E-selectin stimulates tyrosine kinase-dependent adhesiveness of 2 Estegrins in PMN: Role of an unidentified receptor

Licia Totani, Antonio Piccoli, Nicola Martelli, Stefano Manarini, Dietmar Vestweber, Giovanni De Gaetano, Chiara Cerletti, Virgilio Evangelista

Research output: Contribution to journalArticle

Abstract

In this study, using soluble recombinant E-selectin-IgG chimera and E-selectin-transfected CHO cells (CHO-E) we tested the hypothesis that E-selectin promotes 2integrin function in PMN. PMN aggregation or adhesion to CHO-E were evaluated in sheared (1000 rpm) cell suspensions (PMN: CHO-E ratio=5) by optical counting and double color flow cytometry. Soluble E-selectin (1-20 g/ml) dose-dependently induced Mac-1-dependent PMN aggregation, that was accompanied by tyrosine phosphorylation of a protein of 110 kD (PI 10). Genistein (1-100 M) and the specific inhibitor of Src kinases PP1 (1-20 M) dose-dependently inhibited P110 phosphorylation and PMN aggregation. PMN did not adhere to untrasfected CHO cells while 251,5% of PMN adhered to 6010% of CHO-E, at 2 min of shear, by a mechanism 2-integrin dependent. Neuraminidase treatment prevented PMN recruitment. Phosphorylation of PI 10 accompanied PMN adhesion and PP1 blocked both PI 10 phosphorylation and adhesion. Among the selectin ligands, PSGL-1 and L-selectin can trigger 2-integrin activation. In our experimental conditions, neither blockade with specific antibodies (PL 1 and PL2 to PSGL1 and DREG-56 to L-selectin) nor chymotrypsin cleavage of these receptors, significantly modify PMN adhesion to CHO-E. Moreover it was not modified by the combination of PL1 and DREG-56 with Affi-6O, a rabbit policlonal antibody to ESL-1. Unexpectedly, H18/7 and l .2B6, two antibodies against the lectin domain of E-selectin, barely affected PMN adhesion to CHO-E or aggregation induced by soluble E-selectin, suggesting a lectin-site independent effect. PMN did not adhere to resting HUVEC while formed mixed cell conjugates, involving 822% of HUVEC and 761 % of PMN, after 2 min of shear with rIL-1 -stimulated HUVEC; that was accompanied by phosphorylation of PI 10. PMN recruitment was abolished by neuraminidase treatment and was reduced to 152%, 303% and 306,4% of the control by anti-CD 18, a combination of anti-CD 1 la and CD1 Ib antibodies, or PP1 (20 M) respectively . This study indicates that E-selectin binding to sialylated unidentified receptor(s), promotes Src-kinase dependent function of 2 integrins. This mechanism may be relevant for PMN recruitment by the endothelium.

Original languageEnglish
JournalBlood
Volume96
Issue number11 PART II
Publication statusPublished - 2000

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Adhesiveness
E-Selectin
Protein-Tyrosine Kinases
Phosphorylation
Adhesion
Agglomeration
Integrins
L-Selectin
src-Family Kinases
CHO Cells
Antibodies
Neuraminidase
Lectins
Proteins
Selectins
Flow cytometry
Genistein
Chymotrypsin
Endothelium
Tyrosine

ASJC Scopus subject areas

  • Hematology

Cite this

Totani, L., Piccoli, A., Martelli, N., Manarini, S., Vestweber, D., De Gaetano, G., ... Evangelista, V. (2000). E-selectin stimulates tyrosine kinase-dependent adhesiveness of 2 Estegrins in PMN: Role of an unidentified receptor. Blood, 96(11 PART II).

E-selectin stimulates tyrosine kinase-dependent adhesiveness of 2 Estegrins in PMN : Role of an unidentified receptor. / Totani, Licia; Piccoli, Antonio; Martelli, Nicola; Manarini, Stefano; Vestweber, Dietmar; De Gaetano, Giovanni; Cerletti, Chiara; Evangelista, Virgilio.

In: Blood, Vol. 96, No. 11 PART II, 2000.

