- Email: [email protected]
Effect of bursal anti-steroidogenic peptide (BASP) on cortisol biosynthesis in ACTH-stimulated canine adrenocortical carcinoma cells in vitro
Vetetinay immunology and immunopathology
Veterinary Immunology and lmmunopathology 47 ( 1995) 3522
ELSEVIER
Effect of bursal anti-steroidogenic peptide (BASP) on cortisol biosynthesis in ACTH-stimulated canine adrenocortical carcinoma cells in vitro J.A. Byrd”, C.E. Dean”, T.W. Fossumb, B .M. Hargis”** “Departments “Department
of PoultryScience of Veterinary
Small
Texas A&M
and
Veterinary Pathobiology VMS. College St&m. TX Animal
University
Medicine System,
College 77843,
and Surgery. College
($ Veterinary
Medicine,
Room
I19
USA Te.ras A,qricultural
Station.
TX 77843,
Experiment
Stufrrm.
USA
Accepted 14 September I994
Abstract Previous studies from our laboratory have demonstrated that bursal anti-steroidogenic peptide (BASP) inhibits progesterone biosynthesis from ovine luteinizing hormone-stimulated chicken ovarian granulosa cells. In the present investigation, we evaluated the efficacy of BASP for reducing cortisol secretion from normal canine adrenocortical cells and neoplastic adrenocortical cells from a dog with Cushing’s syndrome. Treatment of adrenocortical cells derived from either normal healthy dogs or a cushingoid dog with adrenocorticotropic hormone (ACTH; O-10 nM) caused an approximately two-fold increase in cortisol production from both normal or tumor derived adrenocortical cells. Small but significant decreases (up to 34%) in cortisol production were observed from normal and tumor derived canine adrenocortical cells when exposed to increasing concentrations of BASP ( 0.0-O. 15 bursal equivalents; BEQ). Incubation of adrenocortical carcinoma cells or normal adrenocortical cells with ACTH (O10 nM) and BASP (0.0-0.15 BEQ) increased cyclic AMP formation up to 2.5fold. Interestingly. RASP suppressed basal cortisol production from tumor derived adrenocortical cells to normal levels when compared to the basal cortisol levels from normal derived adrenocortisol cells. Data from the present studies indicate that BASP is capable of suppressing basal and ACTHstimulated cortisol production from normal or tumor derived adrenocortical cells in vitro. The possible clinical efficacy of homologous canine BASP on canine adrenal function or chicken BASP in other species of animals remains to be evaluated. Abbreviations ACTH, adrenocorticotropic hormone; BASP, bursal anti-steroidogenic peptide; BEQ, bursal equivalents; CAMP, cyclic AMP; RIA, radioimmunoassay; SE, standard error.
* Corresponding author. 0165.2427/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSD/O165-2427(94)05388-X
36
J.A. Byrd et al. / Veierinary Immunology and immunopatltology
47 (1995) 35-42
1. Introduction Cushing’s syndrome was first described by Harvey Cushing ( 1932) as a disorder caused by ‘pituitary basophilism’. Cushing’s syndrome is a term used to describe all aspects of the clinical and chemical abnormalities resulting from chronic overproduction of glucocorticoids. It is recognized as one of the most common endocrine disorders in dogs with adrenal tumors accounting for 7-15% of the spontaneous cases of hyperadrenocorticism. In almost all cases, adrenal tumors are unilateral in dogs. Chronically elevated endogenous secretion of corticosteroids from dogs with hyperadrenocorticism can cause a number of clinical signs including polydipsia, polyuria, polyphagia, abdominal distention, muscle weakness, lethargy, alopecia, obesity and increased panting (Feldman, 1983). A serious consequence of hyperadrenocorticism is an impairment of normal immune function. Small elevations of circulating endogenous glucocorticoids have been reported to stimulate an increase in antibody formation with a decrease in Tlymphocyte suppressor cells in vitro (Orson et al., 1983). However, high concentrations of circulating cortisol have been shown to suppress antibody synthesis and kill B-lymphocytes both in vitro and in vivo (Settipane et al., 1978; Grayson et al., 1981). While the negative effects of adrenal corticosteroids on immune function are well described (Warnes et al., 1960; Grayson et al., 1981; Tizard, 1992) a similar ability of the immune system to modulate adrenal corticosteroid biosynthesis has not yet been demonstrated. Recently, our laboratory has partially isolated a low molecular weight ( = 3000-5000) peptide-bursal anti-steroidogenic peptide (BASP)-from the chicken bursa of Fabricius that inhibits the production of chicken gonadal and adrenal steroids to basal levels in vitro (Byrd et al., 1993, 1994). The low molecular weight of BASP suggests that the peptide may be teleologically conserved between species, and this peptide has been postulated to function as a natural suppressor of steroid producing cells (i.e. gonadal and adrenal organs) (Byrd et al., 1993, 1994). However, possible therapeutic efficacy of BASP for hyperadrenocorticism has not been evaluated. The present study was conducted to evaluate the efficacy of BASP (isolated from prepubertal chickens) for reduction of cortisol secretion from canine adrenocortical tumor cells surgically obtained from a dog with a steroid-producing adrenocortical tumor.
