Inhibition of human lymphocyte stimulation by steroid hormones: cytokinetic mechanisms.

The steroid hormones estradiol, progesterone and testosterone, in addition to cortisol, inhibited stimulation of human peripheral blood lymphocytes by phytohaemagglutinin (PHA) and Con A. This effect upon lymphocyte transformation was assayed by three methods: quantitation of [3H]thymidine incorporation into acid precipitable material, microscopic assessment of blastic transformation and determination of the labelling index. Addition of steroid hormones at the initiation of culture resulted in a marked inhibition in all three parameters, which was observed with lower concentrations of cortisol than the other hormones. The inhibition was not attributable to cell death and could be partially reversed by removing hormones from the incubation medium after culture for 48-72 hr. Late addition of steroid hormones, 52 hr after addition of mitogen and 18 hr prior to pulse-labelling with [3H]thymidine, also resulted in reduced [3H]thymidine incorporation, accompanied by a nearly 50% reduction in the labelling indices and only a minimal decrease in the per cent transformed cells. Inhibition of lympohcyte stimulation by steroid hormones operates by the following cytokinetic mechanisms: (1) suppressed recruitment of cells from G3 to G1 phase of the cell cycle, as indicated by the diminished per cent blasts; (2) inhibition of progression from G1 phase into S phase, as evidenced by the reduced ratio [labelling index/blasts]; and, in the case of estradiol and progesterone, (3) reduced rate of DNA replication or altered intracellular [3H]thymidine specific activity as shown by the decreased ([3H]thymidine incorporation/labelling index) ratio. Late addition of steroid hormones to stimulated cultures reduced the per cent of cells in S phase, but did not revert previously transformed cycling lymphocytes to the G3 state.     

Evidence for direct regulation of epidermal growth factor receptors by steroid hormones in human endometrial cells.

Recent evidence suggests that epidermal growth factor (EGF) may serve as a paracrine and/or autocrine mediator of oestrogen action in uterine growth. In this study, the effects of various steroid hormones on EGF receptors in primary cultures of human endometrial cells were examined. Human endometrial cells exhibited a single class of high-affinity binding sites for EGF (Kd: 0.14 nM) with approximately 3600 receptors/cell. The addition of progesterone increased the EGF binding without affecting the Kd value. Cortisol also increased EGF binding and acted additively with a relatively low concentration of progesterone (10(-10) M). Oestradiol alone had no effect on EGF binding. However, oestradiol in combination with progesterone and cortisol further increased EGF binding. These results present evidence for the direct regulation of EGF receptors by steroid hormones in human endometrial cells, and raise the possibility that steroid hormones may act on these cells in part by modulating EGF receptors.     

Sex steroid hormones and circulating IgE levels.

The possible influence of sex steroid hormones on circulating IgE levels in general and IgE anti-Candida antibodies in particular was studied by quantification of plasma levels of progesterone, estradiol and IgE (total and anti-Candida-specific) in females during the follicular and luteal phases of the menstrual cycle, and during pregnancy. IgE levels during the follicular and luteal phases were not significantly different, although the mean values for the luteal phase were slightly lower. This trend was apparent in daily samples from two normal females during one menstrual cycle. During pregnancy, when the levels of circulating sex steroids were high, IgE levels were only slightly higher than in the follicular and luteal phases. In men and in gonadal dysgenetics, circulating progesterone levels were similar to those of women during the follicular phase (i.e., lower than in the luteal phase or in pregnancy), but the IgE levels were not different. The apparently low levels of IgE during the luteal phase may therefore be due to physiological factors other than fluctuations in the sex steroid hormones. From the present studies, it is apparent that sex steroid hormones have little or no effect on humoral IgE levels, in marked contrast to previously described correlations for other immunoglobulins, especially anti-Candida antibodies.     

Inactivation of lysis of oncornaviruses by human serum.

