Authentic 17 kDa tumour necrosis factor alpha is synthesized and released by canine mast cells and up-regulated by stem cell factor

Background Previous studies have suggested varying molecular weights for mast cell derived tumour necrosis factor alpha (TNF alpha) and little data exist upon the factors which may regulate the control of this cytokine in these cells. Objective To determine the molecular weight of canine mastocytoma-derived TNF alpha, to determine whether it is pre-formed within the granule and whether its expression could be up-regulated by stem cell factor (SCF). Methods Molecular sizing was assessed by immunoblot. The cellular localization of the TNF alpha war determined by immunocytochemistry before and after stimulation by A23187 and passive sensitization. Subcellular localization was performed by immunogold immunocytochemistry,Changes in the level of mastocytoma mRNA for TNF alpha in response to stimulation with SCF or fibroblast conditioned media for up to 12 weeks were studied using Northern analysis and changes in the level of TNF alpha protein expression on immunoblot and immunocytochemistry. Results Mast cells contained authentic 17 kDa TNF alpha as identified by immunoblotting. Immunocytochemical studies demonstrated preformed TNF alpha which was released by stimulation with antigen after passive sensitization, or by the calcium ionophore A23187. Further confirmation of the preformed nature of this TNF alpha was provided by immunogold electron microscopy which localized this cytokine to the granule of the inactive mast cell. Northern blotting revealed a constitutive message for TNF alpha, which increased in response to fibroblast conditioned media (FCM) and to recombinant human SCF. Immunocytochemical studies of mast cells cultured long-term with FCM or with recombinant SCF showed increased expression of TNF alpha over the course of 12 weeks incubation with these stimuli. Conclusion Mastocytoma derived-TNF alpha is a preformed, granule-associated 17 kDa cytokine which is released on stimulation with A23187 or passive sensitization. It is up-regulated by stem cell factor and by FCM over the course of 12 weeks.