The integrin alpha9beta1 mediates adhesion to activated endothelial cells and transendothelial neutrophil migration through interaction with vascular cell adhesion molecule-1 - PubMed (original) (raw)
The integrin alpha9beta1 mediates adhesion to activated endothelial cells and transendothelial neutrophil migration through interaction with vascular cell adhesion molecule-1
Y Taooka et al. J Cell Biol. 1999.
Abstract
The integrin alpha9beta1 has been shown to be widely expressed on smooth muscle and epithelial cells, and to mediate adhesion to the extracellular matrix proteins osteopontin and tenascin-C. We have found that the peptide sequence this integrin recognizes in tenascin-C is highly homologous to the sequence recognized by the closely related integrin alpha4beta1, in the inducible endothelial ligand, vascular cell adhesion mole-cule-1 (VCAM-1). We therefore sought to determine whether alpha9beta1 also recognizes VCAM-1, and whether any such interaction would be biologically significant. In this report, we demonstrate that alpha9beta1 mediates stable cell adhesion to recombinant VCAM-1 and to VCAM-1 induced on human umbilical vein endothelial cells by tumor necrosis factor-alpha. Furthermore, we show that alpha9beta1 is highly and selectively expressed on neutrophils and is critical for neutrophil migration on VCAM-1 and tenascin-C. Finally, alpha9beta1 and alpha4 integrins contribute to neutrophil chemotaxis across activated endothelial monolayers. These observations suggest a possible role for alpha9beta1/VCAM-1 interactions in extravasation of neutrophils at sites of acute inflammation.
Figures
Figure 1
Adhesion of α9- and mock-transfected SW480 and CHO cells to TNfn3RAA or VCAM-1. Flow cytometric evaluation of cell surface expression of integrin α9 on CHO cells (A), or SW480 cells (B). Open peaks represent fluorescence of mock-transfected cells and shaded peaks represent fluorescence of α9-transfected cells stained with anti-α9β1 antibody, Y9A2. α9- or mock-transfected CHO (C and E) or SW480 cells (D and F) were added to 96-well plates coated with a range of concentrations of TNfn3RAA (C and D) or VCAM-1/Ig (E and F). Cells were allowed to attach for 1 h, nonadherent cells were removed by centrifugation, and adherent cells were stained with crystal violet and quantified by measurement of absorbance at 595 nm. Data for a typical experiment are shown and are expressed as the mean (+ SD) of triplicate measurements for untreated α9-transfected cells (squares), untreated mock-transfected cells (circles), α9-transfected cells treated with Y9A2 (10 μg/ml; diamonds), and mock-transfected cells treated with Y9A2 (10 μg/ml; triangles). Similar results were obtained in four separate experiments.
Figure 2
Adhesion of α9- or mock-transfected SW480 cells to HUVE cells. (A) Confluent monolayers of HUVE cells were incubated for 24 h with medium alone (no activation), TNF-α (3 ng/ml), or IFN-γ (3 ng/ml). Fluorescently labeled α9- or mock-transfected SW480 cells were allowed to adhere to HUVE cell monolayers for 60 min in the presence or absence of the α9β1 blocking antibody Y9A2 (10 μg/ ml) or the VCAM-1 blocking antibody CD106 (5 μg/ml). Nonadherent cells were removed by gentle washing and the percent of adherent cells was calculated based on fluorescence. Data are presented as the mean (+ SD) of triplicate measurements. Similar results were obtained in two separate experiments. Flow cytometric evaluation of cell surface expression of VCAM-1 on HUVE cells treated with medium alone (B), TNF-α (3 ng/ml; C), or IFN-γ (3 ng/ml; D). Open peaks represent fluorescence of unstained HUVE cells and shaded peaks represent fluorescence of cells stained with the anti–VCAM-1 antibody, CD106.
Figure 3
Expression of α4 or α9 integrins on leukocytes. (A) Whole blood leukocytes were stained with control antibody E7P6 that recognizes αvβ6, an integrin not expressed on leukocytes (unshaded peaks), Y9A2 against α9β1, or 100226 against α4 (shaded peaks). Fluorescence of lymphocytes, neutrophils, eosinophils, and monocytes were analyzed separately by gating on each population on the basis of a plot of forward versus side scattering of light. Fluorescence of eosinophils was analyzed from separate atopic donor by gating on eosinophils based on light scattering and the absence of expression CD16. (B) Western blot with anti-α9 antiserum 1057 of lysates of human neutrophils that had been immunoprecipitated with anti-α9β1 antibody Y9A2 or the control antibody R6G9 against the irrelevant integrin αvβ6 (lane 2). The expected molecular mass of the α9 subunit (160 kD) is shown by the lefthand arrow. The position of molecular size marker (kD) is shown to the right.
Figure 4
Neutrophil migration on VCAM-1 or TNfn3RAA. Neutrophils were allowed to migrate on glass coverslips coated with: (A) 1% BSA or TNfn3RAA (10 μg/ml); or (B) VCAM-1 (10 μg/ml) or ICAM-1 (5 μg/ml), for 3 min in the presence or absence of FMLP (10 nM), and in the presence of: no antibody; α9β1 antibody, Y9A2 (10 μg/ml); α4 antibody, 100226 (10 μg/ml); antibody IB4 (20 μg/ml); or the combination of these antibodies. The percentage of migrating cells was determined by analyzing ≥40 cells from each of three microscopic fields, and is expressed as the mean (+ SD) of triplicate values from two separate experiments.
Figure 5
Transmigration of neutrophils across activated HUVE cell monolayers. Purified human neutrophils that had been incubated with no antibody or antibody to α9β1 (Y9A2, 10 μg/ml), α4β1 (100226, 10 μg/ml), β2 (IB4, 20 μg/ml), a combination of these antibodies, or human MHC (W6/32, 10 μg/ml) were added to the top chambers above microporous chambers containing confluent monolayers of HUVE cells that had been incubated with (A) or without TNF-α (3 ng/ml; B) for 24 h. DMEM containing FMLP (10 nM) or DMEM alone was added to the bottom chamber. After 3 h at 37°C in 5% CO2, neutrophils that had migrated across the monolayer were collected from the bottom chamber and counted. In additional chambers, untreated neutrophils were added to HUVE cells that had been preincubated for 15 min with antibody to VCAM-1 (CD106, 5 μg/ml). Data are expressed as the mean (+ SD) of quadruplicate measurements from two separate experiments.
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