2. Materials and methods 2.1. Hormone and reagents Complete culture medium consisted of Eagle’s minimum essential medium, containing 25 mM HEPES buffer and included 0.1% bovine serum albumin, Fraction V, 50 IU penicillin ml-’ and 0.05 mg streptomycin ml-‘. The final pH of the medium was adjusted to 7.4 with 1 N NaOH. Adrenocorticotropic hormone (ACTH; human fragment l-24) was obtained from Sigma Chemical Co. and diluted immediately before use with cell culture medium. Partially purified BASP was extracted from chicken bursa of Fabricius as previously described (Byrd et al., 1993).
J.A. Byrd et al. /Veterinary
Immunology and Immunopathology
47 (1995) 35-42
17
2.2. Collection of tissue Adrenal tissues were collected from a dog with Cushing’s syndrome and four normal dogs. A 9-year-old female spayed Belgian Sheepdog was referred for evaluation of polyphagia, polydipsia, polyuria, and diarrhea, alopecia, and muscle atrophy. Cushing’s syndrome was suspected and plasma cortisol concentrations were measured. A tentative diagnosis of functional adrenocortical tumor was made. Baseline plasma cortisol concentration was 9.9 pg dl- ‘; 8 h following administration of 0.1 mg dexamethasone kg-‘, cortisol levels were essentially unchanged ( 11.4 pg dll’). Abdominal ultrasonography revealed a hypoechoic soft tissue mass located cranial to the left kidney. At surgery, a 3.5 cm X 3.5 cm encapsulated mass was found associated with the left adrenal gland. The mass was excised, trimmed free of connective tissue, and diced into small pieces (approximately 2 mm) in ice-cold culture medium for cell isolation. A section was taken for histological examination which confirmed the diagnosis of adrenocortical carcinoma. Adrenal glands were collected from four healthy anesthetized male and female adult dogs, trimmed free of connective tissue, and diced into small pieces (about 2 mm) in icecold culture medium for cell isolation. 2.3. Cell isolation and incubation The adrenocortical carcinoma cells from a dog with Cushing’s syndrome and adrenals removed from the four healthy dogs were isolated according to procedures that were previously described (Carsia et al., 1987). Briefly, adrenal tissues were decapsulated, minced with scissors, washed three times with equal volumes of cell culture medium, and recovered by centrifugation (220 X g) . Adrenal pieces were enzymatically dispersed at 37°C for 1 h with gentle shaking in 50 ml of cell culture medium containing 1 mg ml- ’ of collagenase. Following a 1 h incubation, cells were washed three times with cell culture medium. Cells were filtered through nylon mesh to remove any large cell clumps or cellular debris. Viability was determined using trypan blue dye exclusion and cells were counted on a live/dead cell basis (Freshney, 1983). Cells were diluted to a concentration of 100 000 viable cells per 250 ~1 and directly added to borosilicate culture tubes ( 12 mm X 75 mm). Treatment groups consisted of 0.0,0.03,0.075 or 0.15 bursal equivalents (BEQ) of BASP. For each concentration of the test compound, five replicates of 0.0, 0.01, 0.1, 1.0, IO nM ACTH or 1 mM dibutyryl CAMP were added to the appropriate tubes. Treatments were added to the culture tubes in a total volume of 250 ~1 so that the final volume was 0.5 ml. Cultures were incubated at 37OC, saturated humidity, with 5% CO, for 2 h. Following the incubation, 0.1 mM 3-isobutyl-1-methylxanthine was added to each culture tube and the tubes were then immediately frozen at - 20°C prior to radioimmunoassay (RIA) for cortisol and CAMP. Cortisol (content of cells plus medium) was determined by a commercially available RIA kit. The sensitivity for the cortisol assay was 1 ng ml-’ with an interassay coefficient of variation of 7.1%. Cellular CAMP content of succinylated samples was measured by a highly sensitive and specific RIA. The sensitivity for the CAMP assay was 800 fmol ml ~ ’ with an interassay coefficient of variation of 7.4%. Each experiment was repeated twice using two separate cell dispersions.
38
J.A. Byrd et ai. / Veterinary inmunology and Im~~nopat~o~ogy 47 (I9951 35+Q
2.4. Statistical analysis Data obtained from each experiment were subjected to analysis of variance using the SAS Program (Statistical Analytical Systems Institute Inc., 1988). Significantly different means were separated using Duncan’s multiple range test (Duncan, 1955). Significance is reported at P < 0.05.
