Androgen and Androgen Receptor as Enhancers of M2 Macrophage Polarization in Allergic Lung Inflammation.

Allergic asthma is a disease initiated by a breach of the lung mucosal barrier and an inappropriate Th2 inflammatory immune response that results in M2 polarization of alveolar macrophages (AM). The number of M2 macrophages in the airway correlates with asthma severity in humans. Sex differences in asthma suggest that sex hormones modify lung inflammation and macrophage polarization. Asthmatic women have more M2 macrophages than asthmatic men and androgens have been used as an experimental asthma treatment. In this study, we demonstrate that although androgen (dihydrotestosterone) reconstitution of castrated mice reduced lung inflammation in a mouse model of allergic lung inflammation, it enhanced M2 polarization of AM. This indicates a cell-specific role for androgens. Dihydrotestosterone also enhanced IL-4-stimulated M2 macrophage polarization in vitro. Using mice lacking androgen receptor (AR) in monocytes/macrophages (ARfloxLysMCre), we found that male but not female mice exhibited less eosinophil recruitment and lung inflammation due to impaired M2 polarization. There was a reduction in eosinophil-recruiting chemokines and IL-5 in AR-deficient AM. These data reveal an unexpected and novel role for androgen/AR in promoting M2 macrophage polarization. Our findings are also important for understanding pathology in diseases promoted by M2 macrophages and androgens, such as asthma, eosinophilic esophagitis, and prostate cancer, and for designing new approaches to treatment.

Dihydrotestosterone levels at birth associate positively with higher proportions of circulating immature/naïve CD5+ B cells in boys.

Boys present with higher proportions of immature/naïve CD5+ B cells than girls up to 3 years of age. Boys also have higher fractions of regulatory T cells (Tregs) in early infancy, but the mechanisms for these sex-related differences are unknown. In the prospective FARMFLORA follow-up study of 23 boys and 25 girls, we investigated if these immunological differences remained at 8 years of age. We also examined if testosterone or dihydrotestosterone (DHT) levels at birth and at 8 years of age were associated with immune maturation. Immunological variables and androgen levels were examined and measured in blood samples obtained at birth, 3-5 days and at 8 years of age. Boys had higher proportions of CD5+ and immature/transitional CD24hiCD38hi B cells, whereas girls had higher fractions of B cells with a memory phenotype at 8 years of age. School-aged boys also presented with higher frequencies of Tregs, and a greater capacity to produce T-cell-associated cytokines. Among boys, higher cord blood DHT levels were associated with higher proportions of CD5+ B cells in early infancy and at 8 years of life. These results suggest that DHT actions in utero might be involved in the mechanism for delayed peripheral B-cell maturation in boys.

Gender-specific expression of beta1 integrin of VLA-4 in myelin basic protein-primed T cells: implications for gender bias in multiple sclerosis.

Susceptibility to multiple sclerosis is higher in females than males. However, the underlying mechanism behind this gender difference is poorly understood. Because the presence of neuroantigen-primed T cells in the CNS is necessary to initiate the neuroinflammatory cascade of multiple sclerosis, we first investigated how these T cells interacted with astroglia, major resident glial cells of the CNS. Interestingly, we found that myelin basic protein (MBP)-primed T cells from female and castrated male mice, but not from male mice, produced proinflammatory molecules, such as NO, IL-1beta, and IL-6 in astroglia, and these responses were purely via contact between T cells and astroglia. Because T cell:glia contact requires several integrin molecules, we examined the involvement of integrins in this process. Both alpha4 and beta1, subunits of VLA-4 integrin, were found to be necessary for T cell contact-induced generation of proinflammatory molecules in astroglia. Interestingly, the expression of beta1, but not alpha4, was absent in male MBP-primed T cells. In contrast, female and castrated male MBP-primed T cells expressed both alpha4 and beta1. Similarly, we also detected beta1 in spleen of normal young female, but not male, mice. Furthermore, we show that male sex hormones (testosterone and dihydrotestosterone), but not female sex hormones (estrogen and progesterone), were able to suppress the mRNA expression of beta1 in female MBP-primed T cells. These studies suggest that beta1, but not alpha4, integrin of VLA-4 is the sex-specific molecule on T cell surface, and that the presence or absence of beta1 determines gender-specific T cell contact-mediated glial activation.

