Bacterial endotoxin (lipopolysaccharide) stimulates interleukin-6 production and inhibits growth of pituitary tumour cells expressing the toll-like receptor 4.

Members of the Toll receptor (Tlr) family have a crucial role in the innate immune response following bacterial infection. The effects of Gram-negative bacteria-derived endotoxins (lipopolysaccharide, LPS) are predominantly mediated by Tlr4, and we have recently shown that pituitary folliculostellate cells express functional Tlr4. In the present study, we investigated whether Tlr4 is also present in normal and transformed endocrine epithelial pituitary cell types. By reverse transcriptase-polymerase chain reaction, Tlr4 mRNA expression was found in some pituitary epithelial tumour cell lines (AtT20, HP75), whereas others were negative (GH3, alphaT3-1). Tlr4 protein was detected by immunohistochemistry in a few epithelial cells in normal human anterior pituitaries and in 26 out of 67 human pituitary tumours analysed. LPS had no effect on adrenocorticotropic hormone secretion in Tlr4-positive AtT20 cells, but it suppressed the growth of these cells in a dose-dependent manner. As expected, neither hormone secretion, nor growth of Tlr4-negative GH3 cells was affected by LPS. In cell cultures of Tlr4-positive pituitary adenomas, LPS dose-dependently stimulated the production of interleukin (IL)-6, which is known to induce growth and hormone production in pituitary tumours. The LPS-induced IL-6 production was blocked by the specific p38alphaMAP kinase inhibitor, SB203580, and by the synthetic glucocorticoid, dexamethasone. The data suggest that, during Gram-negative bacteria-induced infections or inflammatory processes, LPS could affect pituitary tumour pathophysiology and progression in the subset of Tlr4-expressing pituitary adenomas.

Estradiol stimulates vascular endothelial growth factor and interleukin-6 in human lactotroph and lactosomatotroph pituitary adenomas.

Estrogens are considered to be critically involved in lactotroph and lactosomatotroph pituitary tumor development. In addition to direct effects, estradiol-induced tumor formation may involve alterations in growth factor and cytokine production. We have studied whether estradiol stimulates the production of the angiogenic vascular endothelial growth factor and the potential tumor progression factor interleukin-6 in 5 lactotroph (LA) and 5 lactosomatotroph (LSA) human pituitary adenoma cell cultures. All tumors secreted heterogenous basal amounts of VEGF (18.0 +/- 1.4 to 425 +/- 26 pg/ml per 24 h) and IL-6 (18.1 +/- 1.5 to 604 +/- 17 pg/ml per 24 h). Estradiol (100 nM) significantly enhanced VEGF release in all LA and LSA cell cultures (47 to 168 % above basal). IL-6 secretion was stimulated in 3 out of 5 LA and in all LSA cell cultures (31 to 287 % above basal). In cell cultures obtained from tumors from which sufficient cells could be isolated, a dose-dependent effect of estradiol (1 to 100 nM) on VEGF and IL-6 production was observed. Stimulation of IL-6 and/or VEGF secretion by estradiol in the majority of human lactotroph and lactosomatotroph adenoma cell cultures studied, suggests that estrogens may contribute to adenoma expansion through the stimulation of these auto-/paracrine-acting adenoma progression factors.

Transforming growth factor-beta stimulates vascular endothelial growth factor production by folliculostellate pituitary cells.

