title: Antibody alone is not a stimulator of exocytosis of Weibel-Palade bodies from human endothelial cells.
abstract: BACKGROUND
  The mechanisms of antibody-mediated damage to allografts are not well understood. We have examined the effect of antibodies to human leukocyte antigens on secretion of von Willebrand factor (vWF) from endothelial cells (ECs).
  
  
  METHODS
  The effect of monoclonal antibodies (W6/32, L2, and L243), in the presence and absence of sublytic concentrations of complement, on the release of vWF from Weibel-Palade bodies (WPBs) in human umbilical vein ECs (HUVECs), human aortic ECs (HAECs), and human heart microvascular ECs (HHMECs) was investigated using biochemical and live-cell imaging. Fura-2-loaded ECs expressing the WPB marker proregion-enhanced green fluorescence protein were imaged simultaneously for intracellular Ca(2+) changes ([Ca(2+)](i)) and WPB exocytosis.
  
  
  RESULTS
  Stimulation of ECs with 1- or 10-µg/mL W6/32, L2, or L243 did not evoke significant vWF release above control IgG. In live-cell imaging studies, exposure of proregion-enhanced green fluorescence protein-expressing HAECs to physiologic saline, 10-µg/mL U9F4, or W6/32 alone for 5 to 10 min induced irregular (Ca(2+))(i)\ spiking but no WPB exocytosis. Histamine-evoked WPB exocytosis was not changed by preexposure of HAECs to physiologic saline, U9F4, or W6/32. Stimulation of HUVECs with sublytic complement concentrations evoked WPB exocytosis; however, the addition of W6/32 did not change the amount of vWF released.
  
  
  CONCLUSIONS
  Antibodies to human leukocyte antigen class I or II do not elicit significant WPB exocytosis or vWF secretion from ECs in the absence of exogenous complement.
authors.affiliation: Transplant Immunology, National Heart and Lung Institute, Imperial College, Harefield Hospital, Middlesex, UK.
authors.forename: Athinoula
authors.initials: A
authors.lastname: Meli
authors.forename: Tom
authors.initials: T
authors.lastname: Carter
authors.forename: Ann
authors.initials: A
authors.lastname: McCormack
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Hannah
authors.forename: Marlene L
authors.initials: ML
authors.lastname: Rose
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D042783:Endothelial Cells / Q000648:ultrastructure*; D005089:Exocytosis; D015395:Histocompatibility Antigens Class I / Q000276:immunology*; D000949:Histocompatibility Antigens Class II / Q000276:immunology*; D006801:Humans; D007518:Isoantibodies / Q000502:physiology*; D011817:Rabbits; D020302:Weibel-Palade Bodies / Q000378:metabolism*; D014841:von Willebrand Factor / Q000378:metabolism*
chemical_list: D015395:Histocompatibility Antigens Class I; D000949:Histocompatibility Antigens Class II; D007518:Isoantibodies; D014841:von Willebrand Factor; D002118:Calcium