3. Results 3.1. Normal adrenocortical cell cortisol biosynthesis In Fig. 1, BASP significantly suppressed cortisol production (up to 23.8%) as compared to unstimulated canine adrenal cells. The highest concentration of BASP evaluated (0.15 BEQ) significantly suppressed (up to 18%) cortisol production from adrenocortical cells derived from healthy dogs at all doses of ACTH. Similarly, BASP (0.075 BEQ) suppressed cortisol biosynthesis (up to 13.7%) from normal adrenocortical cells stimulated with high concentrations of ACTH (0. I-10 nM). The lowest dose of BASP evaluated (0.03 BEQ) was only effective in attenuating cortisol secretion from either unstimulated (0 nM ACTH) or the highest concentration of ACTH ( 10 nM) _The test compound BASP did not affect normal cortisol synthesis in adrenal cells stimulated with 1 mM dibutyryl CAMP. 3.2. Adrenocortical carcinoma cell cortisol biosynthesis Fig. 2 shows cortisol production by isolated ACTH-stimulated and unstimulated canine adrenocortical carcinoma cells in response to increasing concentrations of BASP. Cortisol 0
CONTROL
m
0.03 BEQ BASP
00758EQf2 BASP
015BEQ BASP
3
0.01
0.1 ACTH
1
10
F-CAMP
(nM)
Fig. 1. Effects of increasing concentrations of partially purified BASP (0.03-O. 15 BEQ) on cortisol production from 200 000 normal canine adrenocortical cells ml-’ in the absence or presence of ACTH (O-10 nM) or 1 mM dibutyryl CAMP (DB-CAMP) after 2 h of incubation. Vertical lines represent standard error of the mean. The asterisks depict mean values of two separate experiments which were significantly different (P< 0.05) from the control within each secretagogue concentration.
J.A. Byrd et al. /Veterinary
Immunology and Immunopathology
0.075 BASP
0
0.01
0.1 ACTH
1
BEQli
IO
47 (1995) 35-42
39
0 15 BECI BASP _.
DE-CAMP
(nM)
Fig. 2. Effects of increasing : Cloncentrations of partially purified BASP (0.03%0).15 BEQ) on cortisol production from 200 000 canine adrenocortical carcinoma cells ml- ’ in the absence or presence of ACTH (O-IO nM) or 1 mM dibutyryl CAMP (DB-CAMP) after 2 h of incubation. Vertical lines represent standard error of the mean. The asterisks depict mean values of two separate experiments which were significantly different (P < 0.05) from the control within each secretagogue concentration.
production from unstimulated (0 nM ACTH) adrenocortical carcinoma cells (Fig. 2) was observed to be more than ten-fold greater than basal secretion from cells derived from normal dogs (Fig. 1) . Stimulation of adrenocortical carcinoma cells with ACTH alone further increased cortisol production approximately two-fold at all concentrations evaluated (Fig. 2). All concentrations of BASP evaluated (0.03, 0.075 and 0.15 BEQ) significantly inhibited (up to 34%) basal (unstimulated) cortisol biosynthesis. ACTH-stimulatedcortisol production was suppressed by inclusion of 0.075 BEQ BASP (0.01 nM ACTH) or 0.015 BEQ BASP (0.1 nM ACTH) (Fig. 2). Similarly, all doses of BASP significantly suppressed cortisol production when combined with 1 nM ACTH. Furthermore, only 0.075 or 0.15 BEQ significantly suppressed both 10 nM ACTH- and 1 mM dibutyryl CAMP-stimulated adrenocortical carcinoma cell cortisol biosynthesis (Fig. 2). 3.3. Normal adrenocortical cell CAMPformation Fig. 3 illustrates CAMP production from ACTH-stimulated (O-10 nM) normal adrenocortical cells in the presence of increasing concentrations of BASP (0.03-0.15 BEQ). Regardless of the concentration of ACTH (O-10 nM) evaluated, BASP significantly stimulated CAMP formation in a dose dependent manner (Fig. 3). 3.4. Adrenocortical carcinoma cell CAMPformation Fig. 4 shows CAMP production by isolated ACTH-stimulated and unstimulated canine adrenocortical carcinoma cells in response to 0.75 or 0.15 BEQ BASP. ACTH alone stimulated CAMP formation over two-fold in a dose dependent manner. Both concentrations of BASP evaluated (0.075 or 0.15 BEQ) significantly increased (up to two-fold) ACTH-
40
J.A. Byrd et al. / Veterinary Immunology and lmmunopathology
0
CONTROL
m
0.03 BEQ BASP
0.075 BASP
BEQ ti;]
47 (1995) 3542
0.15 BEQ BASP
7
0
0.01
1
0.1 ACTH
10
(nb.4)
Fig. 3. Effects of increasing concentrations of partially purified BASP (0.034L15 BEQ) on CAMP production from 200 000 normal canine adrenocortical cells ml - ’ in the absence or presence of ACTH (O-10 nM) after 2 h of incubation. Vertical lines represent standard error of the mean. The asterisks depict mean values of two separate experiments which were significantly different (P <0.05) from the control within each secretagogue concentration. cl
CONTROL
m
0.075 BASP
BEQ
0.15 BEQ BASP
I
ZOr-p
0
0.01
1
0.1 ACTH
IO
(nM)
Fig, 4. Effects of increasing concentrations of partially purified BASP (0.075-0.15 BEQ) on CAMP production from 200 000 canine adrenocortical carcinoma cells ml-’ in the absence or presence of ACTH (O-10 nM) after 2 h of incubation. Vertical lines represent stanadard error of the mean. The astricks depict mean values of two separate experiments which were significantly different (P < 0.05) from the control within each secretagogue concentration. stimulated
canine
adrenocortical
carcinoma
cell CAMP formation
in a dose dependent
manner (Fig. 4).
