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Subsecond induction of alpha4 integrin clustering by immobilized chemokines stimulates leukocyte tethering and rolling on endothelial vascular cell adhesion molecule 1 under flow conditions.

Grabovsky V, Feigelson S, Chen C, Bleijs DA, Peled A, Cinamon G, Baleux F, Arenzana-Seisdedos F, Lapidot T - J. Exp. Med. (2000)

Bottom Line: We show here that immobilized chemokines can augment not only arrest but also earlier integrin-mediated capture (tethering) of lymphocytes on inflamed endothelium.Furthermore, when presented in juxtaposition to vascular cell adhesion molecule 1 (VCAM-1), the endothelial ligand for the integrin very late antigen 4 (VLA-4, alpha4beta1), chemokines rapidly augment reversible lymphocyte tethering and rolling adhesions on VCAM-1.Chemokines potentiate VLA-4 tethering within <0.1 s of contact through Gi protein signaling, the fastest inside-out integrin signaling events reported to date.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, The Weizmann Institute of Science, Rehovot, 76100 Israel.

ABSTRACT
Leukocyte recruitment to target tissue is initiated by weak rolling attachments to vessel wall ligands followed by firm integrin-dependent arrest triggered by endothelial chemokines. We show here that immobilized chemokines can augment not only arrest but also earlier integrin-mediated capture (tethering) of lymphocytes on inflamed endothelium. Furthermore, when presented in juxtaposition to vascular cell adhesion molecule 1 (VCAM-1), the endothelial ligand for the integrin very late antigen 4 (VLA-4, alpha4beta1), chemokines rapidly augment reversible lymphocyte tethering and rolling adhesions on VCAM-1. Chemokines potentiate VLA-4 tethering within <0.1 s of contact through Gi protein signaling, the fastest inside-out integrin signaling events reported to date. Although VLA-4 affinity is not altered upon chemokine signaling, subsecond VLA-4 clustering at the leukocyte-substrate contact zone results in enhanced leukocyte avidity to VCAM-1. Endothelial chemokines thus regulate all steps in adhesive cascades that control leukocyte recruitment at specific vascular beds.

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Related in: MedlinePlus

Immobilized chemokines augment VLA-4–mediated capture and arrest of T lymphocytes to purified VCAM-1 under shear flow. (A) Frequency of PBTL tethers to purified sVCAM-1 coated at 1.5 μg/ml on polystyrene surface with functional or heat-inactivated SDF-1 (2 μg/ml). The different tether categories were determined in two fields at 1 dyn/cm2 and results are an average and range of each tether category. (B) Frequency and type of tethers formed by naive (CD45RA+) and memory (CD45RO+) subsets of peripheral blood CD3+ T lymphocytes interacting at 1 dyn/cm2 with sVCAM-1 (1.5 μg/ml) coated together with the indicated intact or heat-inactivated chemokines (each at 2 μg/ml). Chemokine-dependent augmentation in total tethering was highly significant (*P < 0.001 and 0.004, for experiments performed on the CD45RA+ CD45RO+ subsets, respectively). (C) Frequency of VLA-4–mediated tethers of PBTLs to sVCAM-1 coated at 0.5 μg/ml, alone, or with inactivated SDF-1, SDF-1, or the non-signaling SDF-1 mutant P2G 16 (each at 2 μg/ml), determined at 0.5 dyn/cm2. VCAM-1 coating density on all substrates was identical as verified by radioimmunodetermination. For PTX treatment, PBTLs were cultured for 15 h with 100 ng/ml of the toxin. In A and D, where indicated, PBTLs were pretreated with the VLA-4 blocking mAb, HP1/2 (10 μg/ml), and were perfused unwashed over the VCAM-1–bearing surfaces. Data in A–C are representative of four independent experiments.
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Figure 2: Immobilized chemokines augment VLA-4–mediated capture and arrest of T lymphocytes to purified VCAM-1 under shear flow. (A) Frequency of PBTL tethers to purified sVCAM-1 coated at 1.5 μg/ml on polystyrene surface with functional or heat-inactivated SDF-1 (2 μg/ml). The different tether categories were determined in two fields at 1 dyn/cm2 and results are an average and range of each tether category. (B) Frequency and type of tethers formed by naive (CD45RA+) and memory (CD45RO+) subsets of peripheral blood CD3+ T lymphocytes interacting at 1 dyn/cm2 with sVCAM-1 (1.5 μg/ml) coated together with the indicated intact or heat-inactivated chemokines (each at 2 μg/ml). Chemokine-dependent augmentation in total tethering was highly significant (*P < 0.001 and 0.004, for experiments performed on the CD45RA+ CD45RO+ subsets, respectively). (C) Frequency of VLA-4–mediated tethers of PBTLs to sVCAM-1 coated at 0.5 μg/ml, alone, or with inactivated SDF-1, SDF-1, or the non-signaling SDF-1 mutant P2G 16 (each at 2 μg/ml), determined at 0.5 dyn/cm2. VCAM-1 coating density on all substrates was identical as verified by radioimmunodetermination. For PTX treatment, PBTLs were cultured for 15 h with 100 ng/ml of the toxin. In A and D, where indicated, PBTLs were pretreated with the VLA-4 blocking mAb, HP1/2 (10 μg/ml), and were perfused unwashed over the VCAM-1–bearing surfaces. Data in A–C are representative of four independent experiments.

