Interleukin-2 induction of ACTH secretion: presence of an interleukin-2 receptor α-chain-like molecule on pituitary cells

Journal of Neuroimmanology, 21 (1989)249-254

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JNI 00754 Short Communication

Interleuldn-2 induction of A C T H secretion: presence of an interleukin-2 receptor a-chain-like molecule on pituitary cells Lawrence R. Smith *, S. Loft Brown and J. Edwin Blalock Department of Physiology and Biophysics, Universityof Alabama at Birmingham, Birmingham, AL 35294, U.S.A.

(Received21 September1988) (Accepted17 October1988) Key words: Interleukin-2receptor; Neuroimmunology;Pituitarycell

Summary Pituitary cells were shown to release corticotropin (ACTH) in response to interleukin-2 (IL-2) and to express a protein that is related to the a-chain of the IL-2 receptor (IL-2R). The a-chain-like molecule was bound by a rat monoclonal antibody to the murine IL-2 receptor as well as to IL-2. Sodium dodecylsulfate-polyacrylamide gel electrophoretic analysis of the affinity-purified material from pituitary c e ~ demonstrated a protein which was similar to that which was isolated from activated splenocytes. Thus, IL-2 and its receptor may be one of several hormone-receptor pairs utilized by both the immune and neuroendocrine systems for intersystem communication.

Introduction. Interleukin-2 (IL-2) and it~ receptor (IL-2R) are pivotal components of T cellmediated immune responses. While IL-2 has historically been characterized with respect to regulation of immune function, it is becoming apparent that the biological activity and target tissues of this molecule are not restricted to the immune system as is evidenced by its influence on neuroendocrine function. For example, IL-2 stimulates ofigodendroglial cell proliferation and maturation (Merrill et al., 1984; Benveniste and Merrill, 1986) and regulates pituitary cell expres-

Addressfor correspondence:J. EdwinBlalock,Department of Physiology and Biophysics, UAB, UAB Station, Birmingham,AL 35294,U.S.A. * Present address: Research Instituteof Scripps Clinic,Immunology Department 1MM3, 10666 North Torrey Pines Road, La Jolla, CA 92037, U.S.A.

sion of pro-opiomelanocortin (POMC) mRNA (Brown et al., 1987) and the POMC gene products, corticotropin (ACTH) and ~-endorphin (Farrar, 1984). IL-2 regulation of POMC gene products may be physiologically important in view of the observations that: (1) ACTH and the adrenal glucocorticoid hormones are mediators of the stress response and may play important roles in stressinduced compromise of immune function (Munck et al., 1984); (2) ACTH and endorphins have various immunomodulatory effects (Blalock et al., 1985; Morley et al., 1987); (3) glucocorticoid hormones, which are released in response to ACTH, can control IL-2 production (Gillis et al., 1979) and IL-2R expression (Reem and Yah, 1984; Reed et al., 1986) in vitro; (4) administration of IL-2 to humans results in elevation of ACTH and 81ucocorticoids (Bi,,don et al., 1983; Lotze et al., 1985). It is hypothesized that such immune-neu-