4. Discussion We have evaluated the in vitro efficacy of bursal anti-steroidogenic peptide (BASP) , a peptide isolated from the bursa of Fabricius of chickens, for ability to attenuate cortisol
J.A. Byrd et al. /Veterinary
immunology and Immunopathology 47 (1995) 35-42
41
production from both canine adrenocortical carcinoma cells and normal adrenocortical cells. BASP, derived from the humoral immune system, is a potent and highly efficacious suppessor of in vitro gonadal and adrenal steroid hormone production in birds (Byrd et al., 1993, 1994). Extensive evaluations of the function of this peptide in gonadal tissue have indicated that progesterone biosynthesis is blocked independent of the gonadotrophin receptor (Byrd et al., 1993). Progesterone is a known precursor for most endproduct steroids (Fantl et al., 1973)) and several laboratories have reported that many tumors secrete only precursors for endproduct steroids (Fukushima and Gallagher, 1963; Fantl et al., 1973). In the present studies, incubation of canine adrenocortical cells with BASP caused significant suppression of cortisol production in both normal and carcinoma cells. Previous investigations have indicated that BASP is capable of total suppression of ACTH-stimulated corticosteroid production in a homologous chicken derived adrenocortical cell culture (Tizard, 1992). Similar to previously reported findings, BASP was only partially efficacious in the suppression of ACTH-stimulated cortisol production from normal canine adrenocortical cells in the present study (Fig. 1) (Byrd et al., 1994). Consistent with BASPmediated effects on normal adrenocortical cells, BASP was observed to only partially attenuate either basal (unstimulated) or ACTH-stimulated cortisol production from canine adrenal carcinoma cells in vitro (Fig. 2). Interestingly, BASP suppressed basal cortisol production from tumor derived adrenocortical cells to normal levels when compared to the basal cortisol levels from normal derived adrenocortisol cells. While many small bioactive peptides have been observed to be highly conserved between animal classes with regard to sequence and structure, some divergence of peptide and/or receptor structure may be anticipated with this 3000-5000 molecular weight peptide (Byrd et al., 1993). It is possible that this type of evolutionary divergence, and resultant incomplete agonist activity, is responsible for the incomplete suppression of cortisol secretion in the heterologous system presently investigated. Alternatively, the mammalian homologue of BASP, if such a peptide exists, may modulate corticosteroid production in a less marked fashion than in birds (Byrd et al., 1994). In support of the latter hypothesis, BASP has been shown to markedly increase CAMP levels in normal chicken, porcine and canine adrenocortical cells (Byrd et al., 1994) and BASP induced CAMP elevations in excess of two-fold in both normal adrenal and carcinoma derived cells in the present experiments (Figs. 3 and 4). Approximately two- to three-fold elevations of CAMP have been observed in chicken adrenocortical cells in which complete suppression of ACTH-stimulated corticosteroid production was noted (Byrd et al., 1994). Definitive elucidation of differences in BASPmediated attenuation of avian and mammalian adrenocortical cell function must await molecular characterization of the peptide in each animal class. The present experiments indicate that avian derived BASP is capable of significant suppression of basal and ACTH-stimulated cortisol production from both normal canine adrenocortical cells and canine adrenocortical carcinoma cells in vitro. However, due to the incomplete suppression of cortisol production observed in both types of cells, anticipation of clinical utility of BASP or BASP analogs for treatment of canine Cushing’s disease must be viewed with some skepticism.
42
LA. Byrd et al. / Veterinary Immunology and Immunopathology 47 (1995) 3542
Acknowledgments The authors would also like to acknowledge support for this research from the Texas Advanced Technology Program. The authors thank Dr. Roberta L. Relford for professional assistance.
