Pituitary neuropeptides and B lymphocyte function.

This review discusses the accumulated evidence that pro-opiomelanocortin (POMC) gene products as well as other pituitary neuropeptides derived from related genes (Proenkephalin, PENK; Prodynorphin, PDYN, and Pronociceptin, PNOC) can exert direct effects on B lymphocytes to modulate their functions. We also review the available data on receptor systems that might be involved in the transmission of such hormonal signals to B cells.

Individual pituitary neuropeptides do not recapitulate the effects of repository corticotropin (Acthar®) on human B cells in vitro.

Repository corticotropin injection (RCI), a complex mixture of adrenocorticotropic hormone (ACTH) analogs and other pituitary peptides, has been found to suppress key aspects of gene expression and cellular function in human B lymphocytes in vitro. The present studies reveal that neither individual POMC peptides (α-MSH, ACTH1-39, ACTH1-24, ß-endorphin) nor other related pituitary neuropeptides are sufficient to elicit these effects, even though specific receptors capable of transmitting signals from these peptides are expressed by human B cells. RCI's direct effects on human B cells may require complementary signals from multiple components of the preparation.

Repository corticotropin injection reverses critical elements of the TLR9/B cell receptor activation response in human B cells in vitro.

We sought evidence for direct effects of repository corticotropin (RCI; an FDA-approved treatment for selected cases of SLE) on isolated human B lymphocytes activated by engagement of TLR9 and B cell receptors. ODN 2395/αIgM treatment was found to result in induction of 162 distinct mRNAs and suppression of 80 mRNAs at 24 h. RCI treatment resulted in suppression of 14 of the ODN 2395/αIgM -induced mRNAs (mean suppression to 23.6 ±â€¯3.1% of stimulated value). The RCI-suppressed mRNAs included two critical regulators of class switch recombination, AICDA and BATF. RCI treatment also resulted in induction of 5 of the ODN 2395/αIgM -suppressed mRNAs (mean induction by RCI = 7.65 ±â€¯2.34-fold). The RCI-induced mRNAs included SLAMF3, a cell surface receptor capable of inhibiting autoantibody responses. These studies reveal that RCI treatment of human B cells reverses key elements of the early mRNA response to TLR9 and B cell receptor engagement.

Direct effects of HP Acthar Gel on human B lymphocyte activation in vitro.

INTRODUCTION: Both clinical experience and experimental evidence have suggested that Adrenocorticotropic hormone (ACTH) might directly exert immunomodulatory effects not dependent on adrenal steroidogenesis. METHODS: The direct effects of H.P. Acthar Gel (Acthar), a repository preparation containing a porcine ACTH analogue, on human B lymphocyte function were studied in vitro using peripheral blood B cells isolated using anti-CD19 coated magnetic beads and activated by interleukin 4 (IL-4) and CD40 ligand (CD40L). Analysis of expression of messenger RNA (mRNA) encoding activation-induced cytidine deaminase (AICDA) was carried out by quantitative real-time polymerase chain reaction (PCR). Cellular proliferation was assessed by a flow cytometric technique using intracellular staining with carboxyfluorescein succinimidyl ester (CFSE). Immunoglobulin G (IgG) production was measured in cell supernatants using an immunoassay. RESULTS: Acthar was found to exert acute, dose-dependent inhibitory effects on IL-4/CD40L-mediated induction of the expression of activation-induced cytidine deaminase (AICDA) after 24 hours, as well as sustained inhibition of B cell proliferation and IgG production during five more days of culture, without deleterious effects on B cell viability. CONCLUSIONS: These experiments demonstrate that Acthar can exert direct effects on the humoral immune system independent of any role in the regulation of adrenal steroidogenesis. Although the impact of these findings on clinical disease was not evaluated in this study, these data support the therapeutic potential of Acthar for the management of autoimmune diseases characterized by B cell activation and aberrant humoral immune function.

Variation in the androgen receptor gene exon 1 CAG repeat correlates with manifestations of autoimmunity in women with lupus.

