Department of Pharmacology, University of Heidelberg, Heidelberg, Federal Republic of Germany HYPERCORTICISM IN RABBITS IMMUNIZED AGAINST CORTICOSTEROIDS By K. H. Gless, M. Hanka, P. Vecsei and F. Gross ABSTRACT In rabbits immunized with hemisuccinate-albumin complexes of cortisol, corticosterone, deoxycorticosterone, and aldosterone, or against all 4 corticoids simultaneously, antibody titres and plasma concentrations of the corresponding hormones were measured by means of radioimmunoassay. In immunized rabbits, plasma concentrations were generally higher than in non-immunized controls. Plasma concentrations of cortisol were above 100 \g=m\g/100ml in the animals with the highest cortisol antibody titres (control below 3.5 \g=m\g/100ml). Plasma corticosterone concentrations reached levels of 20\p=n-\100\g=m\g/100ml (control below 3.5 \g=m\g/100ml) in rabbits immunized against corticosterone, deoxycorticosterone, or against all 4 corticoids. Concentrations of aldosterone in rabbits immunized against this hormone were 100\p=n-\1000times higher than in the control animals (17.9 \m=+-\3.4 ng/100 ml). In all immunized animals the disappearance of intravenously administered labelled corticoids from plasma was delayed as compared with controls, indicating a diminished plasma clearance rate. After administration of ACTH, plasma concentrations of cortisol and corticosterone were the more elevated, the higher the antibody titre was. Despite the high antibody titres, immunized animals had no signs of adrenal insufficiency. On the contrary, some of the animals showed symptoms of hypercorticism. The mechanisms that may be responsible for the elevated plasma concentrations of the various corticoids are discussed.
When animals are immunized against a hormone, circulating antibodies may influence the release, the metabolism, and the actions of the endogenous hormone. In rats the administration of antiserum against cortisol, aldosterone, or testosterone neutralized the effects of the corresponding hormones (Neri et al. 1964). According to these results, symptoms of adrenal insufficiency might be expected in animals actively immunized against various corticoste roids. However, when we immunized rabbits against aldosterone and cortisol to raise antibodies for the radioimmunoassay of these hormones, no symptoms of adrenal insufficiency were apparent. On the contrary, some of the immunized animals showed patchy loss of hair, skin infections (abscesses), muscular weak ness and hypokalaemia, a syndrome that corresponded to hypercorticism rather than to hypocorticism. To obtain more precise information on the secretory activity of the adrenal cortex under conditions of active immunization against adrenal hormones, plasma concentration and metabolism of cortisol, corti costerone, and aldosterone were investigated in rabbits immunized with various corticoid-protein complexes. METHODS AND MATERIAL Immunization procedure White New Zealand rabbits were immunized against cortisol, corticosterone, deoxy corticosterone, and aldosterone respectively, or against all 4 corticoids simultaneously. Antigens were prepared according to the procedure described by Erlanger et al. (1957) as complexes of corticosteroid-21-hemisuccinates and bovine serum albumin. Imme diately before the intramuscular injection, 1 mg of the antigen was suspended in 0.5 ml of 0.9 /o saline solution and 0.5 ml of complete Freund's adjuvant (DIFCO). Corresponding injections were given every 3 to 4 weeks for a total of one year. Control rabbits received a mixture of Freund's adjuvant and isotonic saline. Anti body titres and plasma concentrations of cortisol and corticosterone were determined repeatedly over a period of one year. Between 6 and 8 months after the first immu nization, the plasma concentration of aldosterone and the disappearance rate of labelled corticoids from the plasma were measured. Antibody titres For the estimation of aldosterone antibody titres the antiserum was diluted by a gamma-globulin buffer solution (0.05 M ph 8 borate buffer containing 0.6 /o serum globulin). For the estimation of the titres of cortisol, corticosterone, and deoxycorti costerone a lysozyme buffer solution (0.05 M ph 8 borate buffer containing 0.1 /o lysozyme) was used as diluent. Dilutions of the antiserum ranging from 10-1 to 106 were incubated for 2 h with the corresponding tritiated corticoid in a dose of 6000 to 8000 cpm. Antibody-bound hormone and free radioactive aldosterone were separated as described by Vecsei et al. (1972t"). Separation of free and bound forms of the other 3 et al. 1972c). The titre was defined as the dilution of antiserum that bound 50% of the added labelled hormone. hormones was performed according to the method for cortisol (Vecsei
