 is enhanced within subsecond contact of PBTLs with surface-bound mAbs in the presence of immobilized SDF-1 leading to increased lymphocyte tethering to the surface. Tethering frequency of PBTLs measured at 0.75 dyn/cm² to the VLA-4–, CXCR4-, or L-selectin–specific mAbs (HP1/2, 12G5, and DREG200, respectively), coated onto the substrates at 0.2 μg/ml, together with inactive SDF-1 (−), intact SDF-1, or the P2G SDF-1 mutant (+), or the control chemokine ELC, each at 2 μg/ml. The frequency of transient tethers and of tethers resulting in immediate arrests is depicted. The majority of transient tethers lasted <1 s. The non-PBTL binding mAb 4B9 (anti–VCAM-1) served as negative control. SDF-1–dependent augmentation in total tethering to anti–VLA-4 mAb coimmobilized with intact SDF-1 was significant compared with tethering measured in the presence of P2G (n = 4, P < 0.01). PTX pretreatment of PBTLs abolished 90 ± 5% of SDF-1–triggered PBTL tethering to the anti–VLA-4 mAb HP1/2 coimmobilized with intact SDF-1. (D) Effect of inhibitors to major integrin or GPCR signaling effectors on SDF-1–triggered tethering to VCAM-1. PBTLs were preincubated for 30 min with the PTK inhibitor (genestein; 100 μM), the PI-3K inhibitor (wortmannin; 100 nM), or with control DMSO solution (0.1%). VLA-4–dependent tethers were determined at 1 dyn/cm² on VCAM-1 (1.5 μg/ml) coimmobilized with inactive or active SDF-1 (2 μg/ml, left). The effect of Ca²⁺ chelation on chemokine augmentation of VLA-4 tethering was tested on VCAM-1 (2 μg/ml) coimmobilized with SDF-1 (2 μg/ml) at shear stress of 1.5 dyn/cm² (right). To chelate [Ca²⁺], PBTLs were preloaded with BAPTA-AM (at 25 μM) or control DMSO solution as described in Materials and Methods. The experiments shown in A–D are each representative of four independent experiments.

graphic file with name JEM000195.f5cd.jpg — Immobilized chemokine induces rapid clustering of VLA-4 at adhesive contact zones. (A) Effect of VCAM-1 density on frequency of PBTL tethering, measured at a shear stress of 0.5 dyn/cm². All tethers measured on the indicated VCAM-1 densities were transient and diminished below a threshold VCAM-1 density (180 sites/μm²). (B) Confocal microscopy analysis of immunofluorescence staining of VLA-4 on PBTLs briefly incubated with control (HSA-coated) or SDF-1–coated beads (control and SDF-1, respectively), washed, fixed, and stained with the nonblocking VLA-4–specific mAb, B5G10. For PMA stimulation, cells were incubated with PMA for 2 min before VLA-4 staining. (C) Microclustering of VLA-4 and CXCR4 is enhanced within subsecond contact of PBTLs with surface-bound mAbs in the presence of immobilized SDF-1 leading to increased lymphocyte tethering to the surface. Tethering frequency of PBTLs measured at 0.75 dyn/cm² to the VLA-4–, CXCR4-, or L-selectin–specific mAbs (HP1/2, 12G5, and DREG200, respectively), coated onto the substrates at 0.2 μg/ml, together with inactive SDF-1 (−), intact SDF-1, or the P2G SDF-1 mutant (+), or the control chemokine ELC, each at 2 μg/ml. The frequency of transient tethers and of tethers resulting in immediate arrests is depicted. The majority of transient tethers lasted <1 s. The non-PBTL binding mAb 4B9 (anti–VCAM-1) served as negative control. SDF-1–dependent augmentation in total tethering to anti–VLA-4 mAb coimmobilized with intact SDF-1 was significant compared with tethering measured in the presence of P2G (n = 4, P < 0.01). PTX pretreatment of PBTLs abolished 90 ± 5% of SDF-1–triggered PBTL tethering to the anti–VLA-4 mAb HP1/2 coimmobilized with intact SDF-1. (D) Effect of inhibitors to major integrin or GPCR signaling effectors on SDF-1–triggered tethering to VCAM-1. PBTLs were preincubated for 30 min with the PTK inhibitor (genestein; 100 μM), the PI-3K inhibitor (wortmannin; 100 nM), or with control DMSO solution (0.1%). VLA-4–dependent tethers were determined at 1 dyn/cm² on VCAM-1 (1.5 μg/ml) coimmobilized with inactive or active SDF-1 (2 μg/ml, left). The effect of Ca²⁺ chelation on chemokine augmentation of VLA-4 tethering was tested on VCAM-1 (2 μg/ml) coimmobilized with SDF-1 (2 μg/ml) at shear stress of 1.5 dyn/cm² (right). To chelate [Ca²⁺], PBTLs were preloaded with BAPTA-AM (at 25 μM) or control DMSO solution as described in Materials and Methods. The experiments shown in A–D are each representative of four independent experiments.