TGF-beta isoforms are expressed in the anterior pituitary and modulate the growth and function of endocrine pituitary cells. Recently, TGF-beta has been shown to stimulate growth and basic fibroblast growth factor secretion in nonendocrine folliculostellate (FS) pituitary cells. We therefore studied whether the production of FS cell-derived vascular endothelial growth factor (VEGF), the most important regulator of vascular permeability and angiogenesis, is affected by TGF-beta. We observed by RT-PCR that TtT/GF cells, which are FS mouse pituitary tumor cells, synthesize TGF-beta1, -beta2, and -beta3. They also express TGF-beta receptors types 1 and 2, as well as Smad2, Smad3, and Smad4 proteins, which are essential for TGF-betabinding and signaling. Stimulation of TtT/GF cells with either TGF-beta1 or TGF-beta3 induced a rapid translocation of Smad2 into the cell nuclei. Both TGF-beta isoforms dose dependently stimulated VEGF production in TtT/GF cells, but not in lactosomatotroph GH3 cells. Time-course studies and suppression of TGF-beta-induced VEGF production by cycloheximide suggest that TGF-beta induces de novo synthesis of VEGF in folliculostellate cells, which is completely blocked by dexamethasone. In primary rat pituitary cell cultures, TGF-beta1 and -beta3 stimulated VEGF production. TGF-beta stimulation of VEGF production by folliculostellate cells could modulate intrapituitary vascular permeability and integrity as well as angiogenesis in an auto-/paracrine manner.

The intrapituitary stimulatory effect of lipopolysaccharide on ACTH secretion is mediated by paracrine-acting IL-6.

During infection/inflammation bacterial lipopolysaccharide (LPS) activates the immune system and thus enhances the level of circulating cytokines. These circulating cytokines induce adaptive processes within the endocrine system and in particular stimulate the HPA axis to increase the level of anti-inflammatory-acting glucocorticoids in the circulation. We have shown recently that LPS stimulates intrapituitary IL-6 production in folliculostellate cells via specific receptors and the p38a mitogen-activated protein kinase/nuclear factor-kappa B pathway. To test the physiological relevance of these findings, we studied whether LPS could enhance ACTH secretion via paracrine-acting intrapituitary IL-6. Lipopolysaccharide stimulated IL-6 secretion both in monolayer and aggregate mouse pituitary cell cultures, but only in aggregates, ACTH secretion was significantly enhanced by LPS. Other hormones, such as GH or PRL, were less stimulated by LPS. My4, an antibody that blocks the interaction of LPS with the LPS receptor CD14, suppressed both LPS-induced IL-6 and ACTH secretion in aggregate cultures. A neutralizing antibody against mouse IL-6 also inhibited LPS-induced ACTH secretion in aggregates. In mouse pituitary fragments, LPS-induced ACTH secretion was blocked by My4 and IL-6 antibodies, identically to re-aggregate cell cultures. LPS-induced ACTH secretion, mediated by intrapituitary IL-6, may represent a pituitary-specific mechanism that stimulates the HPA axis during infection/inflammation.

Vascular endothelial growth factor production and regulation in rodent and human pituitary tumor cells in vitro.

Angiogenesis, the formation of a new blood supply, is an essential step in tumorigenesis. Although vascular endothelial growth factor (VEGF) is known to be a very potent angiogenic factor in most solid tumors, little is known about its production and regulation in pituitary adenomas. We have investigated basal and stimulated VEGF production by rodent pituitary tumor cells (mouse corticotrope AtT20, rat lactosomatotrope GH3, mouse gonadotrope alpha T3-1 and mouse folliculostellate TtT/GF cells), and by hormone-inactive (27), corticotrope (9), lactotrope (3) and somatotrope (21) human pituitary adenoma cell cultures. All 4 pituitary cell lines secreted VEGF, which in the case of AtT20, GH3 and TtT/GF cells was inhibited by approximately 50% by dexamethasone. TtT/GF cells were the most responsive to the different stimuli used since basal values were augmented by pituitary adenylate cyclase activating polypeptide-38 (PACAP-38), interleukin-6 (IL-6), transforming growth factor-alpha (TGF-alpha), IGF-I and the somatostatin analogue ocreotide. However, in GH3, AtT20 and alpha T3-1 cells, basal VEGF levels where not enhanced with any of the stimuli tested. The majority of the human adenomas tested (92%) basally secreted measurable VEGF which was inhibited by dexamethasone in most cases (84%). VEGF levels were increased in hormone inactive adenomas, somatotrope tumors and prolactinomas by TGF-alpha, PACAP-38, and 17 beta-estradiol, respectively. In conclusion, pituitary tumor cells are capable of producing VEGF which may be involved in tumoral angiogenesis. Our results concerning the suppression of VEGF by dexamethasone suggest that glucocorticoids may have anti-angiogenic properties and therefore therapeutic relevance for the treatment of pituitary adenomas.

