Testosterone signaling through internalizable surface receptors in androgen receptor-free macrophages.

Testosterone acts on cells through intracellular transcription-regulating androgen receptors (ARs). Here, we show that mouse IC-21 macrophages lack the classical AR yet exhibit specific nongenomic responses to testosterone. These manifest themselves as testosterone-induced rapid increase in intracellular free [Ca(2+)], which is due to release of Ca(2+) from intracellular Ca(2+) stores. This Ca(2+) mobilization is also inducible by plasma membrane-impermeable testosterone-BSA. It is not affected by the AR blockers cyproterone and flutamide, whereas it is completely inhibited by the phospholipase C inhibitor U-73122 and pertussis toxin. Binding sites for testosterone are detectable on the surface of intact IC-21 cells, which become selectively internalized independent on caveolae and clathrin-coated vesicles upon agonist stimulation. Internalization is dependent on temperature, ATP, cytoskeletal elements, phospholipase C, and G-proteins. Collectively, our data provide evidence for the existence of G-protein-coupled, agonist-sequestrable receptors for testosterone in plasma membranes, which initiate a transcription-independent signaling pathway of testosterone.

Estradiol signaling via sequestrable surface receptors.

Estradiol (E(2))-signaling is widely considered to be exclusively mediated through the transcription-regulating intracellular estrogen receptor (ER) alpha and ERbeta. The aim of this study was to investigate transcription-independent E(2)-signaling in mouse IC-21 macrophages. E(2) and E(2)-BSA induce a rapid rise in the intracellular free Ca(2+) concentration ([Ca(2+)](i)) of Fura-2 loaded IC-21 cells as examined by spectrofluorometry. These changes in [Ca(2+)](i) can be inhibited by pertussis toxin, but not by the ER-blockers tamoxifen and raloxifene. The E(2)-signaling initiated at the plasma membrane is mediated through neither ERalpha nor ERbeta, but rather through a novel G protein-coupled membrane E(2)-receptor as revealed by RT-PCR, flow cytometry, and confocal laser scanning microscopy. A special feature of this E(2)-receptor is its sequestration upon agonist stimulation. Sequestration depends on energy and temperature, and it proceeds through a clathrin- and caveolin-independent pathway.

Testosterone induces Ca2+ influx via non-genomic surface receptors in activated T cells.

Using the Fura-2 method we investigated a possible direct action of testosterone on cytosolic free calcium of splenic T cells isolated from female C57BL/10 mice. Testosterone at physiological concentrations of 1-10 nM induces an increase in [Ca2+]i within seconds, which is due to Ca2+ influx and not to Ca2+ release from intracellular stores. In contrast, estradiol induces both Ca2+ influx and Ca2+ release. The testosterone-induced Ca2+ influx is mediated by Ni2+-blockable channels and is not inhibited by cyproterone, a blocker of the classical androgen receptor. Ca2+ influx can also be induced by testosterone conjugated to BSA which is impermeable to the plasma membrane. These data indicate a novel mode of direct action of testosterone on T cells which is not mediated through the classical androgen receptor response, but through unconventional plasma membrane receptors.

Estradiol binding to cell surface raises cytosolic free calcium in T cells.

The Fura-2 method is used to examine a possible action of 17beta-estradiol (E2) on [Ca2+]i of splenic T cells isolated from female C57BL/10 mice. E2 concentrations between 10 fM and 10 nM induce a rapid and dose-dependent increase in [Ca2+]i due to Ca2+ influx and release of Ca2+ from intracellular stores. Ca2+ influx is mediated by Ca2+ channels which are completely blockable by Ni2+ and partly by nifedipine. The antiestrogen tamoxifen does not inhibit the E2-induced rise in [Ca2+]i. Ca2+ influx and Ca2+ release from intracellular stores is also inducible by plasma membrane impermeable E2 conjugated to BSA. E2-BSA-FITC binds to the surface of T cells of both the CD4+ and CD8+ subset. Our data suggest a novel E2-signalling pathway in T cells which is not mediated through the classical nuclear estrogen receptor response but rather through putative plasma membrane receptors for E2.

