Repository corticotrophin injection exerts direct acute effects on human B cell gene expression distinct from the actions of glucocorticoids.

Repository corticotrophin injection (RCI, H.P Acthar® gel) has been approved for use in the management of multiple autoimmune and inflammatory diseases for more than a half-century, but its mechanism of action is not well understood. We used RNA-Seq methods to define RCI-regulated mRNAs in cultured human B cells under conditions of activation by interleukin (IL)-4 and CD40 ligand. Following IL-4/CD40L activation and RCI treatment we found up-regulation of 115 unique mRNA transcripts and down-regulation of 80 unique mRNAs. The effect on these RNA levels was dose-dependent for RCI and was distinct from changes in mRNA expression induced by treatment with a potent synthetic glucocorticoid. RCI down-regulated mRNAs were observed to include a significant over-representation of genes critical for B cell proliferation under activating conditions. These data confirm that RCI exerts direct effects on human B cells to modulate mRNA expression in specific pathways of importance to B cell function and that, at the molecular level, the effects of RCI are distinct from those exerted by glucocorticoids.

Bone marrow stromal cells mediate androgenic suppression of B lymphocyte development.

Castration of normal male mice induces expansion of the bone marrow B cell population, an effect that can be reversed by androgen replacement. We employed in vitro cultures and two in vivo models to investigate whether androgens exert these effects directly on marrow lymphoid precursors or whether actions on marrow stromal elements are required. Immature B cells from normal mouse bone marrow were not responsive to the suppressive effect of androgens unless they were cocultured with marrow stromal cells or with supernatants from androgen-treated stromal cells, suggesting that the androgen effects are exerted through marrow stromal elements by production of a diffusible mediator. Further experiments revealed that bone marrow stromal cells produced TGF-beta in response to dihydrotestosterone (DHT), and neutralization of TGF-beta in the DHT-treated stromal cells reversed the suppressive effects. The stromal cell requirement for androgen-mediated effects was confirmed in vivo by experiments using chimeric animals created by bone marrow transplantation in which androgen receptor expression was restricted to either the stromal or lymphoid cells of the bone marrow. Androgens only affected B cell development in chimeric mice with androgen-sensitive stromal cells. These experiments suggest that effects of androgens on developing B cells are mediated through androgen receptors in bone marrow stromal cells. TGF-beta is a candidate mediator for these hormonal effects.

Purification of brain peroxisomes and localization of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

At least three different subcellular compartments, including peroxisomes, are involved in cholesterol biosynthesis. Because proper CNS development depends on de novo cholesterol biosynthesis, peroxisomes must play a critical functional role in this process. Surprisingly, no information is available on the peroxisomal isoprenoid/cholesterol biosynthesis pathway in normal brain tissue or on the compartmentalization of isoprene metabolism in the CNS. This has been due mainly to the lack of a well-defined isolation procedure for brain tissue, and also to the presence of myelin in brain tissue, which results in significant contamination of subcellular fractions. As a first step in characterizing the peroxisomal isoprenoid pathway in the CNS, we have established a purification procedure to isolate peroxisomes and other cellular organelles from the brain stem, cerebellum and spinal cord of the mouse brain. We demonstrate by use of marker enzymes and immunoblotting with antibodies against organelle specific proteins that the isolated peroxisomes are highly purified and well separated from the ER and mitochondria, and are free of myelin contamination. The isolated peroxisomal fraction was purified at least 40-fold over the original homogenate. In addition, we show by analytical subcellular fractionation and immunoelectron microscopy that HMG-CoA reductase protein and activity are localized both in the ER and peroxisomes in the CNS.

Effects of androgens on T and B lymphocyte development.