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250 roendocrine interactions occur in part through hormones and receptors utilized between as well as within systems (Blalock, 1984). In support of this concept and in view of the above observa6ons, studies were conducted to determine whether IL-2 has a biologic effect on pituitary cells and whether pituitary cells, fike lenkocytes, have an IL-2 receptor. Materials and methods. Cell cultures: The murine anterior pituitary tumor cell line AtT-20 was obtained from American Type Culture Collection (Rockville, MD, U.S.A.). ART-20 cells were cultured at 37°C in 5~ CO 2 in Ham's F-10 medium complete with 15~ horse serum (HS) and 2.5~ fetal calf serum (FCS) (Gibco, Grand Island, NY, U.S.A.). Primary rat pituitary cells were obtained from freshly killed female Sprague-Dawiey rats. Murine pituitaries were obtained from female C57BI/6J (Jackson Laboratory, Bar Harbour, ME, U.S.A.). Pituitaries were diced in Eagle's minimal essential medium (EMEM)containing 1 mg/rn!l trypsin (Difco, Detroit, MI, U.S.A.) and incubated at 37°C for 2 h to disperse the cells. Dispersed cells were washed in F-10 complete and plated overnight in this medium containing 25 p g / m l mycostatin, 100 U / m l penicillin, and 100 p g / m l streptomycin sulfate. Pituitary cells were cultured overnight to allow re.-expressiox~ of any trypsinsensitive surface receptors prior to use in the reverse hemolytic plaque assay. Aff'mity chromatography: For purifying the IL2R from cells, affinity matrixes were coupled with dther monoclonal anti-m-.Lrine IL-2R antibody (Osawa and Diamanstein, 1984) (Boehringer Mannheim, Indianapolis, IN, U.S.A.) or human ultrapure IL-2 (Genzyme, Boston, MAt U.S.A.). 40 /~g of the monoclonal antibody or 140 pg of ultrapure h u m ~ IL-2 was coupled to 1 ml of Reacti-Gel (6 × ) (Pierce Chemical Co., Rockford, IL, U.S.A.) in 0.1 M NaHCO 3 (pH 9.5)/0.5 M NaCI for 20 h at 4 ° C with gentle rocking. "Iris (0.1 M) was added to the gel after this time to block any uureacted sites on the matrix. The gels were pipetted into 3 nd columns (Bio-Rad, Rockville Centre, NY, U.S.A.) and washed extensively with phosphate-buffered sali~le (PBS)-0.02~ NaN 3. Immunoaff'mity purification of IL-2R from ART-20, normal murine pituitaries, or murine

splenocytes was achieved as follows. 20 × 106 to 100 × 10e cells were centrifuged, washed in PBS, and solubilized by the addition of 200-500/~1 of 5 mM (3-[(3fl-cholamidopropyl)dimethylammonio]1-propane-sulfonate (CHAPS), 30 mM NaCI. Protease inhibitors were also used and included: 10 mlVl phenylmethylsulfoliyl fluoride (PMSF), 5 p g / m l aprotinin, 1 pg/ml leupeptin (Sigma). Clarified receptor supernates were diluted in 10-15 nd of PBS-0.02~ NaN 3 and then passaged through an affinity column. Eluates were removed from the column by acid elution with PBS pH 2, neutralized to pH 7, and hydroextracted with polyethylene glycol to reduce the volume approximately 30- to 50-fold. Concentrated eluates were dialyzed extensively for 24-36 h in distilled water at 4°C. Samples were further concentrated to a 100 pl volume by speed-vacuum centrifugation. S~.ples were subsequently analyzed by sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE: 10~ SDS-polyacrylamide gels were cast and run according to the method of Laemmli (1970) and subsequently silver stained (Merrill et al., 1981). Reverse hemolytic plaque assay (RI-IPA): The RHPA was performed as previously described (Smith et al., 1986). Briefly, rat pituitaries were dispersed as described above and used the following day. 1 ml of packed sheep red blood cells (,~RBC, Colorado Serum Co., Denver, CO, U.S.A.) was coupled to 0.5 mg/ml Staphylococcus protein A (Sigma) with chromium chloride hexahydrate (Sigma). After extensive wasl~dngs, the SRBC were resuspended in Dulbecco's minimal essential medium (DMEM) with 0.1~ BSA at a 2~ final stock concentration. 18~ SRBC suspension was mixed with an equal volume of monodispersed pituitary cells (5 × 10S/m1) and loaded into a Cunningham chamber previously coated with 0.25 mg/ml poly-L-lysine (Sigma). After 45 rain incubation at 37 °C to promote cell adherence, the slides w~e rinsed with DMEM and then ACTH antibody (1:50 final), secretagogues (corticotropin releasing factor (CRF) or IL-2), and guinea pig complement (C-ibco) (1 : 50 final) were added. Plaques were enumerated 2-3 h later. Results. The pituitary cell line (AtT-20) was previously reported to release ACTH in response

