High density human neutrophils produce a transferable factor that delays apoptosis

• Main Author: Ahmad, Sharon Ameena
• Language: English
• Published: 2009
• Online Access: http://hdl.handle.net/2429/8103

Pilot experiments performed in this laboratory showed that there was a delay in neutrophil apoptosis in culture at high cell densities. Increasing cell density resulted in a logarithmic decline in the percentage of apoptotic neutrophils when cells were cultured at densities between 3xl0³ and l xl0⁷ cells/cm² (p<0.003, n=3). Investigation of this phenomenon resulted in the detection of a transferable factor produced by high density neutrophils, that was able to delay apoptosis in low density neutrophils when co-cultured. Various soluble mediators were studied to determine the identity of the transferable factor. ELISA assays performed on high density neutrophil-conditioned medium indicated that, of the cytokines tested (GM-CSF, IL-8, IL-6, IL-1β, and TNF-α), neutrophils were producing only IL-8 in significant quantities. Monoclonal neutralizing antibodies to the cytokines G M - CSF, G-CSF, and IL-8 added to neutrophil cultures were not successful in inhibiting the high density delay in neutrophil apoptosis, nor was the platelet-activating factor inhibitor, WEB 2170. The role of cell-cell contact and adhesion molecules CD18, CD11b, CD11a, and L - selectin, were also investigated as high neutrophil density suggests increased cell-cell contact. Blocking antibodies to these cell adhesion molecules added to neutrophil cultures had no effect on the density-dependent delay in neutrophil apoptosis. The CD18/ CD11b upregulator, fMLP, added at either high or low concentrations, also had no effect on neutrophil apoptosis at any density. These data show that there is a delay in neutrophil apoptosis at high cell densities, potentially involving a transferable factor produced by the high density neutrophils, and unlikely to involve intercellular interaction of CD8, CD11b, CD11a, or L-selectin. These findings have important implications for the study of neutrophils both in vitro and in vivo.