BACKGROUND: Heparan sulphate proteoglycans (HSPGs) are important participants in cell surface signalling and critical in controlling cell behaviour. They modulate inflammatory cell maturation and activation, leucocyte rolling, adhesion to endothelium as well as extravasation and chemotaxis. Whether eosinophil's function is affected has not yet been reported. OBJECTIVE: We investigated the effects of transgenic, recombinant anti-thrombin III and Kybernin P, an anti-thrombin III concentrate, as HSPG ligands on spontaneous and chemokine-triggered migration of normal eosinophils from human peripheral blood in modified Boyden chamber micropore filter assays. METHODS: Eosinophils from human peripheral blood were purified using magnetic antibody cell sorting. The signalling mechanisms required for anti-thrombin-dependent migration were studied using signalling enzyme blockers. Expression of HSPG core protein mRNA was studied by PCR. RESULTS: Pre-treatment of eosinophils with anti-thrombin III inhibited chemotaxis toward optimal concentrations of eotaxin or RANTES (regulated upon activation normal T cell expressed and activated). In the absence of the chemokines, direct exposure to gradients of anti-thrombin III stimulated eosinophil migration. The effects of anti-thrombin III were abolished by pre-treating cells with heparinase-1, chondroitinase, sodium chlorate and anti-syndecan-4 antibodies. Syndecan-4 gene expression in eosinophils was confirmed in PCR. In the presence of pentasaccharide, anti-thrombin III lost its effect on the cells. Functional responses were also abrogated by inhibition of protein kinase C, phosphatidylinositol 3-kinase and phosphodiesterase. CONCLUSION: Data indicate that anti-thrombin III affects eosinophil motility via the effects of its heparin-binding site on cell surface syndecan-4. Ligation of syndecan-4 with anti-thrombin III induces eosinophil migration and deactivates motility toward chemokines. These observations suggest that syndecan-4-dependent signalling may control eosinophil locomotion.
BACKGROUND: Heparan sulphate proteoglycans (HSPGs) are important participants in cell surface signalling and critical in controlling cell behaviour. They modulate inflammatory cell maturation and activation, leucocyte rolling, adhesion to endothelium as well as extravasation and chemotaxis. Whether eosinophil's function is affected has not yet been reported. OBJECTIVE: We investigated the effects of transgenic, recombinant anti-thrombin III and Kybernin P, an anti-thrombin III concentrate, as HSPG ligands on spontaneous and chemokine-triggered migration of normal eosinophils from human peripheral blood in modified Boyden chamber micropore filter assays. METHODS: Eosinophils from human peripheral blood were purified using magnetic antibody cell sorting. The signalling mechanisms required for anti-thrombin-dependent migration were studied using signalling enzyme blockers. Expression of HSPG core protein mRNA was studied by PCR. RESULTS: Pre-treatment of eosinophils with anti-thrombin III inhibited chemotaxis toward optimal concentrations of eotaxin or RANTES (regulated upon activation normal T cell expressed and activated). In the absence of the chemokines, direct exposure to gradients of anti-thrombin III stimulated eosinophil migration. The effects of anti-thrombin III were abolished by pre-treating cells with heparinase-1, chondroitinase, sodium chlorate and anti-syndecan-4 antibodies. Syndecan-4 gene expression in eosinophils was confirmed in PCR. In the presence of pentasaccharide, anti-thrombin III lost its effect on the cells. Functional responses were also abrogated by inhibition of protein kinase C, phosphatidylinositol 3-kinase and phosphodiesterase. CONCLUSION: Data indicate that anti-thrombin III affects eosinophil motility via the effects of its heparin-binding site on cell surface syndecan-4. Ligation of syndecan-4 with anti-thrombin III induces eosinophil migration and deactivates motility toward chemokines. These observations suggest that syndecan-4-dependent signalling may control eosinophil locomotion.
Authors: Sandeep Gopal; Samantha Arokiasamy; Csilla Pataki; James R Whiteford; John R Couchman Journal: Open Biol Date: 2021-02-10 Impact factor: 6.411