Journal of Leukocyte Biology Volume 52, August 1992 183 The myeloid leukemia cell line HL-60 produces a factor that induces chloride secretion in cultured epithelial cells Sean P Colgan, Jeffrey B. Matthews, Charles A. Parkos, Charlene DeIp, Christopher S. Awtrey, and James L. Madara Division of Gastrointestinal Pathology, Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School; Department of Surgery, Beth Israel Hospital and Harvard Medical School; and the Harvard Digestive Diseases Center, Boston, Massachusetts Abstract: When activated, polymorphonuclear leuko- cytes (PMNs) produce a small soluble factor, termed neutrophil-derived secretagogue (NDS), that elicits dcc- trogenic Cl secretion - the transport event responsible for hydration of mucosal surfaces. Work toward purifica- tion of this factor has been hampered by variability in ac- tivity of PMN-derived NDS. Using a human-derived in- testinal epithelial cell line (T84) that serves as a model for studies of the regulation of electrogenic Cl secretion, we find that the human promyelocytic leukemia cell line HL-60 secretes a factor with NDS-like activity. Buffer conditioned by HL-60 cells (1O cells/ml for 1 h), when applied to T84 monolayers grown on permeable supports and analyzed by routine electrophysiological techniques, elicited a short-circuit current (I, ) of 11.7 ± 1.02 zA/cm2 This short-circuit current was sensitive to bumetanide, an inhibitor of the basolateral Na-K-2C1 cotransporter, and was dependent on the presence of chlo- ride in the assay buffers. Such data indicate that buffer conditioned by HL-60 cells stimulates electrogenic Cl secretion. Like NDS, the active factor in HL-60-condi- tioned buffer has a nominal molecular weight of less than 500 and was increased by activation of cells with phorbol ester. 1251 and 86Rb efflux assays confirmed that the secretagogue released by stimulated HL-60 cells, similar to PMN-derived NDS, preferentially stimulates opening of Cl rather than K channels in T84 cells. Lastly, release of NDS-like bioactivity increases when HL-60 cells are differentiated toward granulocytes compared to cells differentiated toward monocytes. J. Leukoc. Biol. 52: 183-187; 1992. Key Words: HL-60 cells polymorphonuclear leukocytes . neutrophil-derived secretagogue electrogenic C1 secretion INTRODUCTION Mucosal surfaces at anatomically distinct sites, such as air- way and intestine, share a common mechanism of hydration referred to as electrogenic Cl secretion [1]. During this process, transcellular Cl secretion is accompanied by paracellular Na and H2O flow that results in the transloca- tion of an isotonic salt solution to the mucosal surface. In the intestine, this Cl secretory process is stimulated in many inflammatory conditions and is the basis of secretory diar- rhea [2]. It is now clear that products released by polymor- phonuclear leukocytes (PMNs) migrating into the crypt lu- men stimulate electrogenic Cl secretion and thus contribute to the “flushing” of mucosal surfaces often observed in inflammatory states [3]. For example, we have previously shown [4] that activated PMNs release a small ( < 500 dal- tons), soluble, hydrophiic, acid- and heat-stable solute that elicits secretion in T84 cells - a human-derived intestinal cpithclial cell line now widely utilized as a model for study of the regulation of clcctrogenic Cl secretion [5]. Attempts to purify this neutrophii-dcrivcd secretagogue (NDS) await the identification of a source of NDS that would facilitate large-scale production. Here we report that a soluble com- pound similar in many features to NDS, including the ability to elicit an identical type of clectrogenic C1 secretion, is present in buffer conditioned with the human promyclocytic leukemia cell line HL-60. Like PMN-derivcd NDS, the NDS-like activity of HL-60-conditioncd supernatants is in- creased if cells are activated. Finally, production of HL-60 NDS-like activity is increased by cell treatments that arc known to result in partial differentiation of HL-60 cells toward granuiocytcs. MATERIALS AND METHODS HL-60 cells were grown in RPMI medium with 10% (v/v) heat-inactivated fetal calf serum and 2 mM L-giutaminc (Gibco) in 75-cm2 tissue culture flasks at 37#{176}C with 5% (JO2. Differentiation of HL-60 cells was induced by addition of 60 mM N,N-dimcthylformamidc (DMF, Sigma Chemical Co, St. Louis) for 3 days to induce granulocytes or i0 M phorbol myristate acetate (PMA, Sigma) for 4 days for in- duction of mononuclear cells as described elsewhere [6]. Cells were harvested as described previously [7, 8] and diluted to 107/ml in Hanks’ balanced salt solution (HBSS, Sigma) containing 10 mM HEPES, pH 7.4. Buffer condi- tioned with activated HL-60 cells was obtained by addition of 10 ’ M PMA to the above solution, incubation for 1 h at 37#{176}C,removal of cells by a 400g centrifugation, and collec- tion of supernatants. The NDS-containing supernatants were filtered through a molecular weight (MW) 1000 filter ( Amicon), unless otherwise noted, and stored at - 70#{176}CUn- til use. PMNs were isolated using 2% gelatin sedimentation as described before [9]. Buffer conditioned with PMN- derived NDS was obtained as described above for HL-60 cells and as described in detail elsewhere [4]. Confluent monoiaycrs of the human intestinal epithelial cell line T84 were grown on collagen-coated permeable sup- ports and maintained until steady-state resistance was Abbreviations: ANOVA, analysis of variance; DMF, N,N-dimethylfor- mamide; HBSS, Hanks’ buffered salt solution; MW, molecular weight; NDS, neutrophil-derived secretagogue; PMA, phorbol myristate acetate; PMN, polymorphonuclear leukocyte. Reprint requests: Sean P. Colgan, Department ofPathology, Brigham and Women’s Hospital, 20 Shattuck St., Thom Bldg., Room 1429, Boston, MA 02115. Received January 31, 1992; accepted March 30, 1992.