Clinical and experimental evidence support a role for gonadal steroids in modulating the expression and course of autoimmune diseases such as lupus. Whether or not inherited variation in sensitivity to circulating androgenic hormones could influence the manifestations of such disease is, however, unknown. We sought to determine whether differences in androgen sensitivity conferred by variation in the exon 1 CAG repeat region of the androgen receptor (AR) gene were associated with differences in the clinical or humoral immune manifestations of lupus in a cohort of female subjects. We found that shorter AR CAG repeat lengths in lupus subjects correlated with a higher Systemic Lupus Erythematosus Disease Activity Index score, higher ANA levels, and expression of a broader array of IgG autoantibodies. Our findings of more severe clinical manifestations and more exuberant humoral autoimmunity in women with a shorter AR exon 1 CAG repeat length suggest a role for genetically determined sensitivity to androgens as a modulator of autoimmune processes.

Glucocorticoid inhibition of activation-induced cytidine deaminase expression in human B lymphocytes.

We examined whether glucocorticoids could modulate the expression of activation-induced cytidine deaminase (AICDA), the principal regulator of the processes of immunoglobulin gene somatic hypermutation and class switch recombination in B lymphocytes. Treatment of human B cells with IL-4 and anti-CD40 antibody for 18-20h resulted in induction of expression of AICDA mRNA by over 10-fold. Dexamethasone at 10nM concentration inhibited AICDA induction by an average of 51.8% (p<0.0001). These effects of glucocorticoids were found to be dose dependent in the physiologic range and were reversible by co-treatment with a glucocorticoid receptor antagonist. Human B cell viability and proliferation were unaltered by glucocorticoid treatment. These data demonstrate that physiologic concentrations of glucocorticoids can act on human B lymphocytes through glucocorticoid receptor-mediated mechanisms to diminish the expression of AICDA, a key regulator of humoral immune responses.

Gonadal steroids and humoral immunity.

Humoral immune responses are sexually dimorphic. Female individuals generally exhibit more-robust antibody responses to vaccines and, in the clinical setting as well as in experimental models, are more likely than male individuals to produce autoreactive antibodies of pathogenic potential. A number of differences between the sexes might account for these observations, including differences in the dosage of specific X-chromosome and Y-chromosomal genes, increased exposure of female individuals to antigenic stimulation in childbearing, and differences in circulating concentrations of gonadal steroid hormones. The role of gonadal steroids in modulating such humoral immune responses has been studied for nearly a century, but advances in our knowledge of B-lymphocyte development and function, the mechanisms of immune tolerance, and the molecular basis of gonadal steroid hormone action are now yielding new understanding of the influence of gonadal steroid hormones on the humoral immune system. This Review examines how oestrogens and androgens modulate B-lymphocyte development and function, focusing on the areas of B-cell production in the bone marrow, the maintenance of immune tolerance for self antigens, and the processes of immunoglobulin heavy chain gene somatic hypermutation and class switch recombination during maturation of cells involved in humoral immune responses.

Estrogen and telomerase in human peripheral blood mononuclear cells.

The enzyme telomerase plays an important role in sustaining the capacity of T lymphocytes for homeostatic replication. Recent data have suggested that gonadal steroids might modulate telomerase expression or activity within these cells. We used quantitative assay techniques for both telomerase mRNA expression and telomerase enzymatic activity to systematically examine the effects of physiologic concentrations of estradiol on human peripheral blood mononuclear cells under basal conditions and under conditions that normally enhance telomerase activity in T lymphocytes. Cells from women tended to exhibit higher responsiveness of telomerase activity to induction by T cell receptor engagement. However, we found no evidence of a direct effect of physiologic concentrations of estradiol on human telomerase reverse transcriptase (hTERT) mRNA expression, hTERT protein expression, or telomerase enzymatic activity in cultured PBMCs. While estrogen might exert developmental effects on T cells to alter telomerase responsiveness to T cell receptor engagement, mature peripheral T cells do not respond to estradiol with changes in expression or function of telomerase.

Short stature in a patient with familial glucocorticoid deficiency.

