Suppression of human IgE antibody forming cell responses by IL-6 Gary I. Kleiner, Dominick L. Auci, Altaf Shaikh, Seto M. Chice, and Helen G. Durkin Department of Pathology State University of New York Health Science Center at Brooklyn 168 Journai of Leukocyte Biology Volume 54, August 1993 BRIEF COMMUNICATION Abstract: To study the effects of cytokines on human IgE antibody forming cells (AFCs), log phase U266 myeloma cells (3 x 103/ml), which secrete immunoglobulin E (IgE), were cultured for 0-24 h with and without cyto- kine or with or without antibodies against various cyto- kines. The numbers of IgE AFCs were determined in ELISPOT assay. We found that interleukin-6 (IL-6) sup- pressed (to 95%) whereas anti-IL-6 increased (to 148%) the numbers of IgE AFCs and that both worked in a dose- dependent fashion. IL-4 and interferon-y (IFN-’y) also suppressed IgE AFC responses in a dose-dependent fashion. However, antibodies to these cytokines had no effect. In contrast, IFN-a increased (to fourfold) the num- bers of IgE AFCs in a dose-dependent fashion. The data are the first to show a suppressive effect of IL-6 on human IgE responses and may also suggest a role for IL-6 in the treatment of atopic disease. J. Leukoc. Biol. .54: 168-170; 1993. Key Words: allergy . IgE . cytokines ‘ IL-6 The search for isotype-specific regulators of immunoglobulin E (IgE) antibody forming cell (AFC) responses commands at- tention because these responses form the basis of atopic dis- ease in humans [1]. Earlier studies from our laboratory reported that oral administration of synthetic derivatives of bacterial peptidoglycan [muramyldipeptide (MDP), mura- butide (MB)] to sensitized mice at the peak of the hapten- specific IgE AFC response transiently suppressed these re- sponses in an isotype-specific and dose-dependent fashion [2]. The mechanisms by which MDP and MB exert these effects in vivo remain unknown. However, in those studies, IL-6, IL-4, and IFN-y also suppressed these responses whereas IFN-a and tumor necrosis factor a (TNF-a) aug- mented them [3]. Only IL-6 suppressed IgE AFC responses in an isotype-specific fashion. Although it is well recognized that IL-4 is required for the induction of IgE responses in mice [4] and humans [5] and that IFN-’y plays a role in sup- pressing these responses [6], the ability of these cytokines to directly regulate human IgE AFC responses has not been studied. We investigated the ability of cytokines (IL-6, IL-4, IFN-a, and IFN- y) and ofantibodies against these cytokines to regulate IgE AFCs. Cultures of human myoloma line U266 (ATCC Rockville, MD), which secretes IgE, were defined as log phase cultures when >20% of cells were in S phase as determined by propidium iodide staining of DNA followed by FACscan analysis using CycleTEST ” software (Becton Dickinson, San Jose, CA). Log phase cells (3 x 10’/mi) were cultured for 0-24 h in complete medium (RPMI containing heat macti- vated fetal calf serum, 10%; Gibco, Grand Island, NY; L- glutamine, 2 mM, Gibco; 2 mercaptoethanol, 50 M, Sigma Chemical Co., St. Louis, MO; and streptomycin, 20 j.tg/ml, Eli Lilly and Co., Indianapolis, IN) with or without various concentrations of cytokine. Human recombinant IL-6 (rIL-6) was obtained from Sandoz Inc. (Vienna, Austria); human rIL-4, from Gen- zyme Corp. (Boston, MA); and human nIFN-’y and human nIFN-a from Lee BioMolecular (San Diego, CA). The doses of cytokines were within 1 log of physiologic relevance (IL-6: <100 U/mi; IL-4: <100 U/ml; IFN- y: <200 U/ml; and IFN-a: <50 U/mi) according to bioassay data provided by the suppliers. Cytokines were used within 1 month of receipt. The enzyme-linked immunosorbent spot (ELISPOT) as- say was performed as follows: Plates coated with anti-IgE were prepared by adding 0.1 ml of goat IgG fraction anti-hu- man IgE (5.0 tg/weli; Miles Laboratories, Elkart, IN) to 96-well flat-bottom tissue culture plates (Millipore mutiscreen HA 45) and incubating overnight at 4#{176}C. The wells were then washed once with distilled water after which bovine serum albumin [0.1% in phosphate-buffered saline (PBS)] was added to each well (0.1 mi/well). The plates were incubated for 2 h at room temperature and washed once with PBS. Log phase cells (300 pen well) in complete medium with or without cytokine were added to anti-IgE-coated 96-well nitrocellulose-coated plates (0.1 mi/well). Cells were cultured for 0-24 h at 37#{176}Cin a humidified atmosphere of 7% CO2 in air. The wells were then washed three times with ice-cold PBS-Tween (0.1%, Tween 20; Aldrich, Milwaukee, WI) to en- sure that all cells were removed. Mouse monoclonal anti- body specific for human IgE (2 sg/ml, 0.1 mi/well; Dako, Carpintenia, CA) was added to appropriate wells and the plates were incubated overnight at 4#{176}C. The wells were then washed three times with PBS-Tween, after which alkaline phosphatase donkey anti-mouse IgE (1:1000, 0.1 ml; Cappei) was added and the plates were incubated for 2 h at room temperature. The welis were then washed three times with PBS-Tween and covered with substrate solution (0.1 ml). Substrate solution consisted of 5-bromo-4-chloro-3-indolyl- phosphate p-toluidine salt (BCIP)/nitroblue tetrazollum chloride (NBT) (1:1 M ratio; Gibco). ELISPOTs, representing the sites of antibody secretion by individuai AFCs, appeared on plates by 40 mm after sub- strate solution was added. The plates were then washed twice with distilled H20 and air dried. The individual ELISPOTs were counted by using a dissection microscope (16 x; Wild, Abbreviations: AFC, antibody forming cell; ELISPOT, enzyme-linked immunosorbent spot; IFN, interferon; IgE, immunoglobulin E; IL-6, inter- leukin 6; MB, murabutide; MDP, muramyldipeptide; PBS, phosphate- buffered saline; rIL-6, recombinant IL-6; TNF, tumor necrosis factor. Reprint requests: Dominick L. Auci, Department of Pathology, Box 25, State University of New York, Health Science Center at Brooklyn, 450 Clarkson Avenue, Brooklyn, NY 11203. Received February 18, 1993; accepted April 30, 1993.