The phenomenon of oncornavirus inactivation by human serum was examined. As judged by 50% inhibition of plaque or focus formation, every fresh human serum tested inactivated eight different oncornaviruses at serum dilution endpoints of 1:16. Fresh human serum released RNA-dependent DNA polymerase (RDDP) activity from each of 10 tested oncornaviruses. Thus, at least one mechanism of the human serum-induced virus inactivation was by viral lysis. The RDDP assay provided us with a simple method to screen the virolytic properties of many sera against a number of viruses. Oncornaviruses were lysed by sera from monkeys and cats, but not by sera from guinea pigs, rabbits, chickens, rats, goats, and mice ($\text{BALB}\text{c}$, CBA, NIH Swiss). Heated normal human sera did not inactivate or lyse oncornaviruses, nor did they sensitize virions to guinea pig serum-mediated lysis. However, serum from a human vaccinated against Rauscher leukemia virus did sensitize the virus to guinea pig serum-mediated lysis. This ability of human sera to lyse oncornaviruses may be a cause of the difficulties in finding oncornaviruses in humans.     

Effect of steroid hormones on virus-induced diabetes mellitus.

Female ICR Swiss mice, generally resistant to the diabetogenic effects of the D variant of encephalomyocarditis virus, develop diabetes to the same extent as males if they are pretreated with testosterone. The data suggest that testosterone is one of the factors involved in the development of diabetes in certain strains of mice.     

Influence of steroid hormones on bovine pericardial calcification.

Calcification is a frequent cause of the clinical failure of bioprosthetic heart valves fabricated from glutaraldehyde pretreated bovine pericardium (GABP). The present investigation describes the influence of steroid hormones in the mineralization of GABP, in an extra-circulatory environment. Calcification was studied on GABP incubation in a metastable solution of calcium phosphate containing steroid hormones such as estrone, progesterone, 7(OH) progesterone, testosterone and beta-estradiol. It is interesting to note that certain steroids can variably increase the GABP calcification. Further, the effect of these steroids in an in vitro hydroxyapatite (HA) formation was investigated. In addition, we observed these steroids alter the calcium transport through GABP in diffusion experiments and also in HA formation. Therefore, it is conceivable that prolonged use of steroids or steroids containing oral contraceptive agents may not be advisable for patients having bioprosthetic implants in contact with blood. A better understanding of the mechanism of these drugs under in vivo conditions is needed to develop applications.     

Immunosuppression by sex steroid hormones. The effect upon PHA- and PPD-stimulated lymphocytes.

Progesterone, estradiol, testosterone, cortisol, and 11-desoxycortisol (compound S) were added to cultures of human lymphocytes stimulated with phytohaemagglutinin (PHA) and purified protein derivative (PPD). The immunosuppressive effect of cortisol was verified and the three sex-steroid hormones also were found to inhibit lymphocyte transformation although at concentrations higher than for cortisol. Compound S, a steroid of low biological potency, also had immunosuppressive activity. At concentrations (0-01-1-0 microng/ml), progesterone, oestrogen, testosterone, and Compound S augmented the transformation response to PPD but not to PHA. Marked variation from individual to individual in the suppressive effects of all the steroids were noted. The clinical implications of immunosuppression by the sex steroid hormones are discussed.     

Influence of steroid hormones on the growth fraction of human breast carcinomas

The growth fraction (GF) of human breast adenocarcinomas was studied in order to determine whether it was influenced by certain biologic characteristics of the host and whether it was modulated by the tumor's steroid hormone receptor status. It was also examined whether steroid treatment in vitro stimulated tumor's GF. Thirty-five primary breast adenocarcinomas from patients ranging in age from 26 to 85 years were analyzed for size and estrogen receptor (E2R) and progesterone receptor (PR) status. One portion of each tumor was cultured in medium 199 under four different conditions: addition of insulin alone or with 17 beta-estradiol (E), progesterone (P), or a combination of both hormones (E + P). In six cases, a dose-response curve was established for E (10(-14) to 10(-12) mol/L) and P (10(-11) to 10(-9) mol/L). Tissues were exposed to a continuous pulse of 2.5 microCi/mL (3H)-thymidine for five days before fixation and processing for autoradiography. GF ranged from 0.0074 to 0.3852, median 0.0550, and it was arbitrarily classified as low GF, less than 0.0550; or high GF, equal or greater than 0.0550. GF was significantly higher in patients with lymph nodes free of metastatic tumor than in those with positive lymph nodes. Estradiol treatment at low doses increased GF in 40% of tumors, whereas high doses increased GF in 14%. Progesterone treatment increased GF in 30% of tumors treated with low doses and in 14% of those treated with high doses. Treatment with E + P markedly depressed the GF of 85% of the tumors. It was concluded that the GF of breast carcinomas showed a great variability, which seemed to be independent of host factors such as age, menopausal status, parity history, or smoking habits; a second conclusion was that breast tumors' GF was not stimulated by steroid hormone treatment in vitro, and its response did not correlate with their hormone receptor status.     

