title: Supernatants from stored red blood cell (RBC) units, but not RBC-derived microvesicles, suppress monocyte function in vitro.
abstract: BACKGROUND
  We have previously shown that critically ill children transfused with red blood cells (RBCs) of longer storage durations have more suppressed monocyte function after transfusion compared to children transfused with fresher RBCs and that older stored RBCs directly suppress monocyte function in vitro, through unknown mechanisms. We hypothesized that RBC-derived microvesicles (MVs) were responsible for monocyte suppression.
  
  
  METHODS
  To determine the role of stored RBC unit-derived MVs, we cocultured monocytes with supernatants, isolated MVs, or supernatants that had been depleted of MVs from prestorage leukoreduced RBCs that had been stored for either 7 or 30 days. Isolated MVs were characterized by electron microscopy and flow cytometry. Monocyte function after coculture experiments was measured by cytokine production after stimulation with lipopolysaccharide (LPS).
  
  
  RESULTS
  Monocyte function was suppressed after exposure to supernatants from 30-day RBC units compared to monocytes cultured in medium alone (LPS-induced tumor necrosis factor-α production, 17,611 ± 3,426 vs. 37,486 ± 5,598 pg/mL; p = 0.02). Monocyte function was not suppressed after exposure to MV fractions. RBC supernatants that had been depleted of MVs remained immunosuppressive. Treating RBC supernatants with heat followed by RNase (to degrade protein-bound RNA) prevented RBC supernatant-induced monocyte suppression.
  
  
  CONCLUSIONS
  Our findings implicate soluble mediators of stored RBC-induced monocyte suppression outside of MV fractions and suggest that extracellular protein-bound RNAs (such as microRNA) may play a role in transfusion-related immunomodulation.
authors.affiliation: Division of Critical Care Medicine.|The Research Institute.|Division of Pulmonary and Critical Care, Department of Internal Medicine, The Ohio State University, Columbus, Ohio.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Muszynski
authors.affiliation: The Research Institute.
authors.forename: Justin
authors.initials: J
authors.lastname: Bale
authors.affiliation: The Research Institute.
authors.forename: Jyotsna
authors.initials: J
authors.lastname: Nateri
authors.affiliation: Department of Pathology, Nationwide Children's Hospital.
authors.forename: Kathleen
authors.initials: K
authors.lastname: Nicol
authors.affiliation: Division of Pulmonary and Critical Care, Department of Internal Medicine, The Ohio State University, Columbus, Ohio.
authors.forename: Yijie
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Division of Pulmonary and Critical Care, Department of Internal Medicine, The Ohio State University, Columbus, Ohio.
authors.forename: Valerie
authors.initials: V
authors.lastname: Wright
authors.affiliation: Division of Pulmonary and Critical Care, Department of Internal Medicine, The Ohio State University, Columbus, Ohio.
authors.forename: Clay B
authors.initials: CB
authors.lastname: Marsh
authors.affiliation: Division of Pulmonary and Critical Care, Department of Internal Medicine, The Ohio State University, Columbus, Ohio.
authors.forename: Mikhail A
authors.initials: MA
authors.lastname: Gavrilin
authors.affiliation: Division of Pulmonary and Critical Care, Department of Internal Medicine, The Ohio State University, Columbus, Ohio.
authors.forename: Anasuya
authors.initials: A
authors.lastname: Sarkar
authors.affiliation: Division of Pulmonary and Critical Care, Department of Internal Medicine, The Ohio State University, Columbus, Ohio.
authors.forename: Mark D
authors.initials: MD
authors.lastname: Wewers
authors.affiliation: Division of Critical Care Medicine.|The Research Institute.|Division of Pulmonary and Critical Care, Department of Internal Medicine, The Ohio State University, Columbus, Ohio.
authors.forename: Mark W
authors.initials: MW
authors.lastname: Hall
mesh_terms: D001793:Blood Preservation; D055252:Cell-Derived Microparticles / Q000276:immunology*; D002478:Cells, Cultured; D018920:Coculture Techniques; D003470:Culture Media / Q000494:pharmacology; D017077:Culture Media, Conditioned / Q000494:pharmacology*; D016207:Cytokines / Q000378:metabolism; D004912:Erythrocytes / Q000737:chemistry* / Q000276:immunology / Q000648:ultrastructure; D006358:Hot Temperature; D006801:Humans; D007165:Immunosuppression Therapy; D066298:In Vitro Techniques; D047589:Leukocyte Reduction Procedures; D008070:Lipopolysaccharides / Q000494:pharmacology; D009000:Monocytes / Q000276:immunology*; D012313:RNA / Q000097:blood; D012260:Ribonucleases / Q000494:pharmacology; D013997:Time Factors
chemical_list: D003470:Culture Media; D017077:Culture Media, Conditioned; D016207:Cytokines; D008070:Lipopolysaccharides; D012313:RNA; D012260:Ribonucleases