Mentions: To study chemokine modulation of PBTL adherence to endothelial cells (ECs) under flow, we used a monolayer of TNF-α–activated HUVECs as model ECs. As TNF-activated HUVECs display only minute levels of functional lymphocyte chemokines on their apical surface 23, the monolayer was overlaid with the pleiotropic lymphocyte chemokine SDF-1α 24. PBTL capture by and rolling on TNF-activated HUVECs was largely mediated by both E-selectin and VCAM-1 (Fig. 1 A, top). Surprisingly, EC-bound SDF-1 could dramatically augment the frequency of cells initiating primary capture events (tethers) to TNF-activated HUVECs, in addition to its ability to stimulate firm integrin-dependent arrest of PBTLs already captured and rolling on the ECs (Fig. 1 A). SDF-1 also increased by twofold the frequency of VLA-4–dependent PBTL capture by selectin-blocked TNF-activated HUVECs (Fig. 1 A, top), without altering VCAM-1 expression on these cells (data not shown). SDF-1 also dramatically increased PBTL tethering and firm arrest of lymphocytes on HUVECs activated with TNF-α for a prolonged period, which lacked endothelial selectin activity (Fig. 1 A, bottom, and data not shown). Cell surface–bound SDF-1 could also augment the frequency of PBTLs initiating primary capture events to VCAM-1–transfected CHO cells, and stimulated firm integrin-dependent arrest of nearly all lymphocytes captured on the cell monolayer (Fig. 1 B). Moreover, SDF-1 coimmobilized with purified VCAM-1 coated on polystyrene substrate enhanced PBTL tethering by more than fourfold, along with triggering rapid arrest of tethered lymphocytes on the adhesive substrate (Fig. 2 A). Complete blocking of chemokine-triggered or spontaneous PBTL tethering to VCAM-1 with β1 integrin mAb suggested an exclusive role for VLA-4, rather than the α4β7 integrin in SDF-1–triggered PBTL tethering to VCAM-1 (data not shown).


Subsecond induction of alpha4 integrin clustering by immobilized chemokines stimulates leukocyte tethering and rolling on endothelial vascular cell adhesion molecule 1 under flow conditions.

Grabovsky V, Feigelson S, Chen C, Bleijs DA, Peled A, Cinamon G, Baleux F, Arenzana-Seisdedos F, Lapidot T - J. Exp. Med. (2000)

Immobilized chemokines augment VLA-4–mediated capture and arrest of T lymphocytes to purified VCAM-1 under shear flow. (A) Frequency of PBTL tethers to purified sVCAM-1 coated at 1.5 μg/ml on polystyrene surface with functional or heat-inactivated SDF-1 (2 μg/ml). The different tether categories were determined in two fields at 1 dyn/cm2 and results are an average and range of each tether category. (B) Frequency and type of tethers formed by naive (CD45RA+) and memory (CD45RO+) subsets of peripheral blood CD3+ T lymphocytes interacting at 1 dyn/cm2 with sVCAM-1 (1.5 μg/ml) coated together with the indicated intact or heat-inactivated chemokines (each at 2 μg/ml). Chemokine-dependent augmentation in total tethering was highly significant (*P < 0.001 and 0.004, for experiments performed on the CD45RA+ CD45RO+ subsets, respectively). (C) Frequency of VLA-4–mediated tethers of PBTLs to sVCAM-1 coated at 0.5 μg/ml, alone, or with inactivated SDF-1, SDF-1, or the non-signaling SDF-1 mutant P2G 16 (each at 2 μg/ml), determined at 0.5 dyn/cm2. VCAM-1 coating density on all substrates was identical as verified by radioimmunodetermination. For PTX treatment, PBTLs were cultured for 15 h with 100 ng/ml of the toxin. In A and D, where indicated, PBTLs were pretreated with the VLA-4 blocking mAb, HP1/2 (10 μg/ml), and were perfused unwashed over the VCAM-1–bearing surfaces. Data in A–C are representative of four independent experiments.
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Related In: Results  -  Collection