Testosterone-induced suppression of self-healing Plasmodium chabaudi malaria: an effect not mediated by androgen receptors?

This study investigates whether androgen receptors (AR) mediate the suppressive effect of testosterone on self-healing Plasmodium chabaudi malaria. Our data show the following. (1) Female and castrated male mice of the inbred strain C57BL/10 self-heal and survive infections when challenged with 10(6) P. chabaudi-parasitized erythrocytes. However, self-healing is prevented when circulating testosterone levels are high as in intact males or in females and castrated males pretreated with 0.9 mg testosterone twice a week for 3 weeks. (2) The lethal outcome of P. chabaudi in intact males is not affected by different doses of AR blockers such as cyproterone acetate, cyproterone, flutamide and nilutamide when applied at least 3 weeks before infection and during infection. Also, these AR blockers do not impair the testosterone-induced lethal outcome of infections in testosterone-treated females and castrated males. (3) Tfm mice possessing mutant non-functional ARs and normal 'male' testosterone levels succumb to infection with P. chabaudi. However, the corresponding wild-type mice possessing functioning ARs are able to resist P. chabaudi infections at low circulating testosterone levels. (4) In contrast to testosterone, testosterone metabolites such as 5 alpha-dihydrotestosterone, 5 beta-dihydrotestosterone, androsterone and 1-dehydrotestosterone cannot suppress self-healing in castrated male B10 mice. Our data suggest that testosterone suppresses the development of protective immunity against P. chabaudi malaria, and that this immunosuppressive effect of testosterone is not primarily mediated by the classical AR response.

Testosterone-induced compared with oestradiol-induced immunosuppression against Plasmodium chabaudi malaria.

Testosterone suppresses immunity against malaria caused by Plasmodium chabaudi in B10 mice. Since this effect is probably not mediated through the classical androgen-receptor response, we investigated whether testosterone might act, after aromatization to oestradiol (OE2), through the oestrogen receptor (ER). Indeed, OE2 was found to act immunosuppressively when used at only about 1% of the immunosuppressive dose of testosterone. This becomes evident as an OE2-induced suppression of self-healing of P. chabaudi infections in female and castrated male B10 mice. The immunosuppressive OE2 effect is associated with a 16-fold increase in the circulating level of OE2 and can be prevented by ER blockers such as tamoxifen and clomifene. In contrast, the immunosuppressive effect of testosterone, which is not associated with any changes in the level of OE2, cannot be abolished by ER blockers or by aromatase inhibitors, such as atamestane and drofazar hydrochloride. Moreover, OE2 and testosterone act differently on spleen cells; OE2 induces a decrease in CD(4+)-T-cells, whereas testosterone causes an increase in CD(8+)-T-cells and a decrease in total nucleated spleen cells. The immunosuppressive effect of testosterone, but not that of OE2, can be adoptively transferred to syngeneic mice by nucleated spleen cells, predominantly T-cells. Our data show that the immunosuppressive activity of testosterone, in contrast to OE2, is not mediated through the ER. The immunosuppressive action of testosterone is therefore thought to be primarily mediated through a non-genomic mechanism.

Testosterone-induced susceptibility to Plasmodium chabaudi malaria: persistence after withdrawal of testosterone.