Experimental autoimmune prostatitis: dihydrotestosterone influence over the immune response.

PURPOSE: In experimental autoimmune prostatitis in a rat model of chronic prostatic inflammation of noninfectious origin the prostatic 5alpha-dihydrotestosterone (DHT) concentration decreases because of depressed 5alpha-reductase activity. This decrease in androgens in situ could favor the development of autoimmune status at the same time. We noted that a DHT increase could protect the gland from immune aggression and/or its consequences in regard to prostatic androgenic metabolism. MATERIALS AND METHODS: We analyzed in vitro the (3H)-DHT enzymatic bioconversion of prostate homogenates of male accessory sexual gland extract (MAG) immunized rats and MAG immunized plus DHT implanted rats (DSG rats), and performed ventral prostate histological observations. The specific cell immune response against MAG antigen(s) was studied by delayed type hypersensitivity. RESULTS: In DSG and MAG rats, and controls enzymatic activities (3alpha/3beta-hydroxysteroid oxidoreductases) were 112.7 +/- 11.3, 91.4 +/- 15.0 (not significant) and 147.0 +/- 12.8 pmol per minute per mg protein (p <0.025). Histological findings in DSG rat ventral prostates revealed infiltrating mononuclear cell foci in lower quantity and less magnitude than in MAG rat prostates. Delayed type hypersensitivity values were positive in MAG rats and lower in DSG rats in relation to kidney treated and untreated rats. CONCLUSIONS: Results suggest that constantly elevated DHT levels could decrease the cell immune response but not at significantly. In contrast, androgenic metabolism remains altered in the presence of exogenous androgens.

Immunization against 5alpha-androstane-3alpha,17beta-diol affects ovarian folliculogenesis in Merino ewes.

The 5alpha-reduced androgens have been implicated as antagonists of follicular development. In this experiment, we examined the effect of active immunization against 5alpha-reduced androgen on follicular development in ewes. During the breeding season, cyclic Merino ewes were either actively immunized three times against 5alpha-androstane-3alpha,17beta-diol (3alpha-diol) or served as controls. Six to nine weeks after the last immunization, they were treated with PGF(2alpha) analog (PG, 125mg cloprostenol i.m.) and luteolysis was induced. Fourteen days after the PG treatment, the ewes were either killed (mid-luteal phase) or treated a second time with PG and killed 24h later (early follicular phase). At slaughter, blood samples were collected and ovaries recovered. All CL and follicles larger than 2mm were dissected and their size and appearance were recorded. Follicular fluid was collected and concentrations of estradiol-17beta (E(2)), progesterone (P), androstenedione (A(4)), testosterone (T), dihydrotestosterone (DHT), 5alpha-androstane-3alpha-ol,17beta-one (androsterone: 3alpha-ol) and 3alpha-diol were determined by RIA. Immunization induced antibodies primarily to DHT and its 5alpha-reduced substrates 3alpha-diol and 3alpha-ol but not to E(2), P, A(4) or T. Immunization increased ovulation rate, size of ovulatory follicles and weight of CL. Immunization appeared to increase ovulation rate by decreasing the incidence of atresia in large preovulatory follicles. Regardless of their physiological status follicles contained only low levels of DHT; 3alpha-ol and 3alpha-diol were not detected in most follicles. Immunization did not appear to affect levels of DHT or other steroids in the follicular fluid. In conclusion, the induction of antibodies to 5alpha-reduced androgens increases ovulation rate by enhancing follicular viability during the preovulatory period in ewes. However, this effect is not brought about by the direct immune-neutralization of DHT or its 5alpha-reduced substrates 3alpha-ol and 3alpha-diol at the ovarian level.