Research output: Contribution to journalArticle

Totani, L, Piccoli, A, Martelli, N, Manarini, S, Vestweber, D, De Gaetano, G, Cerletti, C & Evangelista, V 2000, 'E-selectin stimulates tyrosine kinase-dependent adhesiveness of 2 Estegrins in PMN: Role of an unidentified receptor', Blood, vol. 96, no. 11 PART II.
Totani, Licia ; Piccoli, Antonio ; Martelli, Nicola ; Manarini, Stefano ; Vestweber, Dietmar ; De Gaetano, Giovanni ; Cerletti, Chiara ; Evangelista, Virgilio. / E-selectin stimulates tyrosine kinase-dependent adhesiveness of 2 Estegrins in PMN : Role of an unidentified receptor. In: Blood. 2000 ; Vol. 96, No. 11 PART II.
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abstract = "In this study, using soluble recombinant E-selectin-IgG chimera and E-selectin-transfected CHO cells (CHO-E) we tested the hypothesis that E-selectin promotes 2integrin function in PMN. PMN aggregation or adhesion to CHO-E were evaluated in sheared (1000 rpm) cell suspensions (PMN: CHO-E ratio=5) by optical counting and double color flow cytometry. Soluble E-selectin (1-20 g/ml) dose-dependently induced Mac-1-dependent PMN aggregation, that was accompanied by tyrosine phosphorylation of a protein of 110 kD (PI 10). Genistein (1-100 M) and the specific inhibitor of Src kinases PP1 (1-20 M) dose-dependently inhibited P110 phosphorylation and PMN aggregation. PMN did not adhere to untrasfected CHO cells while 251,5{\%} of PMN adhered to 6010{\%} of CHO-E, at 2 min of shear, by a mechanism 2-integrin dependent. Neuraminidase treatment prevented PMN recruitment. Phosphorylation of PI 10 accompanied PMN adhesion and PP1 blocked both PI 10 phosphorylation and adhesion. Among the selectin ligands, PSGL-1 and L-selectin can trigger 2-integrin activation. In our experimental conditions, neither blockade with specific antibodies (PL 1 and PL2 to PSGL1 and DREG-56 to L-selectin) nor chymotrypsin cleavage of these receptors, significantly modify PMN adhesion to CHO-E. Moreover it was not modified by the combination of PL1 and DREG-56 with Affi-6O, a rabbit policlonal antibody to ESL-1. Unexpectedly, H18/7 and l .2B6, two antibodies against the lectin domain of E-selectin, barely affected PMN adhesion to CHO-E or aggregation induced by soluble E-selectin, suggesting a lectin-site independent effect. PMN did not adhere to resting HUVEC while formed mixed cell conjugates, involving 822{\%} of HUVEC and 761 {\%} of PMN, after 2 min of shear with rIL-1 -stimulated HUVEC; that was accompanied by phosphorylation of PI 10. PMN recruitment was abolished by neuraminidase treatment and was reduced to 152{\%}, 303{\%} and 306,4{\%} of the control by anti-CD 18, a combination of anti-CD 1 la and CD1 Ib antibodies, or PP1 (20 M) respectively . This study indicates that E-selectin binding to sialylated unidentified receptor(s), promotes Src-kinase dependent function of 2 integrins. This mechanism may be relevant for PMN recruitment by the endothelium.",
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T2 - Role of an unidentified receptor

AU - Totani, Licia

AU - Piccoli, Antonio

AU - Martelli, Nicola

AU - Manarini, Stefano

AU - Vestweber, Dietmar

AU - De Gaetano, Giovanni

AU - Cerletti, Chiara

AU - Evangelista, Virgilio

PY - 2000

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N2 - In this study, using soluble recombinant E-selectin-IgG chimera and E-selectin-transfected CHO cells (CHO-E) we tested the hypothesis that E-selectin promotes 2integrin function in PMN. PMN aggregation or adhesion to CHO-E were evaluated in sheared (1000 rpm) cell suspensions (PMN: CHO-E ratio=5) by optical counting and double color flow cytometry. Soluble E-selectin (1-20 g/ml) dose-dependently induced Mac-1-dependent PMN aggregation, that was accompanied by tyrosine phosphorylation of a protein of 110 kD (PI 10). Genistein (1-100 M) and the specific inhibitor of Src kinases PP1 (1-20 M) dose-dependently inhibited P110 phosphorylation and PMN aggregation. PMN did not adhere to untrasfected CHO cells while 251,5% of PMN adhered to 6010% of CHO-E, at 2 min of shear, by a mechanism 2-integrin dependent. Neuraminidase treatment prevented PMN recruitment. Phosphorylation of PI 10 accompanied PMN adhesion and PP1 blocked both PI 10 phosphorylation and adhesion. Among the selectin ligands, PSGL-1 and L-selectin can trigger 2-integrin activation. In our experimental conditions, neither blockade with specific antibodies (PL 1 and PL2 to PSGL1 and DREG-56 to L-selectin) nor chymotrypsin cleavage of these receptors, significantly modify PMN adhesion to CHO-E. Moreover it was not modified by the combination of PL1 and DREG-56 with Affi-6O, a rabbit policlonal antibody to ESL-1. Unexpectedly, H18/7 and l .2B6, two antibodies against the lectin domain of E-selectin, barely affected PMN adhesion to CHO-E or aggregation induced by soluble E-selectin, suggesting a lectin-site independent effect. PMN did not adhere to resting HUVEC while formed mixed cell conjugates, involving 822% of HUVEC and 761 % of PMN, after 2 min of shear with rIL-1 -stimulated HUVEC; that was accompanied by phosphorylation of PI 10. PMN recruitment was abolished by neuraminidase treatment and was reduced to 152%, 303% and 306,4% of the control by anti-CD 18, a combination of anti-CD 1 la and CD1 Ib antibodies, or PP1 (20 M) respectively . This study indicates that E-selectin binding to sialylated unidentified receptor(s), promotes Src-kinase dependent function of 2 integrins. This mechanism may be relevant for PMN recruitment by the endothelium.

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