Testosterone induces susceptibility to Plasmodium chabaudi malaria by imposing restrictions on those mechanisms which mediate resistance controlled by genes of the H-2 complex and the non-H-2 background in mice. This study investigated whether these restrictions are abolished after withdrawal of testosterone. Female mice of the inbred strain C57BL/10 were treated with 0.9 mg testosterone twice a week for 3 weeks and testosterone was then withdrawn for 12 weeks. The treatment raised plasma testosterone levels from 0.18 ng/ml to 3.79 ng/ml. After the testosterone treatment, these levels progressively dropped and reached 0.21 ng/ml by week 12 after testosterone withdrawal. Surprisingly, however, the testosterone-induced susceptibility still persisted. When mice were challenged on week 12 after testosterone withdrawal, P. chabaudi infections were still fatal in testosterone-treated mice, in contrast to self-healing infections in resistant, i.e. untreated, control mice. In addition, testosterone caused a persistent decrease in the levels of total IgG antibodies, especially IgG1 and IgG2b isotypes. In contrast, testosterone-induced changes in spleen cells, such as the reduction in number by 50%, the relative increase in CD8+ cells and the decrease in Ig+ cells, as well as the acquisition of the susceptible phenotype, were completely reversed on week 10 after testosterone withdrawal at the latest. Testosterone did not affect the production of the TH1-signalling cytokine interferon-gamma and the TH2-signalling cytokines interleukin (IL)-4 and IL-10 in response to P. chabaudi malaria. Together, our data indicated that the gene-controlled host resistance to P. chabaudi malaria is subject to superior hormonal imprinting: when once induced by testosterone, mechanisms which suppress resistance thus causing susceptibility persist independently of testosterone.

Testosterone-induced susceptibility to Plasmodium chabaudi malaria: variant protein expression in functionally changed splenic non-T cells.

This study investigates the effects of the male sex hormone, testosterone (Te), on self-healing of Plasmodium chabaudi malaria as well as on protein expression and functional properties of total spleen cells and splenic T cells in females of the mouse strain C57BL/10. About 90% of the B10 females survive a challenge with 10(6) P. chabaudi-infected erythrocytes. The percentage of self-healers, however, is reduced to about 60%, 40%, and 0% after pretreatment with Te for 1, 2, and 3 weeks, respectively. The progressive loss of the capability of self-healing is correlated with an increasing expression of five proteins in splenic non-T cells as revealed by two-dimensional fluorography after metabolic labelling of total spleen cells and T cells with [35S]methionine. These have molecular masses (isoelectric points) of about 10 kDa (pI 5.7), 14 kDa (pI 6.3), 14 kDa (pI 6.4), 38 kDa (pI 6.5), and 46 kDa (pI 5.5), respectively. Splenic non-T cells from mice treated with Te for 3 weeks have gained an increased capability to stimulate the concanavalin A-induced proliferative response of T cells. Te induces the changes in functional properties and protein expression of splenic non-T cells only in vivo and not in vitro. This suggests that the changes in splenic non-T cells as well as the prevention of self-healing P. chabaudi malaria are not directly induced by Te but rather indirectly, i.e. by a Te metabolite and/or Te-induced factor(s).

Dietary testosterone suppresses protective responsiveness to Plasmodium chabaudi malaria.

This study investigates the effect of orally administered testosterone on serum testosterone levels and immune responses including outcome of Plasmodium chabaudi malaria. Female C57BL/10 mice were fed on a diet impregnated with 17 alpha-methyl-testosterone for 3 weeks. This raised the circulating testosterone levels from 0.28 ng/ml to 2.69 ng/ml on the average. In these mice, blood-stage infections of P. chabaudi resulted in a lethal outcome, whereas protective immunity developed in about 80% of mice fed on control diet without testosterone. Dietary 17 alpha-methyl-testosterone reduced the capacity of peritoneal cells to generate reactive oxygen intermediates after stimulation with C3b-coated zymosan and phorbol-myristate-acetate. Also, mice fed on dietary 17 alpha-methyl-testosterone responded to heat-killed Salmonella typhimurium with a higher increase in serum TNF, whereas the induced increase in the production of IL-10 by spleen cells was largely suppressed and no effect was found with respect to the production of IFN-gamma and IL-4. Our data indicate that the method of oral administration of 17 alpha-methyl-testosterone raises circulating testosterone to levels that impair protective immune responses to P. chabaudi malaria.

Plasmodium chabaudi: estradiol suppresses acquiring, but not once-acquired immunity.