The sexually dimorphic nature of normal immune responses and the remarkably higher incidence of autoimmune diseases in females have suggested a role for gonadal steroid hormones as modulators of immune system function. We have investigated the effects of androgens on the development of lymphocytes in the thymus and bone marrow. Expression of the androgen receptor, the ligand-activated transcription factor that mediates hormone actions, has been documented in lymphoid and nonlymphoid cells of thymus and bone marrow, but not in mature peripheral lymphocytes. This expression pattern suggests that the major impact of androgens must be on the developmental maturation of T and B lymphocytes rather than on the mature effector cells. Recent experiments have explored whether developing lymphoid precursors are the direct targets of androgen action or whether supporting cells, such as thymic epithelial cells and bone marrow stromal cells, are required for the receptor-mediated effects of androgens on lymphoid cell development. Bone marrow transplantation techniques using an androgen-resistant mouse strain permit the creation of chimeric mice with androgen receptor-defective lymphoid or epithelial/stromal cellular compartments. Hormonal manipulation experiments in these chimeric animals have suggested that thymic epithelial cells and bone marrow stromal cells are mediators of androgenic effects on immature lymphocytes. The long-range goal of these studies is to understand the basis for the disproportionate occurrence of autoimmune diseases in females.

Alterations in the regulation of androgen-sensitive Cyp 4a monooxygenases cause hypertension.

Hypertension is a leading cause of cardiovascular, cerebral, and renal disease morbidity and mortality. Here we show that disruption of the Cyp 4a14 gene causes hypertension, which is, like most human hypertension, more severe in males. Male Cyp 4a14 (-/-) mice show increases in plasma androgens, kidney Cyp 4a12 expression, and the formation of prohypertensive 20-hydroxyarachidonate. Castration normalizes the blood pressure of Cyp 4a14 (-/-) mice and minimizes Cyp 4a12 expression and arachidonate omega-hydroxylation. Androgen replacement restores hypertensive phenotype, Cyp 4a12 expression, and 20-hydroxy-arachidonate formation. We conclude that the androgen-mediated regulation of Cyp 4a arachidonate monooxygenases is an important component of the renal mechanisms that control systemic blood pressures. These results provide direct evidence for a role of Cyp 4a isoforms in cardiovascular physiology, establish Cyp 4a14 (-/-) mice as a monogenic model for the study of cause/effect relationships between blood pressure, sex hormones, and P450 omega-hydroxylases, and suggest the human CYP 4A homologues as candidate genes for the analysis of the genetic and molecular basis of human hypertension.

Function of a truncated glucocorticoid receptor form at a negative glucocorticoid response element in the proopiomelanocortin gene.

ACTH-producing tumors of nonpituitary origin characteristically exhibit insensitivity to the negative feedback effects of glucocorticoids. In the DMS-79 cell line derived from an ACTH-producing small cell lung cancer we have previously identified an aberrantly spliced glucocorticoid receptor (GRDelta) that lacks a ligand-binding domain. We examined the interactions of this truncated form of GR with the proximal human proopiomelanocortin (POMC) promoter. In electrophoretic mobility shift assays GRDelta bound to the negative glucocorticoid response element (nGRE) at position -78 to -50 in the human POMC promoter. Nur77, an orphan nuclear receptor that exerts positive regulatory effects on the POMC gene is also known to bind to this DNA element. The functional properties of GR and GRDelta binding to this DNA element were examined in transient transfection experiments in murine AtT-20 corticotroph tumor cells. Reporter gene expression under the control of proximal POMC promoter elements was stimulated by addition of forskolin to the culture medium or by transfection with expression constructs for human Nak1, the human homologue of Nur77. Treatment of transfected cells with dexamethasone resulted in suppression of forskolin- or Nak1-stimulated POMC-reporter gene expression in the presence of co-transfected GR but not with GRDelta. The experiments indicate that in the human POMC promoter GRDelta is capable of binding to the nGRE but cannot effect trans-repression of POMC-reporter gene expression.

Androgen receptors in thymic epithelium modulate thymus size and thymocyte development.