251

to ILL,-2(Farrar, 1984) as determined by radioimmunoas,my. A reverse hemolytic plaque assay was ~erfo_r~ed to determine whether normal pituitary cells, like ART-20 cells, respond to IL-2 by releasing ACTH. This assay measures hormone secretion by in~vidual pituitary cells several hours

~ter • stimulation with seeretagogues (Smith eta|., 1986). Plaque formation is seen as a zone of hemolysis of sheep red blood cells surrounding an ACTH-secreting pituitary cell (Fig. 1B-D). Staphylococcus protein A-coupled SRBC bind anti-ACTH which interacts with secreted ACTH r i

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Fig- ]. Top pm~ls: The ~'ver~ h~lolyti¢ plaque assay for rat pituitary cell secretion of AC~H. The zones of bemolys~ represent the secretion of A~'~J[~ by mdi~du~ pituitary cells in ~.sponsc to a s~:re~o~ue. (A) Untreated cells; (B) 0.5 ps/ml CRF; (C) 100 U / m l 11-2; ( D ) higher magnification of ( C ) demonstrates the presence of a pituitary cell in each plaque ( A - C 100 × , D 400 × ). Bottom panel: The number of Ac'WH secreting plaque forming cells (PFC) per slide was enumerated from dupll.cate samples and average PFC/slide are depicted in this 8raph. C R F (0.5/~8/ml) or i1-2 (10-500 U / m l ) caused the rapid release of ACTH. I1-3 had

no effect.

252 and eventually lyses the SRBC.s in the presence of guinea pig complement. Fig. 1B and C show pituitary cells treated with 0.5 /Lg/'ml CRF and 100 U / m l IL-2, respectively; compared to unstimulated cells (Figs. 1A), the CRF- and 11.-2treated cells secrete ACTH and thus produce plaques. An enlarged field from Fig. 1C is shown in Fig. 1D where one can see a pituitary cell in the center of each plaque with remnants of SRBC ('ghosts'), indicating specific plaque formation. Enumeration of CRF- and IL-2-stimulated plaque forming cells (PFC) is also shown in Fig. 1. 100-250 U / m l of murine recombinant IL-2 (approximately 100-250 p g / m l based on the specific activity ( > l0 s units/~ag) of this preparation) were essentially equipotent with 0.5 /tg/ml CRF, the positive releaser of ACTH, in causing ACTH release in this assay. Another lymphokine, IL-3, did not cause release. The murine anterior pituitary tumor cell line, ART-20, was previously shown to respond to IL-2 with increases in POMC mRNA (Brown et al., 1987) as well as release of ACTH and /3-endorphin (Farrar, 1984). Becanse IL-2 caused a biological response in these cells, and since IL-2 acts through a specific receptor on lymphocyte.s, it was of interest to determine whether pituitary cells also express glycoproteins wh;ch have IL-2 binding activity. Affinity chromatography and SDSPAGE were utilized to purify and characterize an immunoreactive IL-2R from pituitary cells. The monoclonal IL-2R antibody was employed to aff'mity purify the IL-2R-like moiety from detergent-solubilized pituitary cells. IL-2 itself was also used to affinity purify this molecule. Fig. 2 represents SDS-PAGE of affinity-purified IL-2R. Lane 1 demonstrates a single band of approximately 58 000 Da isolated from concanavalin A (ConA)stimulated splenocytes by passage through an IL2-coupled matrix. II-2R antibody identified an identical species from mitogen-stimulated splenocytes (data not shown). Lanes 2 and 3 represent material from ART-20 cells purified using II.-.2 and monoclonal antibody to IL-21L respectively. Lane 4 represents material purified from normal rnurine pituitary cells using IL-2R antibody. The murine T cell IL-2R has been reported to migrate betwecn 50000 and 65000 Da (Leonard et al., 1985; Farrar et al., 1987). All species isolated from splenocytes