A 10.5-year-old Caucasian girl with familial glucocorticoid deficiency (FGD) is presented. She had a homozygous S74I mutation of the ACTH receptor and her parents were heterozygous for the same mutation. Around 4 years prior to the diagnosis of FGD, she was diagnosed with antibody positive primary hypothyroidism and was on thyroxin supplementation. FGD patients are considered to be tall. Our patient was only 146.5 cm (4' 9.25") tall at age 17 years (-2.21 standard deviations below the mean for her age). The possible mechanism for short stature in FGD is speculated.

Expression of humoral autoimmunity is related to androgen receptor CAG repeat length in men with systemic lupus erythematosus.

We sought to explore whether inherited differences in androgen sensitivity conferred by variation in the length of a CAG repeat in exon 1 of the androgen receptor gene could be correlated with differing manifestations of humoral autoimmunity in men with lupus. In a sample of 15 men with lupus, AR CAG repeat length was linearly correlated with levels of antibodies against extractable nuclear antigens and with the number of diagnostic criteria for lupus. Protein microarrays were used to assess levels of 86 different IgG and IgM autoantibodies in the sera of these patients. IgG autoantibodies were more frequently observed in male lupus patients with longer AR CAG repeat length (>23), while IgM autoantibodies were more prevalent in subjects with shorter CAG repeat length (≤23). These data support a potential role for androgen signaling in the modulation of immunoglobulin class switching processes, with consequent impact on the autoimmune phenotype in men with lupus.

Sexual dimorphism of RA manifestations: genes, hormones and behavior.

Women are more likely than men to develop rheumatoid arthritis (RA), and recent data suggest that they also suffer greater disability than men with this disease. The reasons for these sexually dimorphic patterns of disease incidence and progression are unknown, but investigations into the underlying mechanisms could provide useful insights into RA pathogenesis and may also suggest new treatment approaches. The processes of sexual differentiation involve genetic input, gonadal hormone signaling and responses from target cells and tissues. Layered upon these processes are behavioral characteristics of males and females acquired as a result of their social context. Differences in disease presentation between the sexes could be the result of complex combinations of all these factors. Recent research suggests that the developmental processes of sexual differentiation might render women more susceptible than men to similar levels of immune or inflammatory burden by virtue of sex-specific differences in body composition and structure.

Evidence that androgens modulate human thymic T cell output.

BACKGROUND: The thymus has long been recognized as a target for the actions of androgenic hormones, but it has only been recently recognized that alterations in circulating levels of gonadal steroids might affect thymic output of T cells. We had the opportunity to examine parameters of thymic cellular output in several hypogonadal men undergoing androgen replacement therapy. METHODS: Circulating naive (CD4+CD45RA+) T cells were quantitated by flow cytometric analysis of peripheral blood mononuclear cells. Cells bearing T-cell receptor excision circles were quantitated using real-time polymerase chain reaction amplification of DNA isolated from peripheral blood mononuclear cells from healthy men and from hypogonadal men before and after testosterone replacement therapy. RESULTS: CD4+CD45+ (naive) T cells comprised 10.5% of lymphocytes in healthy males; this proportion was greatly increased in 2 hypogonadal men (35.5% and 44.4%). One man was studied sequentially during treatment with physiologic doses of testosterone. CD4+CD45RA+ cells fell from 37.36% to 20.05% after 1 month and to 12.51% after 7 months of normalized androgen levels. In 2 hypogonadal patients, T-cell receptor excision circle levels fell by 83% and 78% after androgen replacement therapy. CONCLUSIONS: Our observations indicate that the hypogonadal state is associated with increased thymic output of T cells and that this increase in recent thymic emigrants in peripheral blood is reversed by androgen replacement.

Macrophage expression of peroxisome proliferator-activated receptor-alpha reduces atherosclerosis in low-density lipoprotein receptor-deficient mice.

