HUMAN BLOOD AND SYNOVIAL-FLUID NEUTROPHILS CULTURED IN-VITRO UNDERGO PROGRAMMED CELL-DEATH WHICH IS PROMOTED BY THE ADDITION OF SYNOVIAL-FLUID

Objective-To assess the influence of inflammatory synovial fluid (SF) on apoptosis of joint and blood neutrophils with particular reference to levels of colony stimulating factors (CSF) contained therein. Metho ds-Neutrophils were separated from fresh synovial fluid and from perip heral blood by density gradient centrifugation. Apoptosis was assayed by light microscope morphology and DNA degradation. CSFs were assayed using bone marrow bioassay and enzyme linked immunosorbent assays for granulocyte (G-) and granulocyte macrophage (GM-) CSF. Separated neutr ophils were cultured in vitro and exposed to: varying concentrations o f SF in which CSF levels were measured, recombinant G-CSF and GM-CSF, and hyaluronic acid control solutions. Numbers of apoptotic neutrophil s and CSF levels were also measured in fresh SF samples. Results-The a ddition of autologous or heterologous inflammatory SF to blood or join t cavity neutrophils cultured in vitro caused a significant dose depen dent increase in the percentage of cells becoming apoptotic with time as measured morphologically and confirmed by DNA degradation. The effe ct bore no relationship to levels of CSF in joint fluid, despite our f inding that GM-CSF produced inhibition of neutrophil apoptosis in vitr o. Conclusion-These data suggest that SF contains a factor or factors capable of directly or indirectly promoting neutrophil apoptosis and n ormally powerful enough to overcome the apoptosis inhibiting effects o f cytokines such as GM-CSF at concentrations usually found in inflamma tory synovial fluids.