References Byrd, J.A., Hayes, T.K., Wright, M.S., Dean, C.E. and Hargis, B.M., 1993. Detection and partial characterization of an anti-steroidogenic peptide from the humoral immune system of the chicken. Life Sci., 52: 1195-1207. Byrd, J.A., Dean, C.E. and Hargis, B.M., 1994. The effect of the humoral immune system derived bursal antisteroidogenic peptide (BASP) on corticosteroid biosynthesis in avian, porcine, and canine adrenal cortical cells. Comp. Biochem. Physiol., 108C: 221-227. Carsia, R.V., Morin, M.E., Rosen, H.D. and Weber, H., 1987. Ontogenic corticosteroidogenesis of the domestic fowl: response of isolated adenocortical cells. Proc. Sot. Exp. Biol. Med., 184: 43644.5. Cushing, H., 1932. The basophil adenomas of the pituitary body and their clinical manifestations (pituitary basophilism). Bull. Johns Hopkins Hosp., 50: 127-195. Duncan, D.B., 1955. Multiple range and multiple F test. Biometrics, 11: l-42. Fan& V., Booth, M. and Gray, C.H., 1973. Urinary preg-5-ene-3A,6A,20A-triol in adrenal dysfunction. J. Endocrinol., 57: 135-142. Feldman, EC., 1983. The adrenal cortex. In: S.J. Ettinger (Editor), Textbook of Veterinary Internal Medicine: Diseases of the Dog and Cat. Saunders, Philadelphia, pp. 1650-1695. Freshney, R.I., 1983. Culture of Animal Cells. Alan R. Liss Press, New York, pp. 207-209. Fukushima, D.K. and Gallagher, T.F., 1963. Steroid production in ‘nonfunctioning’ adrenalcottical tumor. J. Clin. Endocrinol. Metab., 23: 923-927. Grayson, J., Dooley, N.J., Kolski, 1. and Blasese, R.M., 1981. Immunoglobulin production in vitro by glucocorticoid hormones: T-cell-dependent stimulation of immunoglobulin production without B cell proliferation in cultures of human peripheral blood lymphocytes. J. Clin. Invest., 68: 1539-1547. Orson, F.M., Grayson, J., Pike, S., de Seau, V. and Blaesa, R.M., 1983. Suppression of glucocorticoid induced immunoglobulin production: responders and non-responders to a serum inhibitory factor. Clin. Res., 3 1: 350A. Settipane, G.A., Pudupakkan, R.K. and McGowan, J.H.. 1978. Corticosteroids effect on immunoglobulin. J. Allergy Clin. Immunol., 62: 162-166. Statistical Analytical Systems Institute Inc., 1988. SAS/STAT User’s Guide for Personal Computers. Statistical Analysis Systems Institute Inc., Gary, NC. Tizard. I., 1992. Introduction to Veterinary Immunology. Saunders, Philadelphia, pp. 53-71. Wames, N.L., Uhr, J.W., Thorbecke, J. and Ovary, 2.. 1960. Immunoglobulins, antibodies and the bursa of Fabricius: induction agammaglobulinemia and the loss of all antibody-forming capacity by hormonal bursectomy. J. Immunol., 103: 1317-1330.
Veterinary Immunology and lmmunopathology 47 ( 1995) 3522
ELSEVIER
Effect of bursal anti-steroidogenic peptide (BASP) on cortisol biosynthesis in ACTH-stimulated canine adrenocortical carcinoma cells in vitro J.A. Byrd”, C.E. Dean”, T.W. Fossumb, B .M. Hargis”** “Departments “Department
of PoultryScience of Veterinary
Small
Texas A&M
and
Veterinary Pathobiology VMS. College St&m. TX Animal
University
Medicine System,
College 77843,
and Surgery. College
($ Veterinary
Medicine,
Room
I19
USA Te.ras A,qricultural
Station.
TX 77843,
Experiment
Stufrrm.
USA
Accepted 14 September I994
Abstract Previous studies from our laboratory have demonstrated that bursal anti-steroidogenic peptide (BASP) inhibits progesterone biosynthesis from ovine luteinizing hormone-stimulated chicken ovarian granulosa cells. In the present investigation, we evaluated the efficacy of BASP for reducing cortisol secretion from normal canine adrenocortical cells and neoplastic adrenocortical cells from a dog with Cushing’s syndrome. Treatment of adrenocortical cells derived from either normal healthy dogs or a cushingoid dog with adrenocorticotropic hormone (ACTH; O-10 nM) caused an approximately two-fold increase in cortisol production from both normal or tumor derived adrenocortical cells. Small but significant decreases (up to 34%) in cortisol production were observed from normal and tumor derived canine adrenocortical cells when exposed to increasing concentrations of BASP ( 0.0-O. 15 bursal equivalents; BEQ). Incubation of adrenocortical carcinoma cells or normal adrenocortical cells with ACTH (O10 nM) and BASP (0.0-0.15 BEQ) increased cyclic AMP formation up to 2.5fold. Interestingly. RASP suppressed basal cortisol production from tumor derived adrenocortical cells to normal levels when compared to the basal cortisol levels from normal derived adrenocortisol cells. Data from the present studies indicate that BASP is capable of suppressing basal and ACTHstimulated cortisol production from normal or tumor derived adrenocortical cells in vitro. The possible clinical efficacy of homologous canine BASP on canine adrenal function or chicken BASP in other species of animals remains to be evaluated. Abbreviations ACTH, adrenocorticotropic hormone; BASP, bursal anti-steroidogenic peptide; BEQ, bursal equivalents; CAMP, cyclic AMP; RIA, radioimmunoassay; SE, standard error.