Plasma levels Plasma concentrations of cortisol, corticosterone, and aldosterone were determined by means of radioimmunoassay. Cortisol was assayed according to the method of Vecsei et al. (19726). Corticosterone was extracted with benzene from plasma samples of 0.1 to 0.2 ml. The benzene extract was dried in a vacuum exsiccator and the residue dissolved in 1 ml of 5 /o ethanol. The radioimmunoassay was carried out as for cortisol, and the loss of corticosterone during the extraction procedure was estimated accordingly. When water was extracted instead of plasma, the values were virtually zero (Vecsei et al. 1972a,b). Aldosterone was assayed according to the method of Mayes et al. (1970), which was modified by using water instead of methanol for the elution of the paper chromatograms. The eluate (1.2 ml) was purified by glasswool columns (20 4 mm) ; the results were zero blank values. Aldosterone antibodies had been raised in a sheep immunized with aldosterone- 21-hemisuccinate bovine serum albumin complex. For the radioimmunoassay the antiserum was diluted with gamma-globulin solution (1:220 000). Cross-reactions of this antibody with other corticoids have been reported elsewhere (Vecsei 8c Otting 1973). Disappearance rate of labelled corticoids from plasma Tritiated cortisol, corticosterone, or aldosterone was injected intravenously in doses corresponding to 3-5 IO6 cpm. After 90 min, blood was collected from the ear vessels in heparinized plastic tubes and centrifuged immediately. Prior to the extrac tion with dichlormethane, the respective 14C-labelled corticoid was added to the plasma samples. Repeated paper chromatography was carried out in isoamylacetate/ water, E^B (Eberlein 8c Bongiovanni 1955), and in Bush B5 systems. Identity and purity of the isolated tritiated corticoid were confirmed by the ratio of 3H/14C, which remained unaltered during the last steps of the separation procedure. Losses of 3H-corticoids during the isolation procedure were corrected by a factor calculated from the amounts of added and recovered 14C-radioactivity. The radioactivity was measured in a Packard Tricarb Liquid scintillation spectrometer (Type 3003). Materials Chemicals of analytical grade, when available, and corticoids used as standards were purchased from Merck Co., Darmstadt. Tritiated and 14C-labelled corticoids were obtained from New England Nuclear Co., Boston. Other preparations used were: Lysozyme from Serva Co., Heidelberg; Forschungs-Gamma-Globulin from Kabi Co., München; complete Freund's adjuvant from DIFCO Laboratories, Detroit; ACTH was a1-24 tetracosactin (Cortrosyn Depot ) from Organon, Oss (Netherlands). The results were statistically evaluated by Student's -test; the difference between results was regarded as statistically significant at the 0.01 level. RESULTS Two months after the first immunization, antibody titres of cortisol, corti costerone, deoxycorticosterone, and aldosterone ranged between 1:200 and 1:4000. After 6 months, the values ranged between 1:400 and 1:18 000. Sub sequently, no significant changes of the titres were observed until the 12th month of the immunization period.
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When 3H-cortisol and 3H-corticosterone were injected, plasma concentrations measured after 90 min were much higher in immunized than in control rabbits (Table 1). Similarly, the disappearance rate of tritiated aldosterone was de layed in animals immunized against this hormone. In some rabbits immunized against aldosterone the plasma concentration of tritiated cortisol determined 90 min after the injection was higher than in non-immunized rabbits, but the difference was statistically not significant (P>0.10 but < 0.15). Plasma concentrations of cortisol and corticosterone measured in animals immunized either against one of the 4 hormones studied or against all 4 steroids simultaneously were generally higher than those obtained in nonimmunized animals (Fig. 1). In control animals, plasma levels of cortisol and corticosterone were mostly below 1.5 / g/100 ml, and only in a few cases the values ranged between 1.5 and 3.5 / g/100 ml. Rabbits immunized against cortisol had higher plasma concentrations of cortisol than of corticosterone. On the other hand, in animals immunized against corticosterone the plasma concentration of this hormone was higher than that of cortisol. When im munized against all 4 hormones, the plasma concentrations of cortisol and corticosterone were elevated. In rabbits immunized against deoxycorticosterone or aldosterone the plasma levels of cortisol and corticosterone were similarly elevated. The antibodies of these animals cross-reacted with cortisol and with corticosterone. Plasma concentration of aldosterone In the control animals, plasma levels were between 11 and 32 ng/100 ml, Mg/lOOml 125 IMMUNIZED DOC AGAINST aldo* aldo F*B*DOC 100. 75. 50 25. it»* *-i- : 8 4 4 2 4 3 Fig. 1. Plasma concentrations of cortisol ( ) and of corticosterone (^) in rabbits immunized against various corticosteroids. During the immunization period, repeated estimations were done in the same animal. C: controls; F: cortisol; B: corticosterone; DOC: deoxycorticosterone; aldo: aldosterone.
