title: Sphingomyelinase-induced adhesion of eryptotic erythrocytes to endothelial cells.
abstract: Eryptosis, the suicidal erythrocyte death, leads to cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Eryptotic erythrocytes adhere to the vascular wall by binding of phosphatidylserine to the CXC chemokine ligand 16 (CXCL16). Stimulators of eryptosis include increased cytosolic Ca(2+) activity, energy depletion, and activation of ceramide-producing sphingomyelinase. The present study explored whether sphingomyelinase triggers erythrocyte adhesion to endothelial cells. To this end, human erythrocytes were exposed for 6 h to bacterial sphingomyelinase (1-10 mU/ml) and phosphatidylserine exposure was estimated from fluorescent annexin-V-binding, cell volume from forward scatter in FACS-analysis, erythrocyte adhesion to human umbilical vein endothelial cells (HUVEC) from trapping of labeled erythrocytes in a flow chamber under flow conditions at arterial shear rates, and CXCL16 protein abundance utilizing Western blotting and FACS analysis of fluorescent antibody binding. As a result, sphingomyelinase (≥1 mU/ml) triggered cell shrinkage, phosphatidylserine exposure and erythrocyte adhesion to HUVEC, effects blunted by Ca(2+) removal. Adhesion was significantly blunted by phosphatidylserine-coating annexin-V (5 μl/ml), following addition of neutralizing antibodies against endothelial CXCL16 (4 μg/ml) and following silencing of the CXCL16 gene with small interfering RNA. Pretreatment of HUVEC with sphingomyelinase upregulated CXCL16 protein abundance. Six hours pretreatment of HUVEC with sphingomyelinase (10 mU/ml) or C6-ceramide (50 μM) augmented erythrocyte adhesion following a 30-min treatment with Ca(2+) ionophore ionomycin (1 μM) or following energy depletion by 48-h glucose removal. Thus exposure to sphingomyelinase or C6-ceramide triggers eryptosis followed by phosphatidylserine- and CXCL16-sensitive adhesion of eryptotic erythrocytes to HUVEC.
authors.affiliation: Physiologisches Institut, Eberhard-Karls-Universität Tübingen, Gmelinstr. 5, D-72076 Tübingen, Germany. florian.lang@uni-tuebingen.de
authors.forename: Majed
authors.initials: M
authors.lastname: Abed
authors.forename: Syeda T
authors.initials: ST
authors.lastname: Towhid
authors.forename: Sobuj
authors.initials: S
authors.lastname: Mia
authors.forename: Tatsiana
authors.initials: T
authors.lastname: Pakladok
authors.forename: Ioana
authors.initials: I
authors.lastname: Alesutan
authors.forename: Oliver
authors.initials: O
authors.lastname: Borst
authors.forename: Meinrad
authors.initials: M
authors.lastname: Gawaz
authors.forename: Erich
authors.initials: E
authors.lastname: Gulbins
authors.forename: Florian
authors.initials: F
authors.lastname: Lang
mesh_terms: D017304:Annexin A5 / Q000502:physiology; D057134:Antibodies, Neutralizing / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D002118:Calcium / Q000494:pharmacology; D061207:Calcium Ionophores / Q000494:pharmacology; D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology; D048429:Cell Size; D002478:Cells, Cultured; D002518:Ceramides / Q000494:pharmacology; D000075743:Chemokine CXCL16; D019743:Chemokines, CXC / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology; D004912:Erythrocytes / Q000187:drug effects* / Q000502:physiology; D020868:Gene Silencing; D005947:Glucose / Q000494:pharmacology; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects / Q000502:physiology; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D010718:Phosphatidylserines / Q000502:physiology; D051116:Receptors, Scavenger / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology; D013108:Sphingomyelin Phosphodiesterase / Q000494:pharmacology*
chemical_list: D017304:Annexin A5; D057134:Antibodies, Neutralizing; C423727:CXCL16 protein, human; D061207:Calcium Ionophores; D002518:Ceramides; D000075743:Chemokine CXCL16; D019743:Chemokines, CXC; D010718:Phosphatidylserines; D051116:Receptors, Scavenger; C101954:N-caproylsphingosine; D015759:Ionomycin; D013108:Sphingomyelin Phosphodiesterase; D005947:Glucose; D002118:Calcium