* Corresponding author. 0165.2427/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSD/O165-2427(94)05388-X
36
J.A. Byrd et al. / Veierinary Immunology and immunopatltology
47 (1995) 35-42
1. Introduction Cushing’s syndrome was first described by Harvey Cushing ( 1932) as a disorder caused by ‘pituitary basophilism’. Cushing’s syndrome is a term used to describe all aspects of the clinical and chemical abnormalities resulting from chronic overproduction of glucocorticoids. It is recognized as one of the most common endocrine disorders in dogs with adrenal tumors accounting for 7-15% of the spontaneous cases of hyperadrenocorticism. In almost all cases, adrenal tumors are unilateral in dogs. Chronically elevated endogenous secretion of corticosteroids from dogs with hyperadrenocorticism can cause a number of clinical signs including polydipsia, polyuria, polyphagia, abdominal distention, muscle weakness, lethargy, alopecia, obesity and increased panting (Feldman, 1983). A serious consequence of hyperadrenocorticism is an impairment of normal immune function. Small elevations of circulating endogenous glucocorticoids have been reported to stimulate an increase in antibody formation with a decrease in Tlymphocyte suppressor cells in vitro (Orson et al., 1983). However, high concentrations of circulating cortisol have been shown to suppress antibody synthesis and kill B-lymphocytes both in vitro and in vivo (Settipane et al., 1978; Grayson et al., 1981). While the negative effects of adrenal corticosteroids on immune function are well described (Warnes et al., 1960; Grayson et al., 1981; Tizard, 1992) a similar ability of the immune system to modulate adrenal corticosteroid biosynthesis has not yet been demonstrated. Recently, our laboratory has partially isolated a low molecular weight ( = 3000-5000) peptide-bursal anti-steroidogenic peptide (BASP)-from the chicken bursa of Fabricius that inhibits the production of chicken gonadal and adrenal steroids to basal levels in vitro (Byrd et al., 1993, 1994). The low molecular weight of BASP suggests that the peptide may be teleologically conserved between species, and this peptide has been postulated to function as a natural suppressor of steroid producing cells (i.e. gonadal and adrenal organs) (Byrd et al., 1993, 1994). However, possible therapeutic efficacy of BASP for hyperadrenocorticism has not been evaluated. The present study was conducted to evaluate the efficacy of BASP (isolated from prepubertal chickens) for reduction of cortisol secretion from canine adrenocortical tumor cells surgically obtained from a dog with a steroid-producing adrenocortical tumor.
2. Materials and methods 2.1. Hormone and reagents Complete culture medium consisted of Eagle’s minimum essential medium, containing 25 mM HEPES buffer and included 0.1% bovine serum albumin, Fraction V, 50 IU penicillin ml-’ and 0.05 mg streptomycin ml-‘. The final pH of the medium was adjusted to 7.4 with 1 N NaOH. Adrenocorticotropic hormone (ACTH; human fragment l-24) was obtained from Sigma Chemical Co. and diluted immediately before use with cell culture medium. Partially purified BASP was extracted from chicken bursa of Fabricius as previously described (Byrd et al., 1993).
J.A. Byrd et al. /Veterinary
Immunology and Immunopathology
47 (1995) 35-42
17
2.2. Collection of tissue Adrenal tissues were collected from a dog with Cushing’s syndrome and four normal dogs. A 9-year-old female spayed Belgian Sheepdog was referred for evaluation of polyphagia, polydipsia, polyuria, and diarrhea, alopecia, and muscle atrophy. Cushing’s syndrome was suspected and plasma cortisol concentrations were measured. A tentative diagnosis of functional adrenocortical tumor was made. Baseline plasma cortisol concentration was 9.9 pg dl- ‘; 8 h following administration of 0.1 mg dexamethasone kg-‘, cortisol levels were essentially unchanged ( 11.4 pg dll’). Abdominal ultrasonography revealed a hypoechoic soft tissue mass located cranial to the left kidney. At surgery, a 3.5 cm X 3.5 cm encapsulated mass was found associated with the left adrenal gland. The mass was excised, trimmed free of connective tissue, and diced into small pieces (approximately 2 mm) in ice-cold culture medium for cell isolation. A section was taken for histological examination which confirmed the diagnosis of adrenocortical carcinoma. Adrenal glands were collected from four healthy anesthetized male and female adult dogs, trimmed free of connective tissue, and diced into small pieces (about 2 mm) in icecold culture medium for cell isolation. 2.3. Cell isolation and incubation The adrenocortical carcinoma cells from a dog with Cushing’s syndrome and adrenals removed from the four healthy dogs were isolated according to procedures that were previously described (Carsia et al., 1987). Briefly, adrenal tissues were decapsulated, minced with scissors, washed three times with equal volumes of cell culture medium, and recovered by centrifugation (220 X g) . Adrenal pieces were enzymatically dispersed at 37°C for 1 h with gentle shaking in 50 ml of cell culture medium containing 1 mg ml- ’ of collagenase. Following a 1 h incubation, cells were washed three times with cell culture medium. Cells were filtered through nylon mesh to remove any large cell clumps or cellular debris. Viability was determined using trypan blue dye exclusion and cells were counted on a live/dead cell basis (Freshney, 1983). Cells were diluted to a concentration of 100 000 viable cells per 250 ~1 and directly added to borosilicate culture tubes ( 12 mm X 75 mm). Treatment groups consisted of 0.0,0.03,0.075 or 0.15 bursal equivalents (BEQ) of BASP. For each concentration of the test compound, five replicates of 0.0, 0.01, 0.1, 1.0, IO nM ACTH or 1 mM dibutyryl CAMP were added to the appropriate tubes. Treatments were added to the culture tubes in a total volume of 250 ~1 so that the final volume was 0.5 ml. Cultures were incubated at 37OC, saturated humidity, with 5% CO, for 2 h. Following the incubation, 0.1 mM 3-isobutyl-1-methylxanthine was added to each culture tube and the tubes were then immediately frozen at - 20°C prior to radioimmunoassay (RIA) for cortisol and CAMP. Cortisol (content of cells plus medium) was determined by a commercially available RIA kit. The sensitivity for the cortisol assay was 1 ng ml-’ with an interassay coefficient of variation of 7.1%. Cellular CAMP content of succinylated samples was measured by a highly sensitive and specific RIA. The sensitivity for the CAMP assay was 800 fmol ml ~ ’ with an interassay coefficient of variation of 7.4%. Each experiment was repeated twice using two separate cell dispersions.