BACKGROUND: The peroxisome proliferator-activated receptor-alpha (PPARalpha) plays important roles in lipid metabolism, inflammation, and atherosclerosis. PPARalpha ligands have been shown to reduce cardiovascular events in high-risk subjects. PPARalpha expression by arterial cells, including macrophages, may exert local antiatherogenic effects independent of plasma lipid changes. METHODS AND RESULTS: To examine the contribution of PPARalpha expression by bone marrow-derived cells in atherosclerosis, male and female low-density lipoprotein receptor-deficient (LDLR(-/-)) mice were reconstituted with bone marrow from PPARalpha(-/-) or PPARalpha(+/+) mice and challenged with a high-fat diet. Although serum lipids and lipoprotein profiles did not differ between the groups, the size of atherosclerotic lesions in the distal aorta of male and female PPARalpha(-/-) --> LDLR(-/-) mice was significantly increased (44% and 46%, respectively) compared with controls. Male PPARalpha(-/-) --> LDLR(-/-) mice also had larger (44%) atherosclerotic lesions in the proximal aorta than male PPARalpha(+/+) --> LDLR(-/-) mice. Peritoneal macrophages from PPARalpha(-/-) mice had increased uptake of oxidized LDL and decreased cholesterol efflux. PPARalpha(-/-) macrophages had lower levels of scavenger receptor B type I and ABCA1 protein expression and an accelerated response of nuclear factor-kappaB-regulated inflammatory genes. A laser capture microdissection analysis verified suppressed scavenger receptor B type I and increased nuclear factor-kappaB gene expression levels in vivo in atherosclerotic lesions of PPARalpha(-/-) --> LDLR(-/-) mice compared with the lesions of control PPARalpha(+/+) --> LDLR(-/-) mice. CONCLUSIONS: These data demonstrate that PPARalpha expression by macrophages has antiatherogenic effects via modulation of cell cholesterol trafficking and inflammatory activity.

Quantitation and cellular localization of 11beta-HSD1 expression in murine thymus.

11beta-hydroxysteroid dehydrogenase type I (11beta-HSD1), an NADPH-dependent reductase, functions in intact cells to convert inactive 11-keto metabolites of glucocorticoids into biologically active glucocorticoids. The enzyme is thus capable of amplifying glucocorticoid action in tissues in which it is expressed. In the experiments presented here, we show that 11beta-HSD1 is expressed in the murine thymus and that expression increases from late fetal development to maximal levels in the adult thymus. Quantitative real time-PCR, immunoblots, and assays of enzymatic activity reveal adult thymic expression of 11beta-HSD1 mRNA and protein at levels approximately 6-7% of those observed in liver. Immunofluorescence experiments show that the enzyme is expressed in the medullary thymocytes and thymocytes present at the corticomedullary junction. These experiments extend our recognition of 11beta-HSD1 expression in cells of the immune system and lend support to the notion that glucocorticoid signaling and amplification of those signals by regeneration of active glucocorticoids from inactive 11-keto metabolites might impact intrathymic T cell development and the establishment of the immune repertoire.

Gene expression phenotyping of an ACTH-producing small cell lung cancer line.

DNA microarray techniques were used to compare gene expression in an adrenocorticotropin (ACTH)-producing human small cell lung carcinoma line (DMS-79) with six other small cell lung cancer (SCLC) lines that do not produce ACTH. Twelve genes were expressed at more than five-fold higher levels in DMS-79 cells. Two transcription factors were the genes that exhibited the most remarkable over-expression: T-box 3 mRNA was detected at levels 19.37 +/- 3.78 times those observed in the SCLCs. Thyroid transcription factor (TTF-1, T/ebp, Nkx2.1) was expressed at 14.24 +/- 3.41-fold higher in DMS-79 cells. Seven genes were identified whose expression levels were at least five-fold lower in the ACTH-producing cell line. Variation in culture medium formulation did not significantly affect the gene expression profile of DMS-79 cells and expression data observed in microarray experiments were corroborated by northern blot analysis of RNA from the same cell lines. These experiments reveal new candidate genes that could be involved in the dysregulation of POMC gene expression manifested by ACTH-producing nonpituitary tumors.

A novel mutation in the preprovasopressin gene identified in a kindred with autosomal dominant neurohypophyseal diabetes insipidus.