Sex steroid hormones enhance immune function in male and female Siberian hamsters.

Immune function is better in females than in males of many vertebrate species, and this dimorphism has been attributed to the presence of immunosuppressive androgens in males. We investigated the influence of sex steroid hormones on immune function in male and female Siberian hamsters. Previous studies indicated that immune function was impaired in male and female hamsters housed under short-day photoperiods when androgen and estrogen concentrations were virtually undetectable. In experiment 1, animals were gonadally intact, gonadectomized (gx), or gx with hormone replacement. Females exhibited the expected increase in antibody production over males, independent of hormone treatment condition, whereas male and female gx animals exhibited decreased lymphocyte proliferation to the T cell mitogen, phytohemagglutinin (PHA) compared with intact animals, and this effect was reversed in gx hamsters following testosterone and estradiol treatment, respectively. In experiment 2, testosterone, dihydrotestosterone, and estradiol all enhanced cell-mediated immunity in vitro, suggesting that sex steroid hormones may be enhancing immune function through direct actions on immune cells. In experiment 3, an acute mitogen challenge of lipopolysaccharide significantly suppressed lymphocyte proliferation to PHA in intact males but not females, suggesting that males may be less reactive to a subsequent mitogenic challenge than females. Contrary to evidence in many species such as rats, mice, and humans, these data suggest that sex steroid hormones enhance immunity in both male and female Siberian hamsters.

Testosterone and/or low estradiol: normally required but harmful immunologically for males after trauma-hemorrhage.

BACKGROUND: Previous studies indicate that after severe hemorrhage, immune functions are markedly depressed in males, whereas females do not show any depression. Although androgen depletion by castration of mice before soft-tissue trauma and hemorrhagic shock prevents the depression of cell-mediated immunity, it remains unknown whether testosterone per se is responsible for producing immune depression. METHODS: Female C3H/HeN mice were pretreated with 5alpha-dihydrotestosterone (DHT) or vehicle for 20 days. The mice then underwent soft-tissue trauma (laparotomy) and hemorrhagic shock (blood pressure 35+/-5 mm Hg for 90 minutes) followed by adequate fluid resuscitation (shed blood and lactated Ringer's solution) or sham operation. Two groups of nontreated male C3H/HeN mice were included as controls: one group was subjected to hemorrhagic shock followed by resuscitation, and the second group underwent only sham operation. At 24 hours after trauma-hemorrhage and resuscitation, animals were killed, macrophages harvested from the peritoneum and spleen, and their ability to release interleukin (IL)-1 and IL-6 was evaluated. Plasma DHT, estradiol, and corticosterone levels were measured by radioimmunoassay. RESULTS: Treatment of female mice with DHT produces a significant increase in DHT levels that was comparable with those seen in nontreated male mice. Alternatively, estradiol levels in female mice were significantly depressed by DHT treatment to levels comparable with those observed in control males. In the vehicle-treated female mice, no depression of the macrophage function was evident after trauma hemorrhage. In contrast, testosterone-treated female mice that had experienced hemorrhage showed significant depression in splenic and peritoneal macrophage IL-1 and IL-6 production, comparable with the values seen in macrophages from male mice that had experienced hemorrhage. CONCLUSIONS: These findings indicate that pretreatment of female mice with DHT depresses macrophage function after trauma-hemorrhage, which mimics the changes seen in normal male mice subjected to trauma-hemorrhage. We propose, therefore, that high testosterone and/or low estradiol levels are responsible for producing the immune depression in male mice after trauma-hemorrhage. Testosterone receptor blocking agents, e.g., flutamide, and/or estradiol administration should thus be useful adjuncts for preventing immune depression in male trauma patients.

Ontogeny of immunocytochemically demonstrable androgen- and androgen receptor-containing cells in the hypothalamus and adenohypophysis of the chick embryo.

