Cholesterol synthesis in polyclonally activated cytotoxic lymphocytes and its requirement for differentiation and proliferation ( T cells / plasma membrane / 25 - hydroxycholesterol / compactin / 3 - hydroxy - 3 - methylglutaryl - CoA reductase )

The kinetics of sterol synthesis and DNA synthesis in polyclonally activated, concanavalin A-stimulated spleen cell cultures were analyzed. Inhibition of DNA synthesis by 1-f-Darabinofuranosylcytosine (Ara-C) did not abrogate the formation of cytotoxic effector cells. However, inhibition of sterol synthesis by 25-hydroxycholesterol inhibited formation of cytotoxic effector cells as well as cellular proliferation. The inhibition of cytotoxicity correlated well with the dose of 25-hydroxycholesterol administered and was dependent on the time of administration. The agent had to be present when sterol synthesis occurred normally during the time lapse before DNA synthesis began. Compactin had the same effect as 25-hydroxycholesterol. The effects of inhibition of sterol biosynthesis on cytotoxicity could be counteracted by addition of cholesterol-containing liposomes. Based on these experiments, the links between proliferation and differentiation in lymphocytes are discussed. Lymphocytes stimulated by mitogens such as concanavalin A (Con A) simulate to some degree a synchronized cell population during the first proliferative cycle after stimulation (1-3). Many metabolic phases have been demonstrated to occur during this activation, among them being a distinct cycle of sterol synthesis (4-6), a cycle ofcAMP synthesis (7), and ultimately the S phase ofDNA synthesis (4, 5). Recently the important observation was made that one or more subpopulations of lectin-stimulated splenic lymphocytes differentiate into cytotoxic effector cells. This cytotoxicity appeared to be polyclonal because its detection required a ligand, such as phytohemagglutinin (PHA) (8). MacDonald and Lees (9) subsequently showed that these cells differentiated into cytotoxic lymphocytes, even ifDNA synthesis was completely inhibited. In this paper we demonstrate, using specific inhibitors of sterol biosynthesis, that the polyclonal induction of naive, splenic lymphocytes into differentiated cytotoxic lymphocytes calls for the synthesis ofcholesterol as an absolute requirement, whether DNA synthesis is inhibited or not. We will use this experimental model system to discuss some aspects of the relationship of proliferation and differentiation in immunocompetent cell populations. Our experiments result in the hypothesis that proliferation is not a prerequisite for differentiation but rather appears to be controlled by the latter in normal tissues (as opposed to uncontrolled proliferation in transformed cells) and merely serves to amplify the product of differentiation. MATERIALS AND METHODS Chemicals. Bactophytohemagglutinin M (PHA-M) was purchased from Difco. The hydrochloride salt of 1-13-D-arabinofuranosylcytosine (Ara-C) was purchased from Calbiochem, Con A from Miles, and a-methyl D-mannoside from Sigma. Sterols were purchased from Steraloids (Wilton, NH), recrystallized from methanol three times (10), dissolved in absolute ethanol, and added to Dulbecco's modified Eagle's medium (DME medium) containing 5% bovine serum albumin (Pentex, Miles) (11). Compactin (ML-236B), an allosteric inhibitor of 3-hydroxy3-methylglutaryl-CoA reductase (NADPH) [HMG-CoA reductase; mevalonate:NAPD' oxide reductase (CoA-acylating), EC 1.1.1.34] isolated from Penicillium citrinum, was obtained from A. A. Kandutsch, who received it as a gift from Akira Endo, Tokyo Noko University, Tokyo, Japan. A stock solution of 10 mg in 1 ml of absolute ethanol containing 1% NaOH was diluted appropriately with DME medium prior to addition to the cell cultures. Filipin U-5956 (no. 8393-DEG-11-8), apolyene isolated from Streptomyces fillipineasis, was obtained as a gift from Joseph E. Grady (Upjohn). It was unpacked and dissolved in the dark in absolute ethanol and then further diluted in phosphate-buffered saline before it was added to the cultures. Spleen Cell