whereas in animals immunized against aldosterone, plasma concentrations rose above 1000 ng/100 ml (Table 2). Similar values were measured in 3 of 4 rabbits immunized against aldosterone and the 3 other hormones, whereas in the 4th animal of this group the plasma concentration of aldosterone was not as high but still significantly above that measured in controls. In cortisolimmunized rabbits the plasma concentration of aldosterone was also elevated, but less than in rabbits immunized against aldosterone. After administration of synthetic ACTH (0.3 mg Cortrosyn Depot ), plasma levels of cortisol and corticosterone were the more elevated, the higher the antibody titre was (Fig. 2). When antibody titres were above 1:10 000, cortisol attained plasma levels between 120 and 210 / g/100 ml. In contrast to non-immunized control animals, several of the immunized rabbits exhibited loss of hair and skin abscesses. Two rabbits, immunized against cortisol and corticosterone respectively, and 2 other animals, immunized simultaneously against all 4 hormones, developed severe muscle paralysis and died within a few days. In 4 animals that had raised antibodies against aldo sterone the plasma concentration of potassium decreased from 4.7 ± 0.2 mval/1 to 3.7 mval/100 ml or below. All rabbits that showed symptoms of hyper corticism had antibody titres above 1:5000. DISCUSSION In rabbits immunized against cortisol, corticosterone, aldosterone, or deoxy corticosterone the plasma concentrations of the corresponding hormones were Table 2. Plasma concentration of aldosterone in ng/100 ml in rabbits immunized against corticosteroids. Plasma samples were taken 8 months after first immunization. Immunized against aldo F + DOC 4-4- aldo 9.3 11.2 15.8 15.8 18.2 18.3 37.0 1498 3760 6750 11220 217 1080 1180 1790 36 84 212 675 C: control rabbits; aldo: DOC: deoxycorticosterone. alsosterone; F: cortisol; B: corticosterone;
µ,/iooml 200 150 100 Fig. 2. Increase in plasma concentrations of cortisol ( - ) and of corticosterone (A-A) after injection of 3 mg of synthetic ACTH. Lines connect values determined in individual animals prior to, and 3 h after, giving ACTH. C: controls; F, B: rabbits immunized against cortisol or corticosterone; F 4-4- DOC + aldo: rabbits immunized against all 4 hormones. markedly elevated in comparison to non-immunized controls. In general, plasma concentrations were the higher, the higher the antibody titre of the corresponding hormones was. Although plasma concentrations of cortisol, corticosterone, and aldosterone were most elevated in those rabbits that had raised specific antibodies against each of these hormones, elevated plasma levels of cortisol and corticosterone were also measured in animals immunized against aldosterone. Furthermore, administration of ACTH resulted in a more marked adrenocortical response in immunized rabbits than in control animals. The increase in plasma concentrations of the various cortical hormones may be explained by either a reduced plasma clearance rate or an increased secre tion rate of the corresponding hormones, or by a combination of the 2 me chanisms. It has been demonstrated that in sheep immunized against oestradiol the disappearance of tritiated oestradiol from plasma was delayed (Longcope 1970). In our experiments, disappearance of the various radioactive hormones that had been administered was also delayed, possibly as a consequence of a diminished plasma clearance rate. However, such a mechanism may only in part contribute to the marked increase in plasma concentrations measured in
our studies and would not explain the increased sensitivity of the adrenal cortex to ACTH. In immunized animals, production of adrenocortical hormones may be stim ulated by a change in the ratio between free and protein-bound hormones in plasma. Plasma concentrations of the hormones refer to both free and proteinbound hormones. After immunization the percentage of protein-bound hor mones may rise as demonstrated in rabbits immunized against testosterone and oestradiol (Nieschlag et al. 1973). Since only the plasma concentration of free hormone is active in controlling ACTH release by means of a feed-back concentration of the mechanism, it might be possible that the total plasma cortical hormones is increased without a corresponding inhibition of the secre tion of ACTH. Under these conditions, high total plasma concentrations of adrenocortical hormones would not result in a proportional increase in nonprotein bound hormones. The fact that symptoms of a hypercorticism have been observed simultaneously with an enhanced response of the adrenal cortex to exogenous ACTH makes it probable that by the process of active immuni zation the adrenal cortex is stimulated to produce more hormones than in non-immunized rabbits. This may in part be a compensatory mechanism to overcome the binding of endogenous hormones to increasing concentrations of antibodies. For a more precise analysis of the observations reported it would be neces sary to determine free and protein-bound hormones in plasma and, in addi tion, to study the morphology of the adrenal cortex in immunized and nonimmunized animals. REFERENCES Eberlein W. R. Se Bongiovanni. M.: Arch. Biochem. 59 (1955) 90. Erlanger B., Borek F., Beiser S. M. Se Liebermann S.: J. biol. Chem. 228 (1957) 713. Longcope C. In: Peron F. G. and Caldwell B. F., Eds. Immunological Methods in Steroid Determination. Appleton-Century-Crofts, New York (1970) 428. Mayes D., Furuyama S., Kern D. C. Sc Nugent C..: J. clin. Endocr. 30 (1970) 682. Neri R. O., Tolksdorf S., Beiser S. M., Erlanger., Agate F. J. Se Liebermann S.: Endocrinology 74 (1964) 593. Nieschlag E., Usadel K. H., Schwedes U., Kley H. K., Schaffung K. Se Kriiskemper H. L.: Acta endocr. (Kbh.) 72, Suppl. 173 (1973) 116. Vecsei P. 8c Otting W.: J. int. Res. Commun. 1 (1973) 15. Vecsei P., Akangbou C, Joumaah A. Sc Salloum N. L: Acta endocr. (Kbh.) 69, Suppl. 159 (1972a) 33. Vecsei P., Joumaah., Salloum N. 1. Se Akangbou C: Excerpta med. (Amst.), Inter national Congress Séries No. 256 (19726) 388. Vecsei P., Renke B., Katzy R. 8c Baek L.: Experientia (Basel) 8 (1972c) 1104. Received on June 12th, 1973.