Histologic response of normal human endometrium to steroid hormones in athymic mice

Despite the widespread use of hormonal therapy for menopausal symptoms, oral contraception, and treatment of metastatic breast carcinoma, information concerning the histologic and biologic effects of individual sex steroids on the human endometrium remains incomplete. Repetitive endometrial biopsy is impractical, and ethical constraints limit the dosage and duration of administration for some steroids. The ovariectomized athymic mouse was investigated as a host for human endometrium in which the hormonal milieu may be manipulated and the histologic response determined. Minced proliferative-phase endometrium from hysterectomy specimens of normally cycling women was inserted into the subcutis of 4- to 6-week-old mice. Proliferation of endometrial gland cells occurred in the transplanted endometrium of estradiol-treated mice, while the complete sequence of secretory-phase events, including subnuclear vacuolization, luminal secretion, and decidualization of stroma, was observed during a 14-day period of treatment with estradiol and progestin (medroxyprogesterone acetate). Progestin treatment alone also caused secretory-phase changes, but the response was delayed and appeared weaker. The transplanted endometrium in control mice appeared to be inactive. These observations provide support for the use of this model to study the histologic response of human endometrium to sex steroids.     

Sex steroid hormones and antibodies to Candida albicans.

The effect of changes in progesterone (P) and estradiol (E2) on titres of antibodies to Candida albicans was studied by measurement of these three parameters in the following endocrinologically diverse human groups: normal females, gonadal dysgenetics, users of a sequential oral contraceptive (Oracon) and normal males. In females, C. albicans titres (mean +/- s.e.m.) were significantly higher (P less than 0.05) in the luteal (74 +/- 14) than in the follicular phase (34 +/- 19) of the cycle, and there were similar significant increases in P and E2. In the gonadal dysgenetic group (n = 29), with E2 levels comparable with males, the antibody titres were also equivalent to those in normal males (40 +/- 0.5), but were significantly lower than those of normal females in the follicular phase (P less than 0.05). In contrast, Oracon users, with high blood progestin levels, had C. albicans titres (118 +/- 15) significantly higher (P less than 0.001) than those of control subjects during the follicular phase. A significant correlation (P less than 0.05) was observed between P and C. albicans titres (mainly IgA) in randomly selected samples (n = 112) from normal females during the follicular and luteal phases, and in two subjects from whom blood samples were drawn daily for the entire cycle. In the latter, an increase in E2 but not P in the late follicular phase was accompanied by a marked decrease in C. albicans titres. No changes were observed in total immunoglobulin levels or antibodies to SRBC or Herpes virus in response to the marked changes in hormones. These results indicate that the production of antibodies to C. albicans may be specifically influenced by sex steroid hormones, being enhanced by P and E2 at low levels but depressed by E2 at high levels.     

Transport of protein-bound steroid hormones into liver in vivo.

The unidirectional influx of 3H-gonadal (progesterone, dihydrotestosterone, testosterone, estradiol) and adrenal (aldosterone, cortisol) steroid hormones into liver was studied with a tissue-sampling single-injection technique in barbiturate-anesthetized rats. Liver uptake of the steroid hormone was measured relative to [14C]butanol, a highly diffusible reference, after a single pass through the liver. Portal flow (1.4 ml.min-1.g-1) under the experimental conditions was measured with 3H2O. The extraction of unidirectional influx of all six steroid hormones was 70-100% after a bolus portal injection of labeled steroid in Ringer solution (0.1% albumin). Steroid transport was nonsaturable because 35 muM concentrations of unlabeled hormone resulted in no inhibition of liver transport. The plasma proteins (albumin and specific globulins) in serum from human (male, female, pregnancy), rat (male), and guinea pig (pregnancy) sources inhibited the liver clearance of the respective steroid hormones to a variable extent. In all cases albumin-bound steroid hormone was freely cleared by liver and, in the case of cortisol or estradiol, the fraction bound to a specific globulin was also transported into liver.     