This study investigates the effect of estradiol (E) on self-healing of Plasmodium chabaudi malaria in mice of the inbred strain C57BL/10. Our data show: (1) Female mice and male castrates are capable of self-healing infections when challenged with 10(6) P. chabaudi-infected erythrocytes. Self-healing is completely suppressed after pretreatment of mice with 12 micrograms E injected sc twice a week for 3 weeks. (2) The suppressive effect of E is prevented by the estrogen receptor blockers tamoxifen and clomiphene. (3) The nonsteroidal E-agonist diethylstilbestrol (DES) also suppresses self-healing. This suppressive DES effect is prevented by tamoxifen. (4) In mice immune to P. chabaudi, neither survival rate nor the course of parasitemia is affected by E, even at 10-fold higher E doses. Our data suggest that the immunosuppressive action of E is a specific genomic effect, i.e., E-induced gene products prevent the development of protective immunity against P. chabaudi.

Testosterone impairs efficacy of protective vaccination against P. chabaudi malaria.

Vaccination with surface membranes isolated from Plasmodium chabaudi-infected erythrocytes can protect B10.A mice from the lethal outcome of P. chabaudi malaria. However, the efficacy depends on gender and testosterone levels. Thus, vaccination protects over 90% of female mice, but only about 55% of male mice and only about 34% of female mice when pretreated with testosterone for 4 weeks. The suppressive testosterone effect remains imprinted in females even at 10 weeks after the testosterone treatment. These data indicate that not only genetic but also environmental factors restrict the host's immune response to a malaria vaccine.

Testosterone-induced abrogation of self-healing of Plasmodium chabaudi malaria in B10 mice: mediation by spleen cells.

This study investigates the suppressive effect of testosterone (Te) on the self-healing of Plasmodium chabaudi malaria in female mice of the strain C57BL/10, and, in particular, the possible role of spleen cells in mediating this Te effect. Our data show the following. (i) About 80% of B10 mice infected with 10(6) P. chabaudi-infected erythrocytes are capable of self-healing the infections. This capability is progressively impaired and finally abrogated after pretreating the B10 mice with Te for 3 weeks. (ii) The spleen is Te responsive. This becomes evident in a reduction of total spleen cells from 1.05 x 10(8) to 0.54 x 10(8) on average after Te treatment for 3 weeks. Moreover, Te treatment causes an increase in the relative proportion of CD8+ cells by about 4% and a decrease of Ig+ cells by about 4.5%, as revealed by flow cytometry. (iii) Spleen cells mediate the suppressive Te effect as revealed by adoptive transfer experiments. The percentage of self-healing mice dramatically decreases to about 8% when they receive, just prior to infection, nucleated spleen cells isolated from mice treated with Te for 3 weeks. This suppressive effect can be transferred by T cells in particular but also by non-T cells, though to a lesser extent. (iv) The adoptively transferred cells mediate their suppressive effect on self-healing only if the recipient mice receive Te during infection. Our data suggest that spleen cells become functionally changed by the Te treatment for 3 weeks. Particularly T cells, but also non-T cells, gain P. chabaudi-specific suppressive activities, and the cells require a Te-induced factor(s) to mediate these activities.

Testosterone and other gonadal factor(s) restrict the efficacy of genes controlling resistance to Plasmodium chabaudi malaria.

