title: Increased resistance of PIG-A- bone marrow progenitors to tumor necrosis factor a and interferon gamma: possible implications for the in vivo dominance of paroxysmal nocturnal hemoglobinuria clones.
abstract: OBJECTIVE
  Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal disorder due to a PIG-A gene mutation, resulting in deficient expression of GPI-anchored-proteins. Both immune-mediated suppression of hematopoiesis and cytokine alterations have been reported in aplastic anemia, a disease closely related to PNH whereas no data are available on PNH itself. The aim of this study was to investigate the effect of exogenous cytokines on clonogenic activity in PNH.
  
  
  METHODS
  We evaluated burst-forming units-erythroid (BFU-E) and colony-forming units-granulocyte-macrophage (CFU-GM) in bone marrow mononuclear cells (BMMC) from 5 PNH patients and 5 controls, alone or in the presence of transforming-growth-factor (TGF)-beta, interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, and specific antibodies. Molecular analysis of the PIG-A gene was performed by polymerase chain reaction (PCR) and direct sequencing on every single colony.
  
  
  RESULTS
  Patients' cells showed less clonogenic activity than did control cells. In PNH, addition of TGF-beta inhibited both BFU-E and CFU-GM; IFN-gamma and TNF-alpha inhibited BFU-E alone. In patients cytokines modulated normal and mutated clones differently: TGF-b reduced the number of PIG-A- and PIG-A+ colony-forming-cells (CFC), whereas TNF-alpha and IFN-gamma reduced PIG-A+ CFC only. BMMC from patients showed higher TGF-beta production than did BMMC from controls.
  
  
  CONCLUSIONS
  TGF-beta could contribute to the genesis of the unfavorable bone marrow microenvironment but does not seem to play a role in the in vivo dominance of PIG-A deficient cells. Mutated clones were more resistant to the inhibitory effects of IFN-gamma and TNF-alpha, suggesting that PNH cells may have a growth advantage in an unfavorable microenvironment.
authors.affiliation: Department of Hematology, IRCCS Ospedale Maggiore Policlinico di Milano, Milan, Italy. wbarcel@policlinico.mi.it
authors.forename: Wilma
authors.initials: W
authors.lastname: Barcellini
authors.forename: Elisa
authors.initials: E
authors.lastname: Fermo
authors.forename: Francesca
authors.initials: F
authors.lastname: Guia Imperiali
authors.forename: Anna
authors.initials: A
authors.lastname: Zaninoni
authors.forename: Paola
authors.initials: P
authors.lastname: Bianchi
authors.forename: Carla
authors.initials: C
authors.lastname: Boschetti
authors.forename: Alberto
authors.initials: A
authors.lastname: Zanella
mesh_terms: D000328:Adult; D001854:Bone Marrow Cells; D016022:Case-Control Studies; D002478:Cells, Cultured; D002999:Clone Cells / Q000187:drug effects / Q000473:pathology; D016207:Cytokines / Q000494:pharmacology; D004351:Drug Resistance; D005260:Female; D006412:Hematopoietic Stem Cells / Q000737:chemistry / Q000187:drug effects*; D006457:Hemoglobinuria, Paroxysmal / Q000473:pathology*; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology*; D008297:Male; D008565:Membrane Proteins / Q000032:analysis / Q000172:deficiency / Q000235:genetics*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D016207:Cytokines; D008565:Membrane Proteins; D014409:Tumor Necrosis Factor-alpha; C080086:phosphatidylinositol glycan-class A protein; D007371:Interferon-gamma