l-:.\p Dermaiid t994: .<: t76 /,SW Prinied in Dennuirk • .til rlgliis reserved Copyright •( Munksgaard 1994 Expmmenbd Dermatology ISSN W9H6-6 70? TNFa-induced activation of eosinophil oxidative metabolism and morphology Comparison with IL-5* Zeck-Kapp G, Czech W, Kapp A. TNFc/.-induced activatioti of eosinophil oxidative tnetabolism and tnotphology Cotnparison with IL-5. Exp Det-tnatol 1994: 3: 176-188. c Munksgaard, 1994 Abstract: Hutnati deinial tnasf cells arc capable of releasing cyfokines, particularly prelortned TNFa, t.tpon appropriate stitnulafion. Masf eell activatioti //; vivo was shown fo be assoeiafed with ati inllux and aetivafion of innatntnafory cells, initially PMN (polytnorphonuclear neufrophilic granuloeytes) theti eosinophils. In order to learn tnote about the tnechan- isms by whieh TNFa is capable of activating eosinophils, in the presetit study the effect of TNFa on morphology and function of highly purified tioftnal eosinophils (>95'Vi.) was examined. As estitnated by transmission and scanning electron tiiicroscopy, TNFa-stitnulated eosinophils appeared to be strictly adheretit atid Hattened exhibiting a characteristie "hetiii- spheric" shape. TNFa indueed a dose-dependent, long-lasting produetioti of reactive oxygen species as measured by lucigenin-dependetif chetni- lutnitiescenee (CL), eveti at a concentration of 0.001 U/tnl. The tnaximal response upon stitnulafion wifh TNFa, however, was significantly lower fhan optitnal effects induced by IL-5. TNFa-induced respotises were completely inhibited by cytoehalasin B atid staurosporin, and partially blocked by pertussis toxin. Separation of eosinophils by discontinuous density gradients revealed the existence of at least two liypodense eositio- phil populations wifh a distinct susceptibility to stitnulation with TNFa. Based on funetional assay systetns, iti eontrast to a significant exfraeellu- lar, only a stnall intraeellular H^OT produetion was detected. Accordingly, HiOi production, deteeted by an ultrastructural technique, was observed only on the outer surface of the plastna tnembrane in the contact zones in between adjaeent eells. Fxfracellular as well as infracellular producfion of HiO; was eotnplefely inhibifed by eytochalasiti EJ. TNFa-indueed acti- vation of eosinophils is most probably tnediated by binding fo fhe 55 kD and the 75 kD TNF-receptor since both receptor molecules could be deteeted by FACS analysis and itntnune electron tnieroseopy using recep- tor-speeifie antibodies. However, in eontrast to its effect on eosinophil oxidafive respotise, TNFa did tiot induce fhe relea.se of sigtiiftcanf coticeti- trations of eosinophil eationie protein or eosinophil peroxidase in super- tiatanfs of cytokine-stimulated eosinophils, as detected by functional as well as immunological assay systems. These results clearly indieate that TNFa represenfs a potent eosinophil-activating cyfokine which may be of relevance in the allergie inllatntiiatory respotise. Gahriele Zeck-Kapp, Wolfgang Czech and Alexander Kapp Depatttnent ot Dermatology. University of Fteiburg, Germany Key wotds: eosinophils - cytokines - interleukin- 5 - TNFa - reactive oxygen species - hydro- gen peroxide - electron microscopy Professor A, Kapp. MD, Department ot Dermatology. Universify of Freibtirg, Haupfstr. 7, D-79104 Freiburg, Germany Accepted for publication f5 April 1994 * The paper was presented iti part at 19fh Sytnposium of the Collegium Internationale Allei'gologieum. "Chemical mediators and eellular intetactions in clinical immunology". Capti, 1992, and the XVth Congress of the European Academy of Allergolo- gy and Clinical Immunology, f'aris. f992. Introduction Mast cells are a heterogenous populatioti of proin- flammatory eells distributed particularly at the mu- cosal surfaces and within eonnective tissues (1, 2). Besides their involvement in host tesponses to parasites, non-specific itillammatioti, angiogenesis. 176