38
J.A. Byrd et ai. / Veterinary inmunology and Im~~nopat~o~ogy 47 (I9951 35+Q
2.4. Statistical analysis Data obtained from each experiment were subjected to analysis of variance using the SAS Program (Statistical Analytical Systems Institute Inc., 1988). Significantly different means were separated using Duncan’s multiple range test (Duncan, 1955). Significance is reported at P < 0.05.
3. Results 3.1. Normal adrenocortical cell cortisol biosynthesis In Fig. 1, BASP significantly suppressed cortisol production (up to 23.8%) as compared to unstimulated canine adrenal cells. The highest concentration of BASP evaluated (0.15 BEQ) significantly suppressed (up to 18%) cortisol production from adrenocortical cells derived from healthy dogs at all doses of ACTH. Similarly, BASP (0.075 BEQ) suppressed cortisol biosynthesis (up to 13.7%) from normal adrenocortical cells stimulated with high concentrations of ACTH (0. I-10 nM). The lowest dose of BASP evaluated (0.03 BEQ) was only effective in attenuating cortisol secretion from either unstimulated (0 nM ACTH) or the highest concentration of ACTH ( 10 nM) _The test compound BASP did not affect normal cortisol synthesis in adrenal cells stimulated with 1 mM dibutyryl CAMP. 3.2. Adrenocortical carcinoma cell cortisol biosynthesis Fig. 2 shows cortisol production by isolated ACTH-stimulated and unstimulated canine adrenocortical carcinoma cells in response to increasing concentrations of BASP. Cortisol 0
CONTROL
m
0.03 BEQ BASP
00758EQf2 BASP
015BEQ BASP
3
0.01
0.1 ACTH
1
10
F-CAMP
(nM)
Fig. 1. Effects of increasing concentrations of partially purified BASP (0.03-O. 15 BEQ) on cortisol production from 200 000 normal canine adrenocortical cells ml-’ in the absence or presence of ACTH (O-10 nM) or 1 mM dibutyryl CAMP (DB-CAMP) after 2 h of incubation. Vertical lines represent standard error of the mean. The asterisks depict mean values of two separate experiments which were significantly different (P< 0.05) from the control within each secretagogue concentration.
J.A. Byrd et al. /Veterinary
Immunology and Immunopathology
0.075 BASP
0
0.01
0.1 ACTH
1
BEQli
IO
47 (1995) 35-42
39
0 15 BECI BASP _.
DE-CAMP
(nM)
Fig. 2. Effects of increasing : Cloncentrations of partially purified BASP (0.03%0).15 BEQ) on cortisol production from 200 000 canine adrenocortical carcinoma cells ml- ’ in the absence or presence of ACTH (O-IO nM) or 1 mM dibutyryl CAMP (DB-CAMP) after 2 h of incubation. Vertical lines represent standard error of the mean. The asterisks depict mean values of two separate experiments which were significantly different (P < 0.05) from the control within each secretagogue concentration.
production from unstimulated (0 nM ACTH) adrenocortical carcinoma cells (Fig. 2) was observed to be more than ten-fold greater than basal secretion from cells derived from normal dogs (Fig. 1) . Stimulation of adrenocortical carcinoma cells with ACTH alone further increased cortisol production approximately two-fold at all concentrations evaluated (Fig. 2). All concentrations of BASP evaluated (0.03, 0.075 and 0.15 BEQ) significantly inhibited (up to 34%) basal (unstimulated) cortisol biosynthesis. ACTH-stimulatedcortisol production was suppressed by inclusion of 0.075 BEQ BASP (0.01 nM ACTH) or 0.015 BEQ BASP (0.1 nM ACTH) (Fig. 2). Similarly, all doses of BASP significantly suppressed cortisol production when combined with 1 nM ACTH. Furthermore, only 0.075 or 0.15 BEQ significantly suppressed both 10 nM ACTH- and 1 mM dibutyryl CAMP-stimulated adrenocortical carcinoma cell cortisol biosynthesis (Fig. 2). 3.3. Normal adrenocortical cell CAMPformation Fig. 3 illustrates CAMP production from ACTH-stimulated (O-10 nM) normal adrenocortical cells in the presence of increasing concentrations of BASP (0.03-0.15 BEQ). Regardless of the concentration of ACTH (O-10 nM) evaluated, BASP significantly stimulated CAMP formation in a dose dependent manner (Fig. 3). 3.4. Adrenocortical carcinoma cell CAMPformation Fig. 4 shows CAMP production by isolated ACTH-stimulated and unstimulated canine adrenocortical carcinoma cells in response to 0.75 or 0.15 BEQ BASP. ACTH alone stimulated CAMP formation over two-fold in a dose dependent manner. Both concentrations of BASP evaluated (0.075 or 0.15 BEQ) significantly increased (up to two-fold) ACTH-
40
J.A. Byrd et al. / Veterinary Immunology and lmmunopathology
0
CONTROL
m
0.03 BEQ BASP
0.075 BASP
BEQ ti;]
47 (1995) 3542
0.15 BEQ BASP
7
0
0.01
1
0.1 ACTH
10
(nb.4)
Fig. 3. Effects of increasing concentrations of partially purified BASP (0.034L15 BEQ) on CAMP production from 200 000 normal canine adrenocortical cells ml - ’ in the absence or presence of ACTH (O-10 nM) after 2 h of incubation. Vertical lines represent standard error of the mean. The asterisks depict mean values of two separate experiments which were significantly different (P <0.05) from the control within each secretagogue concentration. cl