Castration of normal male rodents results in significant enlargement of the thymus, and androgen replacement reverses these changes. Androgen-resistant testicular feminization (Tfm) mice also show significant thymus enlargement, which suggests that these changes are mediated by the androgen receptor (AR). The cellular targets of androgen action in the thymus are not known, but may include the lymphoid cells (thymocytes) as well as nonlymphoid epithelial cells, both of which have been believed to express AR. In the present study immunohistochemical analysis and hormone binding assays were used to demonstrate the presence of AR in thymic epithelial cells. The physiological significance of this epithelial cell AR expression was defined by further studies performed in vivo using chimeric mice, produced by bone marrow transplantation, in which AR expression was limited to either lymphoid or epithelial components of the thymus. Chimeric C57 mice engrafted with Tfm bone marrow cells (AR(+) epithelium and AR(-) thymocytes) had thymuses of normal size and showed the normal involutional response to androgens, whereas chimeric Tfm mice engrafted with C57 bone marrow cells (AR(-) epithelium and AR(+) thymocytes) showed thymus enlargement and androgen insensitivity. Furthermore, phenotypic analyses of lymphocytes in mice with AR(-) thymic epithelium showed abrogation of the normal responses to androgens. These data suggest that AR expressed by thymic epithelium are important modulators of thymocyte development.

An ACTH-producing small cell lung cancer expresses aberrant glucocorticoid receptor transcripts from a normal gene.

ACTH production by non-pituitary tumors is generally not suppressible by exogenous glucocorticoid administration. We had postulated that defects in the glucocorticoid receptor (GR) signaling system might be responsible for this apparent glucocorticoid resistance and had previously demonstrated that DMS-79 cells, derived from an ectopic ACTH-producing tumor, express an abnormal GR mRNA. In this DMS-79 cell GR the sequence normally derived from exons 8 and 9 is replaced by sequence unmatched in the DNA databases. The protein encoded by this mRNA lacks the steroid-binding domain and does not function as a ligand-activated transcription factor. In the present work, we sought to identify the origin of the novel GR mRNA sequence. Southern blot analysis of DMS-79 genomic DNA showed no major structural alteration of the GR gene. Southern blotting of cosmid clones of the normal GR gene revealed that the novel DMS-79 GR mRNA sequence is derived from intron G, between exons 7 and 8. No splice site mutations were found in PCR-amplified DMS-79 DNA fragments surrounding the downstream splice junctions. Further sequencing indicated that the aberrant GR transcript appears to be generated by use of a consensus cleavage/polyadenylation signal found 3650 base pairs into the normal intron G. We conclude that abnormal GR pre-mRNA processing rather than a GR gene mutation confers glucocorticoid resistance on DMS-79 cells.

Androgens accelerate thymocyte apoptosis.

Mechanisms of androgen-induced thymic involution are largely undefined. We have found that significant decreases in thymic size occur 2-4 h after a dose of testosterone is administered to castrated male mice. This rapid rate of change suggests a role for androgen-induced apoptosis in modulating the size and composition of the thymus. Using thymic organ cultures to define these effects of androgens, we found that dihydrotestosterone treatment of thymus tissues from females or from castrated males results in enhancement of thymocyte apoptosis. Intact (androgen-replete) or testicular feminization, Tfm/Y (androgen-resistant) mice failed to show apoptotic change with androgen treatment, although the apoptotic response to glucocorticoids was present, suggesting a requirement for a functional androgen receptor. Acceleration of thymocyte apoptosis by androgens may mediate processes of thymocyte selection, with the potential to impart gender-specific characteristics on the peripheral T cell repertoire.

Expression of 11beta-hydroxysteroid dehydrogenase type 2 in an ACTH-producing small cell lung cancer.