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Figure 2: Immobilized chemokines augment VLA-4–mediated capture and arrest of T lymphocytes to purified VCAM-1 under shear flow. (A) Frequency of PBTL tethers to purified sVCAM-1 coated at 1.5 μg/ml on polystyrene surface with functional or heat-inactivated SDF-1 (2 μg/ml). The different tether categories were determined in two fields at 1 dyn/cm2 and results are an average and range of each tether category. (B) Frequency and type of tethers formed by naive (CD45RA+) and memory (CD45RO+) subsets of peripheral blood CD3+ T lymphocytes interacting at 1 dyn/cm2 with sVCAM-1 (1.5 μg/ml) coated together with the indicated intact or heat-inactivated chemokines (each at 2 μg/ml). Chemokine-dependent augmentation in total tethering was highly significant (*P < 0.001 and 0.004, for experiments performed on the CD45RA+ CD45RO+ subsets, respectively). (C) Frequency of VLA-4–mediated tethers of PBTLs to sVCAM-1 coated at 0.5 μg/ml, alone, or with inactivated SDF-1, SDF-1, or the non-signaling SDF-1 mutant P2G 16 (each at 2 μg/ml), determined at 0.5 dyn/cm2. VCAM-1 coating density on all substrates was identical as verified by radioimmunodetermination. For PTX treatment, PBTLs were cultured for 15 h with 100 ng/ml of the toxin. In A and D, where indicated, PBTLs were pretreated with the VLA-4 blocking mAb, HP1/2 (10 μg/ml), and were perfused unwashed over the VCAM-1–bearing surfaces. Data in A–C are representative of four independent experiments.
Mentions: To study chemokine modulation of PBTL adherence to endothelial cells (ECs) under flow, we used a monolayer of TNF-α–activated HUVECs as model ECs. As TNF-activated HUVECs display only minute levels of functional lymphocyte chemokines on their apical surface 23, the monolayer was overlaid with the pleiotropic lymphocyte chemokine SDF-1α 24. PBTL capture by and rolling on TNF-activated HUVECs was largely mediated by both E-selectin and VCAM-1 (Fig. 1 A, top). Surprisingly, EC-bound SDF-1 could dramatically augment the frequency of cells initiating primary capture events (tethers) to TNF-activated HUVECs, in addition to its ability to stimulate firm integrin-dependent arrest of PBTLs already captured and rolling on the ECs (Fig. 1 A). SDF-1 also increased by twofold the frequency of VLA-4–dependent PBTL capture by selectin-blocked TNF-activated HUVECs (Fig. 1 A, top), without altering VCAM-1 expression on these cells (data not shown). SDF-1 also dramatically increased PBTL tethering and firm arrest of lymphocytes on HUVECs activated with TNF-α for a prolonged period, which lacked endothelial selectin activity (Fig. 1 A, bottom, and data not shown). Cell surface–bound SDF-1 could also augment the frequency of PBTLs initiating primary capture events to VCAM-1–transfected CHO cells, and stimulated firm integrin-dependent arrest of nearly all lymphocytes captured on the cell monolayer (Fig. 1 B). Moreover, SDF-1 coimmobilized with purified VCAM-1 coated on polystyrene substrate enhanced PBTL tethering by more than fourfold, along with triggering rapid arrest of tethered lymphocytes on the adhesive substrate (Fig. 2 A). Complete blocking of chemokine-triggered or spontaneous PBTL tethering to VCAM-1 with β1 integrin mAb suggested an exclusive role for VLA-4, rather than the α4β7 integrin in SDF-1–triggered PBTL tethering to VCAM-1 (data not shown).

Bottom Line: We show here that immobilized chemokines can augment not only arrest but also earlier integrin-mediated capture (tethering) of lymphocytes on inflamed endothelium.Furthermore, when presented in juxtaposition to vascular cell adhesion molecule 1 (VCAM-1), the endothelial ligand for the integrin very late antigen 4 (VLA-4, alpha4beta1), chemokines rapidly augment reversible lymphocyte tethering and rolling adhesions on VCAM-1.Chemokines potentiate VLA-4 tethering within <0.1 s of contact through Gi protein signaling, the fastest inside-out integrin signaling events reported to date.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, The Weizmann Institute of Science, Rehovot, 76100 Israel.

ABSTRACT
Leukocyte recruitment to target tissue is initiated by weak rolling attachments to vessel wall ligands followed by firm integrin-dependent arrest triggered by endothelial chemokines. We show here that immobilized chemokines can augment not only arrest but also earlier integrin-mediated capture (tethering) of lymphocytes on inflamed endothelium. Furthermore, when presented in juxtaposition to vascular cell adhesion molecule 1 (VCAM-1), the endothelial ligand for the integrin very late antigen 4 (VLA-4, alpha4beta1), chemokines rapidly augment reversible lymphocyte tethering and rolling adhesions on VCAM-1. Chemokines potentiate VLA-4 tethering within <0.1 s of contact through Gi protein signaling, the fastest inside-out integrin signaling events reported to date. Although VLA-4 affinity is not altered upon chemokine signaling, subsecond VLA-4 clustering at the leukocyte-substrate contact zone results in enhanced leukocyte avidity to VCAM-1. Endothelial chemokines thus regulate all steps in adhesive cascades that control leukocyte recruitment at specific vascular beds.

Show MeSH
Related in: MedlinePlus