Brain sections of male chick embryos, 6.5-18.5 days of age, were examined immunocytochemically for the presence of androgen- and androgen receptor-containing cells in the hypothalamus and adenohypophyseal pars distalis. Using antibodies (Ab) against both androgens (T-Ab) and the androgen receptor (AR-Ab), single- and double-immunostained cells were located in a total of five nuclei of the anterior-, mid-, and posterior-hypothalamus, as well as in the rostral and caudal lobes of the adenohypophyseal pars distalis. From Days 9.5-12.5, the mean number of androgen-immunostained cells within the hypothalamus and pars distalis increased significantly (P < 0.01), while from Days 12.5-18.5 there was no further statistically significant increase. The results of the present investigation support previous findings which suggest that in the chick embryo the negative feedback loop of the hypothalamo-adenohypophyseal-testicular (HATest) axis is functional by the 13th day of development (Woods et al., 1989a,b). They also agree with the observations of Wilson and Glick (1970) that in the male chick embryo testosterone organizes masculine mating behavior prior to Day 13.0.

Estradiol and testosterone in minor salivary glands of Sjögren's syndrome.

Nine patients with Sjögren's syndrome were studied in terms of estradiol, testosterone, and dihydrotestosterone in the labial minor salivary glands using the peroxidase-antiperoxidase method. In normal controls in women, estradiol was positive in the epithelial cells of duct, but testosterone and dihydrotestosterone were negative or doubtfully positive by case. Thus, it seems that there is a sex difference of receptors in the ductal epithelia. In the labial minor salivary glands of the patients, all estradiol, testosterone, and dihydrotestosterone were positive. As the background of Sjögren's syndrome, it seems that there is an influence of sex hormones.

Influence of different combinations of antibodies and penicillinase-labeled testosterone derivatives on sensitivity and specificity of immunoassays.

Three antisera raised against bovine serum albumin (BSA) conjugates of testosterone-3-(O-carboxy-methyl)-oxime (T-3-CMO), 11 beta-hydroxytestosterone-11-carboxymethyl ether (T-11 beta-O-CME) and 19-hydroxytestosterone-19-carboxymethyl-ether (T-19-O-CME) were evaluated in enzyme immunoassays (EIAs) in combinations with penicillinase-labeled T-3-CMO, T-11 beta-O-CME, T-19-O-CME, and testosterone-17 beta-hemisuccinate (T-17 beta-HS) for their influence on the sensitivity and specificity of EIAs. Of the various combinations, anti-T-3-CMO antiserum along with T-11 beta-O-CME-penicillinase showed no cross-reaction with any of the closely related steroids, although the same antibody had 21.6% binding to 5 alpha-dihydrotestosterone (5 alpha-DHT) in radioimmunoassay. All the homologous combinations appeared to be less sensitive due to their low affinity for testosterone. It was also apparent that of all the heterologous systems tested, only two combinations, (a) anti-T-19-O-CME antiserum and T-3-CMO-penicillinase and (b) anti-T-3-CMO antiserum and T-11 beta-O-CME-penicillinase, were found to be more sensitive. The former was less specific; it showed 70% cross-reaction with 5 alpha-DHT. The ability of testosterone to displace the hapten-enzyme conjugate and the specificity of the assay appear to depend on the position of the enzyme label on the steroid molecule as well as on the availability of antigenic sites in particular combinations of antibody and hapten-enzyme conjugates.

Characterisation of monoclonal and polyclonal antibodies to 19-linked conjugates of testosterone with corresponding radioligands.