Non-pituitary tumors that produce adrenocorticotropic hormone (ACTH) exhibit resistance to the normal feedback effects of glucocorticoids on proopiomelanocortin (POMC) gene expression. This glucocorticoid resistance is typically complete, although some tumors show only relative glucocorticoid resistance in the clinical setting. The molecular mechanisms responsible for these clinical pathophysiologic observations are unknown, but might include glucocorticoid receptor defects or aberrant expression of enzymes or transporters that exclude glucocorticoids from access to their intracellular receptors. We examined whether ACTH-producing non-pituitary tumor cells might express 11beta-hydroxysteroid dehydrogenase (11beta-HSD), the principal 'gatekeeper' enzyme known to metabolize glucocorticoids. 11Beta-HSD mRNA and enzyme activity were assessed in DMS-79 cells, a line derived from an ACTH-producing small cell lung cancer. RT-PCR studies showed expression of mRNA encoding 11beta-HSD2 but not 11beta-HSD1 in DMS-79 cells. Control human fibroblasts expressed predominantly 11beta-HSD1 but also had detectable 11beta-HSD2 mRNA, while HepG2 hepatoma cells also expressed only 11beta-HSD2 mRNA. Whole cell assays in DMS-79 cells revealed 11beta-HSD activity with a Km for cortisol of 26.1 +/- 9.0 nM and Vmax of 57.0 +/- 5.9 pmol/h/mg protein. HepG2 cells expressed a similar high affinity enzyme activity, while control fibroblasts expressed 11beta-HSD activity with a Km for cortisol of 652 nM. Conversion of cortisol to cortisone in DMS-79 cells was inhibited to 7% of baseline by addition of 10 microM glycyrrhetinic acid. Dexamethasone (20 nM) was converted to a single product in DMS-79 cells at a rate of 17.2 pmol/h/mg protein; this activity was also inhibited by glycyrrhetinic acid. We conclude that DMS-79 cells express 11beta-HSD2. While DMS-79 cells harbor additional defects in glucocorticoid signaling, these data suggest that expression of 11beta-HSD2 might contribute to the development of the glucocorticoid-resistant phenotype of some ACTH-producing tumors.

Schistosoma mansoni: susceptibility differences between male and female mice can be mediated by testosterone during early infection.

In murine Schistosoma mansoni infections, fewer adult worms develop in male than in female mice infected with the same number of cercariae. To evaluate a potential role for testosterone in this phenomenon, testosterone levels were manipulated in groups of CBA/J mice that were then infected and monitored for survival rates, worm burdens, organomegaly, and egg production. By 16 weeks of infection, more than 80% of mice in groups with low levels of testosterone (untreated females, castrated males, or carrier-treated castrates) were dead, while less than 40% of those in groups with high levels of testosterone (sham-castrated males, testosterone-treated castrates, or testosterone-treated female mice) succumbed to infection. The mean number of worms recovered from mice in the low testosterone level groups was comparable among groups, and significantly greater than that from those in high-testosterone-level groups. The degree of organomegaly observed correlated strongly with worm burden, but the number of hepatic eggs per female worm did not differ significantly between groups. When male mice were castrated or sham-castrated 5 weeks after S. mansoni infection, no significant differences in host survival occurred. Furthermore, female mice treated with testosterone demonstrated reduced worm burdens if the testosterone was given 10 days prior to infection but not if the testosterone was given 10 days or 5 weeks after infection. Thus, the host sex bias observed in parallel-infected male and female mice appears to be related to the presence of male gonadal tissue or testosterone early in infection, during the development of immature schistosomules.

Androgens alter B cell development in normal male mice.

Castration of normal male mice leads to splenic enlargement and expansion of the B cell population. Since the spleen does not express receptors for androgens, these changes are most likely mediated by effects of androgens on other target organs. Two potential sites of androgen-mediated effects on B cells are evaluated in these studies: thymus and bone marrow. We first confirmed other findings indicating that castration of normal male mice results in expansion in the numbers of bone marrow B cells and then extended these observations by showing that these changes were reversible following androgen replacement. B cell expansion in castrate marrow and spleen was not altered by prior thymectomy, suggesting that thymic androgen receptors are not involved in the observed effects. Androgen receptors were found to be present in both immature B cells and marrow stromal cells by immunoblotting and ligand binding assays. The results suggest a direct modulatory role for androgens on B cells within the bone marrow compartment.

Case report: testosterone treatment of systemic lupus erythematosus in a patient with Klinefelter's syndrome.

Systemic lupus erythematosus occurs with much greater frequency in females than in males, and in some reports, researchers suggested that treatment with androgenic hormones might have therapeutic effects in this disease. The authors report a case of systemic lupus erythematosus in a hypogonadal male with Klinefelter's syndrome who was treated with testosterone in doses sufficient to normalize the serum level of this hormone to the adult male range. Hematologic and serologic abnormalities, including elevated levels of anti-DNA antibodies and depressed complement levels, returned to normal within 9 months of increasing the testosterone dose. The findings in this patient indicate that androgenic steroids can exert significant effects on immune parameters, and suggest that effects of androgens on the immune system may contribute to the sexual dimorphism of autoimmune disease.