Lipopolysaccharide directly stimulates the intrapituitary interleukin-6 production by folliculostellate cells via specific receptors and the p38alpha mitogen-activated protein kinase/nuclear factor-kappaB pathway.

Bacterial lipopolysaccharide (LPS) activates the immune system and induces increases in peripheral cytokines, which, in turn, affect the endocrine system. In particular, LPS-induced cytokines stimulate the hypothalamic-pituitary-adrenal axis to increase levels of antiinflammatory-acting glucocorticoids. In the present work, we show that LPS directly stimulates interleukin (IL)-6 release by mouse pituitary folliculostellate (FS) TtT/GF tumor cells and FS cells of mouse pituitary cell cultures. The stimulatory effect of LPS was strongly enhanced in the presence of serum, suggesting that LPS is only fully active as a complex with LPS-binding protein (LBP). Both TtT/GF cells and mouse pituitaries expressed CD14, which binds the LPS/LBP complex, and Toll-like receptor type 4, which induces LPS signals. LPS increased phospoinositol turnover in TtT/GF cells and induced phosphorylation of p38alpha mitogen-activated protein kinase and the inhibitor (IkappaB) of nuclear factor-kappa B. Nuclear factor-kappa B was activated by LPS in TtT/GF cells. Functional studies demonstrated that My4 (an antibody blocking the interaction between LPS/LBP and CD14), SB203580, (a specific inhibitor of p38alpha mitogen-activated protein kinase phosphorylation), dexamethasone, and the messenger RNA translation inhibitor cycloheximide all inhibited LPS-induced IL-6 production by TtT/GF cells and mouse pituitary FS cells. LPS-induced intrapituitary IL-6 may modulate the function of anterior pituitary cells during bacterial infection/inflammation.

Interleukin-6 is inhibited by glucocorticoids and stimulates ACTH secretion and POMC expression in human corticotroph pituitary adenomas.

Interleukins and their receptors are expressed intrinsically in the anterior pituitary and regulate hormone production and cell proliferation. It has previously been shown that interleukin-6 (IL-6) regulates hormone secretion in normal pituitary cells and cell lines. Here we examined the effects of IL-6 on propiomelanocortin (POMC) expression and ACTH production in corticotroph adenoma cells in vitro. We found that IL-6 stimulates both ACTH secretion and POMC gene expression in corticotroph adenoma cell cultures. This first demonstration of the stimulatory action of IL-6 on human corticotroph adenoma cell function provides further evidence for a direct action of IL-6 on corticotroph pituitary cells. We have confirmed previous reports of IL-6 production by corticotroph adenoma cells and in addition, demonstrated for the first time that the synthetic glucocorticoid dexamethasone is a potent suppressor of intratumoral IL-6 production. This intratumoral produced IL-6 may be in part responsible, in an autocrine manner, for the stimulation of ACTH synthesis and secretion. Our results suggest that IL-6 might play a role in the pathogenesis of Cushing's disease. However, elevated glucocorticoid levels in patients with Cushing's disease may prevent excessive action of IL-6 on ACTH production and tumor progression of corticotroph adenomas in vivo.

Assessment of diabetes-related distress.

