A B S T R A C T The pyrogenic properties of some C-19 and C-21 steroids were examined by in vitro incubation of human blood leukocytes with serum-buffer solutions of the steroids and injection of the 18-hr supernatants into rabbits. In previous studies this method demonstrated release of leukocyte endogenous pyrogen by etiocholanolone. With two exceptions, steroids known to cause fever in man, such as 11p-OH etiocholanolone and 3a-hydroxy-5P-pregnane-20-one were also pyrogenic in vitro. All steroids tested which are nonpyrogenic in man, such as androsterone, 3,-OH etiocholanolone, and 3a, 1 7a-dihydroxy-5P-pregnan-20-one were also nonpyrogenic in vitro. Solubility in aqueous solution did not correlate with pyrogenic capacity. INTRODUCTIONIn 1956, Kappas, Hellman, Fukushima, and Gallagher (1) first reported that injection of the naturally occurring steroid etiocholanolone (5fi-androstane-3a-ol, 17-one)1 produced fever in man. This finding has been confirmed in many subsequent reports (2-5). In addition, recent studies have shown that incubation of a serum-buffer solution of etiocholanolone with human blood leukocytes in vitro causes release of an endogenous pyrogen (6, 7). This in vitro reaction has many features which closely resemble those described for the experimental fever caused by injection of etiocholanolone and related steroids into normal subjects (5, 8).The pyrogenicity of various steroids structurally related to etiocholanolone has been investigated in man and certain features of steroid structure have been correlated with pyrogenic activity (8, 9). Both C-19 (3-5, 10) and C-21 (4, 5, 11) steroids, as well as certain bile acids (12), have been studied. It seemed of interest to examine the action of some of these steroids on leukocytes in vitro, to determine whether similar structural features would also be required for pyrogenic activity in this system. Inhibition of etiocholanolone fever was observed when subjects had received cortisone injections on preceding days (2) hydroxy-5fi-androstan-17-one; 11,11 -dihydroxy-5g-androstan-17-one; androsterone, 3a-hydroxy-5a-androstan-17-one; 3p-OH etiocholanolone, 3fi-hydroxy-5,f-androstan-17-one; hydrocortisone, 11i, 17a,21-trihydroxy-4-pregnene-3,20-dione; estradiol, 1,3,5(10) -estratriene-3,17#-diol; progesterone, 4-pregnene-3,20-dione; lithocholic acid, 3a-hydroxy-5fi-cholan-24-oic acid; deoxycholic acid, 3a, 12a-dihydroxy-5,8-cholan-24-oic acid; dehydroepiandrosterone (DHEA), 3a-hydroxy-5-androsten-17-one; 3,17-etiocholanedione, 5ft-androstane-3,17-dione. 2418The Journal of Clinical Investigation Volume 49 1970 lanolone into the same intramuscular site (13). Etiocholanolone produces less fever in women than in men (14,15), and in one study, administration of estrogen to women appeared to diminish febrile responses to etiocholanolone, but not to endotoxin (7). In view of these observations, the possible inhibitory effects of hydrocortisone and estradiol on pyrogen release in vitro were investigated. METHODS Leukocytes. Preparations of leukocytes and method...