Glucocorticoid receptor structure and function in an adrenocorticotropin-secreting small cell lung cancer.

ACTH secretion by tumors of nonpituitary origin is characteristically resistant to negative feedback regulation by glucocorticoids. One possible mechanism for the phenomenon could be a structural defect in the intracellular glucocorticoid receptor (GR). We studied the GR in DMS-79 cells derived from a human ACTH-secreting small cell lung cancer. Compared with control cells, DMS-79 cells were found to have greatly diminished GR ligand-binding activity and immunoreactive 94-kilodalton (kDa) GR content. Northern blot analysis revealed expression of GR transcripts that appeared to be slightly larger than those in control cells. A DMS-79 cell GR cDNA was cloned by reverse transcription/polymerase chain reaction amplification of mRNA using primers specific for full-length normal GR. The derived sequence of this full-length GR differed from the reported sequence by a single altered codon (G to A; Asn to Ser at codon 363) outside the steroid-binding domain. This N363S DMS-79 GR functioned normally to activate a target gene [mouse mammary tumor virus-chloramphenicol acetyl transferase (MMTV-CAT)] in transient transfection experiments in COS cells. Evidence for expression of a second type of GR mRNA was obtained by screening a DMS-79 cell cDNA library. This GR cDNA contained normal GR sequence up to nucleotide 2155, corresponding exactly to the end of exon 7 in the normal GR gene. The sequence appended to the GR sequences was not matched by any known sequence in DNA databases and included an in-frame termination codon after only 6 bases. The predicted truncated GR protein product (GR delta) has a mol wt of 73,740 and lacks most of the ligand-binding domain. Transient transfection of the GR delta form into COS cells did not reveal any dominant negative effect on the function of a cotransfected normal GR.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunochemical and flow cytometric analysis of androgen receptor expression in thymocytes.

A variety of evidence suggests that the cells of the immune system are targets for the actions of gonadal steroids. Experiments in both normal animals and in autoimmune disease models have established that androgens exert immunomodulatory effects at the level of the thymus. We have attempted to define precisely the potential target cells for androgen action in the thymus using recently developed antibodies to the androgen receptor. We report here that these antibodies reveal AR expression in all classes of thymocytes defined by surface markers CD4 and CD8. The highest levels of AR expression were observed in the CD4-CD8+ and CD4-CD8- subsets that include the most immature cells. These experiments establish that thymocytes are potential targets for direct actions of androgens. The data further suggest AR expression in thymocytes may be developmentally regulated in these cells, and that androgen effects early in the process of thymocyte selection may contribute to the sexual dimorphism of immune responsiveness.

Castration alters peripheral immune function in normal male mice.

While it is generally recognized that females show enhanced cell-mediated and antibody responses to antigenic stimulation, the physiological basis for this observed sexual dimorphism of the immune response is not well understood. We report here studies on the effects of androgen deficiency on the peripheral immune system. Intact male mice were compared to animals castrated 3-4 months previously. Phenotypic characterization of thymocyte and lymphocyte subpopulations was carried out using dual-colour flow cytometry. In vitro production by spleen cells of interleukin-2 (IL-2), IL-4 and interferon-gamma (IFN-gamma), and levels of total immunoglobulin and autoreactive antibodies was measured by specific immunoassays. In addition to thymic hypertrophy, castrated animals showed significant splenic enlargement, which was largely owing to expansion of the B-cell population. The castrated spleens contained relatively fewer mature T cells than intact controls (P < or = 0.001), but culture supernatants from these spleen cells contained higher levels of IL-2 and IFN-gamma than control cultures (P < 0.04). Levels of in vitro antibody synthesis (IgM, IgG, IgA) were not higher in castrated animals compared to controls, but the castrate spleen cell cultures showed increased levels of production of two autoreactive antibodies, anti-IgG (rheumatoid factor) and anti-thyroglobulin. These data suggest that androgen deprivation results in a relative decrease in the number of mature peripheral T cells, but those which reach the spleen have functional characteristics suggestive of enhanced activation. Dysregulation in the B-cell compartment may be the result of altered effects of T-cell-mediated control.

