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Ex vivo modelling of the formation of inflammatory platelet-leucocyte aggregates and their adhesion on endothelial cells, an early event in sepsis.

Authors
  • Alharbi, Azzah1, 2
  • Thompson, Jonathan P3
  • Brindle, Nicholas P4, 5
  • Stover, Cordula M6
  • 1 Department of Infection, Immunity and Inflammation, College of Life Sciences, University of Leicester, Leicester, LE1 9HN, UK.
  • 2 King Abdulaziz University, Jeddah, Saudi Arabia. , (Saudi Arabia)
  • 3 Department of Cardiovascular Sciences, Division of Anaesthesia, Critical Care and Pain Management, University of Leicester, Robert Kilpatrick Clinical Sciences Building, Leicester Royal Infirmary, Leicester, LE2 7LX, UK.
  • 4 Department of Cardiovascular Sciences, College of Life Sciences, University of Leicester, Leicester, LE1 9HN, UK.
  • 5 Department of Molecular & Cell Biology, College of Life Sciences, University of Leicester, Leicester, LE1 9HN, UK.
  • 6 Department of Infection, Immunity and Inflammation, College of Life Sciences, University of Leicester, Leicester, LE1 9HN, UK. [email protected]
Type
Published Article
Journal
Clinical and experimental medicine
Publication Date
Aug 01, 2019
Volume
19
Issue
3
Pages
321–337
Identifiers
DOI: 10.1007/s10238-018-0526-1
PMID: 30191349
Source
Medline
Keywords
Language
English
License
Unknown

Abstract

Septicaemia is an acute inflammatory reaction in the bloodstream to the presence of pathogen-associated molecular patterns. Whole blood stimulation assays capture endotoxin-induced formation of aggregates between platelets and leucocytes using flow cytometry. We wanted to assess extent of spontaneous aggregate formation in whole blood stimulation assays and compare the effects of endotoxin and heat-killed, clinically relevant, bacterial pathogens on aggregate formation and then on adhesion of aggregates to TNFα-stimulated endothelial cells. We found that endotoxin (from Escherichia coli or Salmonella enteritidis) was not a suitable stimulus to provoke platelet-leucocyte aggregates in vitro, as it did not further increase the extent of aggregates formed spontaneously in stasis of hirudin-anticoagulated blood. Specifically, whole blood samples stimulated with or without LPS produced aggregates with a mean surface area of 140.97 and 117.68 μm2, respectively. By contrast, incubation of whole blood with heat-killed Klebsiella pneumoniae or Staphylococcus aureus produced significantly enhanced and complex cellular aggregates (with a mean surface area of 470.61 and 518.39 μm2, respectively) which adhered more frequently to TNFα (and free fatty acid)-stimulated endothelial cells. These were reliably captured by scanning electron microscopy. Adhesion of cellular aggregates could be blocked by incubation of endothelial cells with a commercial P-selectin antibody and an angiopoietin-2 ligand trap. In conclusion, we have developed an in vitro method that models the acute inflammatory reaction in whole blood in the presence of sepsis-relevant bacterial pathogen surfaces.

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