CONTROL
m
0.075 BASP
BEQ
0.15 BEQ BASP
I
ZOr-p
0
0.01
1
0.1 ACTH
IO
(nM)
Fig, 4. Effects of increasing concentrations of partially purified BASP (0.075-0.15 BEQ) on CAMP production from 200 000 canine adrenocortical carcinoma cells ml-’ in the absence or presence of ACTH (O-10 nM) after 2 h of incubation. Vertical lines represent stanadard error of the mean. The astricks depict mean values of two separate experiments which were significantly different (P < 0.05) from the control within each secretagogue concentration. stimulated
canine
adrenocortical
carcinoma
cell CAMP formation
in a dose dependent
manner (Fig. 4).
4. Discussion We have evaluated the in vitro efficacy of bursal anti-steroidogenic peptide (BASP) , a peptide isolated from the bursa of Fabricius of chickens, for ability to attenuate cortisol
J.A. Byrd et al. /Veterinary
immunology and Immunopathology 47 (1995) 35-42
41
production from both canine adrenocortical carcinoma cells and normal adrenocortical cells. BASP, derived from the humoral immune system, is a potent and highly efficacious suppessor of in vitro gonadal and adrenal steroid hormone production in birds (Byrd et al., 1993, 1994). Extensive evaluations of the function of this peptide in gonadal tissue have indicated that progesterone biosynthesis is blocked independent of the gonadotrophin receptor (Byrd et al., 1993). Progesterone is a known precursor for most endproduct steroids (Fantl et al., 1973)) and several laboratories have reported that many tumors secrete only precursors for endproduct steroids (Fukushima and Gallagher, 1963; Fantl et al., 1973). In the present studies, incubation of canine adrenocortical cells with BASP caused significant suppression of cortisol production in both normal and carcinoma cells. Previous investigations have indicated that BASP is capable of total suppression of ACTH-stimulated corticosteroid production in a homologous chicken derived adrenocortical cell culture (Tizard, 1992). Similar to previously reported findings, BASP was only partially efficacious in the suppression of ACTH-stimulated cortisol production from normal canine adrenocortical cells in the present study (Fig. 1) (Byrd et al., 1994). Consistent with BASPmediated effects on normal adrenocortical cells, BASP was observed to only partially attenuate either basal (unstimulated) or ACTH-stimulated cortisol production from canine adrenal carcinoma cells in vitro (Fig. 2). Interestingly, BASP suppressed basal cortisol production from tumor derived adrenocortical cells to normal levels when compared to the basal cortisol levels from normal derived adrenocortisol cells. While many small bioactive peptides have been observed to be highly conserved between animal classes with regard to sequence and structure, some divergence of peptide and/or receptor structure may be anticipated with this 3000-5000 molecular weight peptide (Byrd et al., 1993). It is possible that this type of evolutionary divergence, and resultant incomplete agonist activity, is responsible for the incomplete suppression of cortisol secretion in the heterologous system presently investigated. Alternatively, the mammalian homologue of BASP, if such a peptide exists, may modulate corticosteroid production in a less marked fashion than in birds (Byrd et al., 1994). In support of the latter hypothesis, BASP has been shown to markedly increase CAMP levels in normal chicken, porcine and canine adrenocortical cells (Byrd et al., 1994) and BASP induced CAMP elevations in excess of two-fold in both normal adrenal and carcinoma derived cells in the present experiments (Figs. 3 and 4). Approximately two- to three-fold elevations of CAMP have been observed in chicken adrenocortical cells in which complete suppression of ACTH-stimulated corticosteroid production was noted (Byrd et al., 1994). Definitive elucidation of differences in BASPmediated attenuation of avian and mammalian adrenocortical cell function must await molecular characterization of the peptide in each animal class. The present experiments indicate that avian derived BASP is capable of significant suppression of basal and ACTH-stimulated cortisol production from both normal canine adrenocortical cells and canine adrenocortical carcinoma cells in vitro. However, due to the incomplete suppression of cortisol production observed in both types of cells, anticipation of clinical utility of BASP or BASP analogs for treatment of canine Cushing’s disease must be viewed with some skepticism.
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Acknowledgments The authors would also like to acknowledge support for this research from the Texas Advanced Technology Program. The authors thank Dr. Roberta L. Relford for professional assistance.
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