Induction of immature thymocyte proliferation after castration of normal male mice.

The physiological basis and immunological significance of thymic enlargement in castrate male animals is not known. We used normal male C57 Bl/6 mice to examine the contribution of in situ thymocyte proliferation to castration-induced enlargement of the thymus. Animals castrated at 8-10 weeks of age were compared to normal intact males. Thymocytes were examined 4-120 days after castration using flow cytometry to determine DNA content and thus the number of cells in active phases of the cell cycle. These properties were examined in unseparated thymocytes and in phenotypic subpopulations defined by expression of CD3, CD4, and CD8. For thymocytes obtained from intact control glands, a mean of 11.0 +/- 1.0% were in active phases of the cell cycle. The percentage of cycling thymocytes was increased to a mean of 22.5 +/- 1.9% in the week after castration (P < 0.001). This change occurred in the absence of significant thymic enlargement. At 8-10 days after castration, thymic weight increased abruptly to a new steady state which was double that of intact controls (78.0 +/- 4.1 vs. 39.1 +/- 2.6 mg; P < 0.001). In these enlarged glands, only 9.9 +/- 0.8% of cells were cycling, which was not significantly different than controls (P > 0.3). Proliferating cells identified in fixed thymus tissue sections after in vivo administration of bromodeoxyuridine were located in the subcapsular cortex and medulla. Analyses of thymocyte subpopulations indicated that most cycling cells had immature phenotypes (CD4+CD8+, CD4-CD8+, and CD3lo or CD3-). Castrate glands studied in the steady state period 8-120 days after surgery contained significantly fewer CD3+ cells than intact controls (P < or = 0.045). The findings suggest an intrathymic role for androgens in affecting generation of the mature T cell repertoire.

Determination of the lethal dose of dexamethasone for early passage in vitro human glioblastoma cell cultures.

Previous investigators have supported the idea that glucocorticoids may be oncolytic. In this study, the percentage of cell death in two human glioblastoma cell cultures was related to the concentration of dexamethasone that was administered. It was determined that for Cell line 1, the median lethal dose was approximately 500-800 micrograms/ml and the completely lethal dose was about 900-1000 micrograms/ml; the 3H-thymidine uptake to approximate the mitotic rate was 16,607 cpm, and the dexamethasone receptor activity was 228 fmol/mg protein. The median lethal dose and completely lethal dose for Cell line 2 was approximately 500-600 micrograms/ml and 700-1000 micrograms/ml, respectively; the 3H-thymidine uptake was 8402 cpm, and the dexamethasone receptor activity was 137 fmol/mg protein. These lethal concentrations of dexamethasone are probably higher than can be tolerated by systemic delivery. However, it remains to be seen whether the interstitial administration of dexamethasone could achieve local concentrations resulting in the oncolysis of malignant gliomas. The clinical significance of these findings will depend on the local tolerance of normal brain parenchyma to very high doses of dexamethasone. A review of some of the literature is included.

Testosterone induces expression of transforming growth factor-beta 1 in the murine thymus.

Castration of adult male mice results in enlargement of the thymus and diminution of peripheral suppressor T cell function. Replacement therapy with physiologic doses of androgens reverses these phenomena. Although the mediators involved are unknown, these effects of androgens on the thymus and peripheral immune system are reminiscent of those reported for transforming growth factor-beta (TGF-beta 1). We examined expression of TGF-beta 1 mRNA and bioactive protein in thymuses from castrate and androgen-replaced animals. Steady-state levels of thymic TGF-beta 1 mRNA fell slightly after castration, but rose 2.3-fold after testosterone replacement. Bioactive TGF-beta 1 production by cultured thymic explants also fell following castration to approx. 50% of the levels observed in intact animals. Following 1 week of testosterone replacement in castrate animals, TGF-beta 1 bioactivity produced in culture was restored to levels indistinguishable from those observed with explants from intact animals. Reverse transcription/polymerase chain reaction amplification of RNA revealed that thymocytes are a source of the androgen-modulated TGF-beta 1. These results suggest that TGF-beta 1 may mediate effects of androgens on the immune system.