TNFα‐induced activation of eosinophil oxidative metabolism and morphology ‐ Comparison with IL‐5*

Abstract Human dermal mast cells are capable of releasing cytokines, particularly preformed TNFα, upon appropriate stimulation. Masl cell activation in vivo was shown to be associated with an influx and activation of inflammatory cells, initially PMN “polymorphonuclear neutrophilic granulocytes” then eosinophils. In order to learn more about the mechanisms by which TNFα is capable of activating eosinophils, in the present study the effect of TNFα on morphology and function of highly purified normal eosinophils “≥95%” was examined. As estimated by transmission and scanning electron microscopy, TNFα‐stimulated eosinophils appeared to be strictly adherent and flattened exhibiting a characteristic “hemispheric” shape. TNFα induced a dose‐dependent, long‐lasting production of reactive oxygen species as measured by lucigenin‐dependent chemiluminescence (CL). even at a concentration of 0.001 U/ml. The maximal response upon stimulation with TNFα, however, was significantly lower than optimal effects induced by IL‐5. TNFα‐induced responses were completely inhibited by cytochalasin B and staurosporin, and partially blocked by pertussis toxin. Separation of eosinophils by discontinuous density gradients revealed the existence of at least two hypodense eosinophil populations with a distinct susceptibility to stimulation with TNFα. Based on functional assay systems, in contrast to a significant extracellular, only a small intracellular H2O2: production was detected. Accordingly, H2O2 production, detected by an ultrastructural technique, was observed only on the outer surface of the plasma membrane in the contact zones in between adjacent cells. Extracellular as well as intracellular production of H2O2; was completely inhibited by cytochalasin B. TNFα‐induced activation of eosinophils is most probably mediated by binding to the 55 kD and the 75 kD TNF‐receptor since both receptor molecules could be detected by FACS analysis and immune electron microscopy using receptor‐specific antibodies. However, in contrast to its effect on eosinophil oxidative response, TNFα did not induce the release of significant concentrations of eosinophil cationic protein or eosinophil peroxidase in supernatants of cytokine‐stimulated eosinophils, as detected by functional as well as immunological assay systems. These results clearly indicate that TNFα represents a potent eosinophil‐activating cytokine which may be of relevance in the allergic inflammatory response.