Immunology Letters, 28 (1991) 175-180

Elsevier IMLET 01570

A monoclonal anti-peptide antibody mimics adrenocorticotropic hormone activity B. L. C l a r k e a n d K. L. Bost University of Alabama at Birmingham, Department of Physiology and Biophysics, Birmingham, AL, US.A.

(Received 9 July 1990; accepted 29 January 1991)

1. Summary A monoclonal antibody that specifically recognizes the adrenocorticotropic receptor (ACTH) on rat adrenal cells was tested for hormonal activity. The antibody behaved as an agonist based on three different biological activities associated with ACTH. An antibody concentration of 16/zg/ml stimulated isolated rat adrenal cells to secrete 800 ng/104 cells of corticosterone with a concomitant 10-fold increase of cAMP to 30 pmol/105 cells. Antibody concentrations above 16 izg/ml inhibited mitotic activity in mouse Y-1 adrenal cells. A radioimmunoassay using an anti-ACTH antibody showed that the monoclonal anti-adrenocorticotropic receptor antibody and ACTH are antigenically related. These findings suggest that the anti-receptor antibody recognizes the ligand binding domain of the ACTH receptor. 2. Introduction Recently we developed a monoclonal antibody to the complementary peptide for ACTH, termed HTCA [1]. This HTCA peptide was designed by using the complementary transcript from the bovine pro-opiomelanocortin mRNA segment that encodes ACTHl_24 [2]. The monoclonal anti-HTCA antiwords:Complementary peptide; Steroidogenesis; Monoclonalantibody; ACTH receptor;Adenylatecyclase;Mitosis

Key

Correspondence to: B. L. Clarke, Universityof Alabama at Birmingham, Dept. of Physiology and Biophysics, Birmingham, AL, U.S.A.

0165-2478 / 91 / $ 3.50 © 1991 ElsevierScience Publishers B.V.

body was found to recognize a restricted domain on the HTCA molecule; in addition, the anti-HTCA antibody could bind to the surface of Y-1 mouse adrenal cells and isolate rat adrenal cells. AntiHTCA antibody binding to adrenal cells was shown to be specific, competitive, and saturable; an observed KTd at 1.9 nM was determined from Y-1 cells. In particular, both ACTH and HTCA could block cell surface binding, These observations suggested that the antibody recognizes cell surface ACTH receptors. In order to address this possibility, we tested the antibody for effects on A C T H activity. The principal biological response to pituitary release of ACTH is the secretion of glucocorticoids by the adrenals. In turn, adenylate cyclase activity was found to be stimulated by the cell surface binding of subnanomolar concentrations of ACTH. Production of cAMP increases following ACTH binding, and is the putative initial signal transducing event towards the production of corticosteroids [3]. Steroidogenesis increases simultaneously with the increase of bound cAMP and preceding the appearance of a significant amount of free cAMP. Other signal transducing events have also been proposed as well, including the involvement of G protein regulatory elements [4], Ca + influxes [5], and K ÷ effluxes [6] to explain the early events in steroidogenesis. Subsequent to stimulation, the adrenal cells enter a refractory state particularly when exposed to nanomolar concentrations of ACTH. Under this condition, the adrenal cells lose ACTH-induced steroidogenic activity and decrease in mitotic activity [7]. In this study, we show that anti-HTCA monoclonal antibodies stimulate adrenal cells to in175

crease adenylate cyclase activity with a concomitant secretion of corticosterone. The anti-HTCA monoclonal antibodies could also mimic the ACTH-dependent refractory state by blocking mitotic activity in Y-1 cells. These findings demonstrate that the anti-complementary peptide antibody that recognizes HTCA is capable of mimicking ACTH biological activity by binding to the ACTH receptor. 3. Materials and Methods

3.1. Peptide probes The peptides P H E 2-NLE4-ACTH l 24 (ACTHI 24) and HTCAl_24 were synthesized as previously described [8]. Iodinated ACTH1_24 was prepared using iodo-beads (Pierce Chemical) and Na125I (NEN DuPont) as previously described [8]. -

3.2. Monoclonal antibody production The immunogen was produced by conjugating HTCA to keyhole limpet hemocyanin (Calbiochem) using gluteraldehyde [8]. BALB/c mice were inocu-" lated with 100 #g of conjugated peptide prepared in an emulsification of Freund's complete adjuvant (Sigma). Splenic lymphocytes from mice developing antibodies capable of binding H T C A in an ELISA, rounding Y-1 cells [9] and blocking [125I]ACTH binding were fused to SP2/0-Agl4 (ATCC CRL1581) myeloma cells using polyethylene glycol (Boehringer Mannheim) and then selected for successful fusion as previously described [1]. Cells were cloned in soft agar, and supernates from clones were retested for reactivity against H T C A and further tested for their ability to block [125I]ACTH binding to Y-1 cells. Selected clones were injected into pristane primed BALB/c mice to produce ascitic fluid. The antibody was then purified using a protein G-Sepharose column which gave a 180-fold enrichment of antiHTCA antibody titers [1]. One hybridoma subclone, designated 3/1-8, was chosen for further characterization of agonist activity based on optimal induction of Y-1 cell rounding and is the subject of this report. 3.3. Cell culture The adrenal 176

glands

from decapitated

male

Sprague-Dawley rats (150-200 g) were rapidly excised and stored in sterile RPMI-1640 plus 10%0 fetal calf serum (FCS) on ice. The adipose tissue was removed prior to squeezing the medulla and fasciculata-reticularis layers away from the capsule. The inner adrenal tissue was suspended in 1 ml of RPMI-1640/10%0 FCS per two glands, then minced by opposing slices with two razor blades, and then collagenase type IV (Sigma) and DNase I (Sigma) were added to a final concentration of 1 mg/ml and 0.1 mg/ml, respectively. The digestion mix was incubated for 15 min at 37 °C with mild agitation, then spun to a pellet and resuspended in fresh RPMI1640/10% FCS, and finally poured through a sterile (50 mesh) sieve. Viable cells were isolated by centrifugation on Hypaque-Ficoll and cultured for 3 - 5 days in RPMI-1640/10% FCS prior to use in binding assays. Binding assays using Y-1 cells were performed in 24-well plates (Costar, Mark II). Cells were grown in RPMI-1640/10%0 FCS to a density of approximately 500000 per well prior to measuring antibody binding. 3.4. Quantification of corticosterone, 3 ',5' cyclic

A M P and [3H]thymidine uptake Corticosterone in culture media was measured by using a radioimmunoassay kit (ICN Biomedicals, Inc.). Total cAMP was isolated at various times after stimulation by washing the cells twice with ice-cold RPMI-1640 and solubilizing the cells with 0.1%0 Triton X-100 (Sigma). The solubilized material (0.5 ml) was transferred to 0.25 ml of ice-cold 10%0perchloric acid and spun at 3000×g for 10 min to pellet the precipitate. A 0.5-ml portion of the supernate was transferred to 0.25 ml of ice-cold 1.5 N KOH and spun at 3 0 0 0 × g for 10 min to pellet KC104. The neutralized supernate was then assayed for cAMP using a radioimmunoassay kit (Rainin Assay System, NEN DuPont). Mitotic activity in stimulated Y1 cells was measured by adding 0.5 /zCi of [3H]thymidine (NEN DuPont) to each chamber of a 96-well plate containing approximately 5)

A monoclonal anti-peptide antibody mimics adrenocorticotropic hormone activity.

A monoclonal antibody that specifically recognizes the adrenocorticotropic receptor (ACTH) on rat adrenal cells was tested for hormonal activity. The ...
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