Cultures. Spleens ofC57BL/6J mice (The Jackson Laboratory) were aseptically removed and placed into DME medium. Cell suspensions were prepared with a Dounce tissue homogenizer as described (12) and transferred into flat-bottomed 16-mm wells (COSTAR) in 2 ml of DME medium supplemented with 5% (vol/vol) fetal bovine serum, 50 AuM 2-mercaptoethanol, additional amino acids (12), and Con A at 2 pug/ ml. Each culture well contained 5-7 X 10' cells. Cultures were maintained in humidified 5% CO2/95% air. Prior to testing for cytolytic activity, the cells were washed once in DME medium containing 50 mM a-methyl D-mannoside and 5% fetal bovine serum and then were incubated in the same medium for 30 min before being used for the cytotoxic assay (8, 9). Target Cells. P815 mastocytoma cells (EG & G Mason Research Institute, Worcester, MA) were maintained in DME medium supplemented with 5% fetal bovine serum and were labeled prior to use as targets with 100 tkCi of Na251CrO4 (200-500 Ci/g of Cr, New England Nuclear; 1 Ci = 3.7 X 1010 becquerels) for 45 min as described (11). Assay for Cytolytic Activity. The cytolytic activity of Con Astimulated spleen cells was assayed as described by the method of MacDonald and Lees (9). Briefly, varying numbers ofeffector cells were mixed with 104 radiolabeled P815 cells in 0.2 ml of Abbreviations: Ara-C, 1-,B3D-arabinofuranosylcytosine; PHA, phytohemagglutinin; PHA-M, bactophytohemagglutinin M: DME medium, Dulbecco's modified Eagle's medium; Con A, concanavalin A; HMGCoA reductase, 3-hydroxy-3-methylglutaryl CoA reductase (NADPH); MLR, mixed lymphocyte reaction; CPR, cholesterol/phospholipid ratio. 3823 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 3824 Immunology: Heiniger and Marshall DME medium (with 5% fetal bovine serum and amino acids) containing 50 mM a-methyl D-mannoside and 2.5 Al of PHAM per ml. The assay was performed in round-bottomed microtiter plates (Flow Laboratories, McLean, VA), and the radioactivity released in the supernatant was assayed in a Nuclear Chicago well-type y spectrophotometer. The specific release percentage was calculated by using the formula of Cerottini et al. (12). Lytic units per 106 cells were calculated from the dose-response curves, 1 unit corresponding to the number of lymphocytes required to lyse 50% of the target cells within 3 hr. Analysis of Sterol Synthesis. Three to six replicate samples of the Con A-stimulated lymphocytes were pooled from the COSTAR culture wells, and the number of viable cells were determined by the trypan blue dye-exclusion test. Cells were gently suspended in 5 ml of DME medium containing 25 ACi of [1-'4C]acetate (59.7 Ci/mol; Amersham) in 25-ml Erlenmeyer flasks and incubated for 1 hr in a 37VC shaking water bath. Labeled sterols, precipitated by digitonin, from the nonsaponifiable fraction were determined as described (10). Analysis ofDNA Synthesis. Replicate samples were removed from three COSTAR culture wells, and the viable, dye-excluding cells therein were determined. The cells were transferred in 1-ml aliquots to tubes containing 1 ACi of [5-nethyl3H]thymidine (specific activity, 20 Ci/mmol; New England Nuclear) and were incubated for 1 hr in a 370C shaking water bath. Incorporation of [3H]thymidine was measured in the perchloric acid-insoluble fraction of the cells as described (2). Preparation of Liposomes. To prepare cholesterol-containing liposomes, L-a-phosphatidylcholine (L-a-lecithin) from egg yolk (Sigma) was mixed with pure, recrystallized cholesterol in chloroform in a cholesterol/phospholipid molar ratio (CPR) of 0.5. The solvent was evaporated under nitrogen with a rotary evaporator apparatus. Then a "swelling solution" (0.29 M glucose/0.05 M phosphate, pH 7.4) was added and allowed to stand 2-3 hr at room temperature under nitrogen. An aliquot was examined by electron microscope to ascertain the morphological features of the liposomes, and the remaining mixture was stored at 40C under nitrogen for not longer than 24 hr prior to use. Liposomes were added to the cells to give a phospholipid concentration of 15 x ,uM.