Inactivation of Escherichia coli penicillin-binding proteins by human neutrophils.

Neutrophils use a variety of microbicidal mechanisms in their role as one of the primary arms of the human host defense system. We have previously observed that a cell-free system containing myeloperoxidase (MPO), one of the major components of the neutrophil's oxidative antimicrobial systems, inactivated microbial penicillin-binding proteins (PBPs), which mediate the formation of the peptidoglycan layer of eubacterial cell walls. This is a potentially important mechanism of MPO-mediated bacterial toxicity. Since numerous other microbicidal systems, both oxidative and nonoxidative, are used by whole neutrophils, we investigated the effect of intact neutrophils on Escherichia coli PBPs. Penicillin binding activity was progressively reduced by neutrophil exposure for all PBPs. Loss of penicillin binding activity correlated well with loss of microbial viability for almost all PBPs. Azide-treated neutrophils, MPO-deficient neutrophils, and chronic granulomatous disease neutrophils inactivated E. coli PBPs in a manner similar to that of normal neutrophils, suggesting that MPO-independent, and even oxygen-independent, microbicidal systems are also involved in inactivation of PBPs. PBP inactivation, an antimicrobial strategy used by beta-lactam-producing molds (and now by physicians), may be an important microbicidal mechanism used by human neutrophils.     

Inactivation of human cytomegalovirus by phytohemagglutinin

Summary Human cytomegalovirus (CMV) was inactivated by treatment with phytohemagglutinin (PHA) in contrast to herpes simplex virus (HSV), which was not. Approximately 90 per cent of infectivity was lost following exposure of CMV to PHA. Greater reduction of infectivity, more than 99 per cent, was obtained following pretreatment of cells with PHA than by direct mixture of the virus and the lectin. Protection of cells was still observed 48 hours after pretreatment of cells with PHA. No difference was found in sensitivity to PHA with 3 strains of CMV tested. PHA preparations from different sources were almost equally effective in inactivation of CMV, whereas leucoagglutinin had minimal effect. Our data suggest that the site of action of PHA occurs at the step(s) of penetration and/or uncoating. Resistance of CMV to trypsin was confirmed, and the enzyme had no effect on sensitivity to lectins.With 1 FigurePresented in part at the Annual Meeting, American Society for Microbiology, New York, N.Y., 1975.     

Co-expression of CXCR4 and CXCR7 in human endometrial stromal cells is modulated by steroid hormones

Endometrium modulated by estrogen (E) and progesterone (P) is important for implantation and pregnancy. The present study compared the expression of chemokine CXCL12 and chemokine receptor CXCR4 and CXCR7 between human cycling and early pregnant endometria by immunohistochemistry (IHC). Then the modulation of E and P on expression of CXCL12, CXCR4 and CXCR7 in human endometrial stromal cells (ESCs) was explored at both mRNA and protein level. The result of IHC showed that human ESCs of the menstrual period did not express CXCL12, CXCR4 or CXCR7 protein, however, the expression of CXCR4 and CXCR7 but not CXCL12 in ESCs increased in the proliferative and secretory phase, and the expression intensity for CXCR4 and CXCR7 in ESCs was the highest in the first trimester. Moreover, E and P were able to up-regulate the mRNA and protein expression of CXCR4 and protein expression of CXCR7 in ESCs (P<0.01). Thus, ESCs spatiotemporally co-express CXCR4 and CXCR7 rather than CXCL12, and E and P are able to regulate the expression of CXCR4 and CXCR7 in ESCs, suggesting the modulation of steroid hormones on chemokine receptor expression in ESCs.