Autosomal dominant neurohypophyseal diabetes insipidus (ADNDI) is a defect in free water conservation caused by mutations in the single gene that encodes both vasopressin (VP) and its binding protein, neurophysin II (NP II). Most of the human mutations in this gene have been in the portion encoding the NP molecule; the resultant abnormal gene products are believed to cause cellular toxicity as improperly folded precursor molecules accumulate in the endoplasmic reticulum. We identified a new American kindred with ADNDI and found a novel mutation in the VP molecule. A 78-yr-old man was noted to have hypotonic polyuria and plasma hyperosmolarity; the urinary concentration defect was reversed by administration of VP. His symptomatology dated to childhood, and his family history was consistent with autosomal transmission of the polyuric syndrome, with affected members in three generations, including several females. Affected individuals were found to be heterozygous for a 3-bp deletion in exon 1 of arginine VP (AVP)-NP II, predicting a deletion of phenylalanine 3 (known to be critical for receptor binding) in the VP nonapeptide. Neuro 2A cells stably transfected with the mutant AVP-NP construct showed increased rates of apoptosis as assessed by flow cytometric methods. These observations support the concept that cellular toxicity of abnormal AVP-NP gene products underlies the development of ADNDI, and the data further demonstrate that mutations affecting the AVP moiety can result in initiation of these pathological processes.

Epidermal growth factor increases cortisol production and type II 3 beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4)-isomerase expression in human adrenocortical carcinoma cells: evidence for a Stat5-dependent mechanism.

We tested the ability of epidermal growth factor (EGF) to regulate a key enzyme in the adrenal synthesis of glucocorticoids: human type II 3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4)-isomerase (3 beta HSD). EGF treatment (25 ng/ml) of human adrenocortical carcinoma cells (H295R) resulted in a 5-fold increase in cortisol production and a corresponding 2-fold increase in 3 beta HSD mRNA. Experiments were performed to determine whether EGF is acting through a previously identified signal transducer and activator of transcription 5 (Stat5)-responsive element located from -110 to -118 in the human type II 3 beta HSD promoter. A Stat5 expression construct was cotransfected with a 3 beta HSD-chloramphenol acetyltransferase (CAT) reporter construct comprised of nucleotides -301-->+45 of the human type II 3 beta HSD promoter linked to the CAT reporter gene sequence. The addition of EGF at doses as low as 10 ng/ml resulted in an 11- to 15-fold increase in CAT activity. The introduction of 3-bp point mutations into critical nucleotides in the Stat5 response element obviated the EGF response. Either Stat5a or Stat5b isoforms induced CAT reporter expression upon treatment with EGF. These results demonstrate the ability of EGF to regulate the expression of a critical enzyme (3 beta HSD) in the production of cortisol and suggest a molecular mechanism by which this regulation occurs.

Glucocorticoids enhance activation of the human type II 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4 isomerase gene.

Glucocorticoids indirectly alter adrenocortical steroid output through the inhibition of ACTH secretion by the anterior pituitary. However, previous studies suggest that glucocorticoids can directly affect adrenocortical steroid production. Therefore, we have investigated the ability of glucocorticoids to affect transcription of adrenocortical steroid biosynthetic enzymes. One potential target of glucocorticoid action in the adrenal is an enzyme critical for adrenocortical steroid production: 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4 isomerase (3beta-HSD). Treatment of the adrenocortical cell line (H295R) with the glucocorticoid agonist dexamethasone (DEX) increased cortisol production and 3beta-HSD mRNA levels alone or in conjunction with phorbol ester. This increase in 3beta-HSD mRNA was paralleled by increases in Steroidogenic Acute Regulatory Protein (StAR) mRNA levels. The human type II 3beta-HSD promoter lacks a consensus palindromic glucocorticoid response element (GRE) but does contain a Stat5 response element (Stat5RE) suggesting that glucocorticoids could affect type II 3beta-HSD transcription via interaction with Stat5. Transfection experiments show enhancement of human type II 3beta-HSD promoter activity by coexpression of the glucocorticoid receptor (GR) and Stat5A and treatment with 100nM dexamethasone. Furthermore, removal of the Stat5RE either by truncation of the 5' flanking sequence in the promoter or introduction of point mutations to the Stat5RE abolished the ability of DEX to enhance 3beta-HSD promoter activity. These studies demonstrate the ability of glucocorticoids to directly enhance the expression of an adrenal steroidogenic enzyme gene albeit independent of a consensus palindromic glucocorticoid response element.