CELLULAR IMMUNOLOGY 122,405-415 (1989) A Flow Cytometric and lmmunofluorescence Microscopic Study of Tumor Necrosis Factor Production and Localization in Human Monocytes EVA HOFSLI,’ ODDMUND BARRE,~ UNNI NONSTAD, AND TERJE ESPEVIK Institute of Cancer Research, University of Trondheim, N-7006 Trondheim. Norway Received September 28,19X3; acceptedMay 7, I989 The production and localization of tumor necrosis factor (TNF) in human monocytes were investigated by using monoclonal and polyclonal antibodies against recombinant human TNF together with flow cytometry and immunofluorescence microscopy. Lipopolysaccharide (LPS) induced a rapid and transient accumulation of TNF in perinuclear vesicles which was detected 20 min after the addition of LPS. The fluorescence intensity of the vesicles peaked at 40 min of LPS exposure, concomitantly with the release of TNF into the medium. Thus, our results indi- cate that the secretion of TNF is typical for secretory proteins as it involves passage through the secretory apparatus. Additional studies demonstrated that plasma membrane-associated TNF could not be detected in live monocytes not exposed to LPS. However, alter 90 min with LPS, a small population of monocytes expressed membrane-associated TNF, and by 24 hr approxi- mately 50% of the monocytes displayed TNF on the plasma membrane. Furthermore, our re- sults indicate that plasma membrane-associated TNF does not represent released TNF bound back to its own receptor. Thus, our findings support the view that TNF exists as a surface trans- membrane protein in LPS-stimulated monocytes. 0 1989 Academic PESS, IX. INTRODUCTION Tumor necrosis factor (TNF) (also called TNF-cz or cachectin) was initially de- scribed as an antitumor activity present in the serum of endotoxin-treated mice (1). It is now known that, in addition to its direct cytotoxic/cytostatic effect against tumor cells, TNF can expressmultiple immunomodulatory activities (2-6). Monocytes pro- duce large quantities of TNF in response to lipopolysaccharide (LPS), which seems to stimulate both translation and transcription of TNF-mRNA (7). Previous studies have shown that TNF activity is associated with the plasma membrane of activated monocytes (8), and that TNF may exist as a 26-kDa cell surface transmembrane ’ To whom correspondence should be addressed. ’ Present address: European Molecular Biology Laboratory, Postfact 10.2209, D-6900 Heidelberg, Ger- many. 3Abbreviations used: TNF, tumor necrosis factor; rTNF, recombinant human tumor necrosis factor; rLT, recombinant human lymphotoxin; LPS, lipopolysaccharide; nRIgG, normal rabbit IgG; FITC, fluo- rescein isothiocyanate. 405 0008~8749189 $3.00 Copyright 0 1989 by Academic Press,Inc. All rights of reproduction in any form reserved.