The effect of circulating concentrations of testosterone (Te) on resistance to Plasmodium chabaudi malaria was investigated in the H-2 congenic mouse strains C57BL/10, B10.A, B10.A(3R), B10.A(4R), and B10.D2. Te-levels were determined by radioimmunoassay and resistance was expressed in terms of percent self-healers after challenge with 10(6) P. chabaudi-infected erythrocytes. Our data indicate: (i) Females and castrated males reveal very similar interstrain variations of resistance. These do not correlate with the interstrain variations of the Te-levels. This is consistent with the view that resistance to P. chaubaudi is controlled by genes of the H-2 complex and genes of the non-H-2 B10-background, (ii) The polygenic control of resistance is inefficacious at high Te-levels. This is evident as high susceptibilities of males, Te-treated females and Te-treated castrated males. Moreover, high Te-levels correlate with susceptibilities to P. chabaudi within mice of the same sex of a given strain, (iii) B10-males chemically castrated using buserelin display the same low Te-level as those surgically castrated. The latter become resistant, while the former remain as highly susceptible to P. chabaudi as untreated B10-males. Obviously, other gonadal factor(s), besides Te, impose restrictions on genes controlling resistance to P. chabaudi malaria.

Testosterone-unresponsiveness of existing immunity against Plasmodium chabaudi malaria.

Testosterone (Te) is known to suppress immunity and to increase host susceptibility to many parasites. This study investigates the action of Te on immunity acquired against blood-stages of the malaria parasite Plasmodium chabaudi in female mice of the inbred strain C57BL/10. Our data show: (i) About 90% of mice infected with 10(6) P. chabaudi-infected erythrocytes are able to develop protective immune mechanisms which become evident in self-healing the infection. The capability of self-healing is lost when mice are pretreated with Te for 3 weeks. (ii) Mice which have self-healed infections acquire immunity to homologous rechallenge. Concomitantly, mice become Te-unresponsive in that their acquired immunity is not suppressible by Te-treatment. (iii) Flow cytometry reveals that Te-pretreatment entails an increase of CD8+ cells and a decrease of Ig+ cells by about 4% in spleens of non-immune mice. In immune mice, however, there is a Te-unresponsiveness of the percental distribution of splenic cell populations. (iv) Adoptive transfer experiments indicate that immunity is conferred by spleen cells, presumably non-T-cells. These cells are Te-unresponsive, since they exert their effect in Te-pretreated mice in the presence of Te. (v) Te-unresponsive immunity can be also transferred by serum, especially the IgG-fraction, obtained from immune mice. Our data demonstrate that Te prevents the development of protective immunity against P. chabaudi infections. However, when once established, protective immunity becomes unresponsive to Te. Our data suggest that the effector mechanisms of protective immunity involve Te-unresponsive B cells secreting protective IgG-antibodies.

Functional testosterone receptors in plasma membranes of T cells.

T cells are considered to be unresponsive to testosterone due to the absence of androgen receptors (AR). Here, we demonstrate the testosterone responsiveness of murine splenic T cells in vitro as well as the presence of unconventional cell surface receptors for testosterone and classical intracellular AR. Binding sites for testosterone on the surface of both CD4(+) and CD8(+) subsets of T cells are directly revealed with the impeded ligand testosterone-BSA-FITC by confocal laser scanning microscopy (CLSM) and flow cytometry, respectively. Binding of the plasma membrane impermeable testosterone-BSA conjugate induces a rapid rise (<5 s) in [Ca2+]i of Fura-2-loaded T cells. This rise reflects influx of extracellular Ca2+ through non-voltage-gated and Ni2+-blockable Ca2+ channels of the plasma membrane. The testosterone-BSA-induced Ca2+ import is not affected by cyproterone, a blocker of the AR. In addition, AR are not detectable on the surface of intact T cells when using anti-AR antibodies directed against the amino and carboxy terminus of the AR, although T cells contain AR, as revealed by reverse transcription-polymerase chain reactions and Western blotting. AR can be visualized with the anti-AR antibodies in the cytoplasm of permeabilized T cells by using CLSM, though AR are not detectable in cytosol fractions when using the charcoal binding assay with 3H-R1881 as ligand. Cytoplasmic AR do not translocate to the nucleus of T cells in the presence of testosterone, in contrast to cytoplasmic AR in human cancer LNCaP cells. These findings suggest that the classical AR present in splenic T cells are not active in the genomic pathway. By contrast, the cell surface receptors for testosterone are in a functionally active state, enabling T cells a nongenomic response to testosterone.