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Fig. 2. Affinity.purifiedIL-2Rfrom murinespleenor pituitary cells. Clarified receptor supemates were passed over affinity matrices coup|ed with either IL-2 or with a rat monoclonal antibody to the routine IL-2R. Lane 1: ConA-sfimulated splenocytepreparationpassagedover an 11.-2e.olunm;lane 2: ART-20cell preparation passagedover an IL-2 column, and lane 3: over an anfi-ILo2Rcolumn; lane 4: murine pituitary cellpreparationpassagedoveran anti-IL-2Rcolumn. and pituitary cells by selection with either IL-2R antibody or IL-2 migrated under nonreduced conditions at approximately 56000-58000 Da. In several preparations, an approximately 36000 Da species was also identified which represents an immature IL-2R undergoing glycosylation (Leonard et al., 1985; Farrar et al., 1987). These studies strongly suggest that splenocytes and pituitary cells contain a molecule identical in molecular weight and amenability to purification by IL-2 or IL-2R antibody-coupled affinity matrices. To further characterize the immunoaffinity-purified IL-2R from ART-20 cells and normal pituitary cells, purified preparations were run under reduced and nonreduced conditions. Samples reduced with 2-mercaptoethanol demonstrated a shift in apparent molecular weight to approximately 67000 Da compared to the molecular weight of 55 000 Da in nonreduced samples (data not shown). The mobility shift seen in reduced samples is typical of many membrane receptors

253 ( M a l b o n et ai., 1987), including IL-2R (Osawa a n d Diamanstein, 1984). Discussion. The IL-2R consists of a- a n d /~chains which together b i n d IL-2 with high affinity a n d signal a proliferative response to the cell (Tsudo et ai., 1986). Either chain alone binds IL-2 with low affinity a n d apparently does not deliver a proliferative signal to the nucleus. Although the studies presented here on]y provide evidence for a n a-chain (58000 Da, equivalent to the Tac antigen), a ~-chain or 'converter protein' may also b e present. T h e IL-2 concentrations used in these studies to achieve A C T H release were in the range of 100-250 p g / m l based o n the specific activity of this IL-2 preparation. Such low IL-2 concentrations suggest high affinity binding b y pituitary IL-2R. This in turn suggests the presence of a ~-chain equivalent in pituitary cells. Studies are currently being conducted to determine the binding characteristics of this receptor b y radioreceptor assay. T o date, however, we have been unable to demonstrate high affinity binding a n d assume this is due to a very low n u m b e r of high affinity receptors o n each cell. These observations imply exquisite economic utilization of IL-2 for regulating i m m u n e a n d neuroendocrine activities. IL-2 may participate not only as a T cell growth factor during a n i m m u n e response, b u t may also be one signal which comm u n i c a t ~ the changes in i m m u n e status to the p i t u i t a r y - a d r e n a l axis. IL-1, a monokine which potentiates IL-2 production in association ~ t h antigen, also causes A C T H release from pituitary cells (Farrar, 1984; Woloski et al., 1985; Bernton et al., 1987), as well as increases in P O M C m R N A levels. Thus, two essential leukocytic hormones, IL-2 a n d IL-1, appear capable of activating a neuroendocrine stress axis. Perhaps the i m m u n e system detects immunostressors, such as viruses or bacteria, which are imperceivable to the neuroendocrine system, a n d communicates such information b y IL-2 and IL-1. Activation of stress response hormones may be a means by which a n i m m u n e response can be controlled a n d prevented from overreacting to antigenic challenge. Thus, IL-2 may stimulate A C T H production in order to regulate certain aspects of a n immune response. Overall, this study provides support for the concept that the neuroendccr:me and immune systems

express similar genes and utilize their products for communicating between systems, in addition to performing effector functions intrasystematically. Acknowledgements. W e t h a n k Diane Weigent for typing the manuscript. This work was supported by N I H grant DK38024, Cancer Center Core grant CA131d8, and N I H grant GM40176.

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