Monoclonal antibodies to testosterone T were produced using testosterone 19-O-carboxymethyl ether (T19C) and testosterone 19-hemisuccinate (T19H) immunogens. All antibodies were characterised with iodinated derivatives of both T19C and T19H. Monoclonal antibodies derived from the T19C immunogen had similar titres and assay sensitivities with both T19-tracers. In contrast antibodies derived from the T19H immunogen bound the homologous but not the heterologous tracer. Individual antibodies showed a wide variation in cross-reactivity with 5 alpha-dihydrotestosterone, DHT (4.4-100%), androstenedione AN (0.5-100%) and progesterone, Po (0.08-5.4%). One antibody 3F11 derived from a T19C immunogen gave 50% displacement of tracer with 180 pgT/tube and low cross-reactivity of 12% with DHT, 3.0% with AN and 1.1% with Po. In general, assay sensitivity and antibody specificity were poorer with an [125I]-histamine conjugate of T-3-carboxymethyloxime than with T19 tracers. Radioimmunoassays for T in extracted human serum were developed with [125I]T19C as tracer and monoclonal antibody 3F11 (T19C immunogen) and rabbit antiserum T19H3R1 (T19H immunogen). Sensitivities of the extracted assays were 43 and 20 pg/tube respectively and results correlated well with those obtained after chromatographic separation of testosterone (r = 0.97 for both antibodies). We conclude that 19-linked derivatives of T are highly immunogenic for the production of specific testosterone antibodies. Selection of the appropriate iodinated tracer is essential to achieve optimal titre, assay sensitivity and specificity, since these characteristics vary widely with individual monoclonal antibodies, and classical bridge recognition is not observed.

Interference of danazol with the radioimmunoassay of steroid hormones.

The crossreactivity of Danazol, a synthetic steroidal drug with antigonadotropic and impeded androgenic properties, with 12 different antisera against 8 different steroids was studied either by conventional competitive inhibition assay or by competitive saturation assay, the latter of which was found to be more sensitive. It was shown that Danazol strongly reacted with an antiserum against 5 alpha-dihydrotestosterone and also, albeit to a much lesser extent, with antisera against testosterone, cortisol and progesterone in decreasing order. It did not interfere with RIA-systems for 11-desoxycortisol, 17 alpha-hydroxyprogesterone, estradiol-17 beta and estriol. However, out of four antisera against estradiol-17 beta two were found to considerably crossreact with Danazol which might explain apparent increases in plasma levels of this hormone during Danazol treatment as recently reported.

Preparation of monoclonal antibodies able to discriminate between testosterone and 5 alpha-dihydrotestosterone.

A procedure for production of monoclonal antibodies to testosterone is described. The method involves immunization of rats with a bovine serum albumin conjugate of testosterone 3-(0-carboxymethyl) oxime followed by polyethylene glycol induced hybridization of the immune lymphocytes with mouse myeloma cells. The resulting hybridomas were cloned and the antibodies produced by each clone were characterized. All the antibodies obtained showed high affinity for testosterone, (Ka = 10(10) 1/mol), but clones differed widely in the degree of cross-reaction of the antibodies with other steroids, such as 5 alpha-dihydrotestosterone (range 2-100%) and androstenedione (less than 0.1-4%). Large quantities of the selected specific antibodies can be obtained by mass growth of the hybridoma line in culture or as tumors in irradiated or nude mice. Monoclonal antibody preparations may improve standardization of immunoassay methods.

The determination of five steroids in avian plasma by radioimmunoassay and competitive protein-binding.

A method has been developed for the simultaneous determination of testosterone, 5alpha-dihydrotestosterone and corticosterone, or of estrone, estradiol-17beta and corticosterone, after separation on a Celite:propylene glycol:ethylene glycol column (6:1.5:1.5 w/v/v). The lower quarter of the column was packed with a Celite: water mixture (3:1 w/v) as a stationary phase (glycol) 'trap'. This effectively prevented leaching of the glycols into the eluate as the concentration of ethyl acetate in the mobile phase was increased to elute the more polar steroids. In addition, a second system utilizing a Celite: ethylene glycol column (2:1 w/v) for the separation of estrone and estradiol-17beta is described. Testosterone, 5alpha-dihydrotestosterone, estrone and estradiol-17beta were measured by radioimmunoassay and corticosterone by a competitive protein-binding technique. Reliability criteria are presented showing that the assay systems used are accurate and reproducible. Plasma-steroid levels of eight avian species are also presented and compared with those found by other investigators.