OBJECTIVE: To describe a new measure of psychosocial adjustment specific to diabetes, the Problem Areas in Diabetes Survey (PAID), and to present initial information on its reliability and validity. RESEARCH DESIGN AND METHODS: Before their routine clinic appointments, 451 female patients with type I and type II diabetes, all of whom required insulin, completed a self-report survey. Included in the survey was the PAID, a 20-item questionnaire in which each item represents a unique area of diabetes-related psychosocial distress. Each item is rated on a six-point Likert scale, reflecting the degree to which the item is perceived as currently problematic. A total scale score, hypothesized to reflect the overall level of diabetes-related emotional distress, is computed by summing the total item responses. To examine the concurrent validity of the PAID, the survey also included a series of standardized questionnaires assessing psychosocial functioning (general emotional distress, fear of hypoglycemia, and disordered eating), attitudes toward diabetes, and self-care behaviors. All subjects were assessed for HbA1, within 30 days of survey completion and again approximately 1-2 years later. Finally, long-term diabetic complications were determined through chart review. RESULTS: Internal reliability of the PAID was high, with good item-to-total correlations. Approximately 60% of the subject sample reported at least one serious diabetes-related concern. As expected, the PAID was positively associated with relevant psychosocial measures of distress, including general emotional distress, disordered eating, and fear of hypoglycemia, short- and long-term diabetic complications, and HbA1, and negatively associated with reported self-care behaviors. The PAID accounted for approximately 9% of the variance in HbA1. Diabetes-related emotional distress, as measured by the PAID, was found to be a unique contributor to adherence to self-care behaviors after adjustment for age, diabetes duration, and general emotional distress. In addition, the PAID was associated with HbA1 even after adjustment for age, diabetes duration, general emotional distress, and adherence to self-care behaviors. CONCLUSIONS: These findings suggest that the PAID, a brief, easy-to-administer instrument, may be valuable in assessing psychosocial adjustment to diabetes. In addition to high internal reliability, the consistent pattern of correlational findings indicates that the PAID is tapping into relevant aspects of emotional distress and that its particular feature, the measurement of diabetes-related emotional distress, is uniquely associated with diabetes-relevant outcomes. These data are also consistent with the hypothesis that diabetes-related emotional distress, separate from general emotional distress, is an independent and major contributor to poor adherence. Given that the study was limited to female patients using insulin, further examination of the clinical usefulness of the PAID will need to focus on more heterogeneous samples.

Insulin omission in women with IDDM.

OBJECTIVE: To describe the extent of intentional insulin omission in an outpatient population of women with insulin-dependent diabetes mellitus (IDDM) and examine its relationship to disordered eating, attitudes toward diabetes, other psychosocial factors, long-term complications, and glycemic control. RESEARCH DESIGN AND METHODS: Before their routinely scheduled clinic appointments, female IDDM patients who were 13-60 years of age completed a self-report survey (final n = 341). The survey included standardized questionnaires assessing disordered eating attitudes and behaviors, psychological functioning (general distress, diabetes-specific distress, and hypoglycemic fear), attitudes toward diabetes, and self-care behaviors. All subjects were assessed for glycosylated hemoglobin within 30 days of survey completion. Long-term complications were determined through chart review. RESULTS: Approximately 31% of the subject sample, representing women of all ages, reported intentional insulin omission, but only 8.8% reported frequent omission. Compared with non-omitters, omitters reported more disordered eating, greater psychological distress (general and diabetes-specific), more hypoglycemic fear, poorer regimen adherence, and greater fears concerning improved diabetes management (which may lead to weight gain). Omitters evidenced poorer glycemic control, more diabetes-related hospitalizations, and higher rates of retinopathy and neuropathy. Multivariate examination revealed only two variables that independently predicted omission: diabetes-specific distress and fear of improved glycemic control ("because I will gain weight"). Of the omitters, approximately half reported omitting insulin for weight-management purposes (weight-related omitters). These subjects evidenced significantly greater psychological distress, poorer regimen adherence (including more frequent omission), poorer glycemic control, and higher rates of complications than did non-weight-related omitters as well as non-omitters. Non-weight-related omitters tended to fall between weight-related omitters and non-omitters on most measures of psychological functioning, adherence, and glycemic control. CONCLUSIONS: These findings suggest that insulin omission is common, that it is not limited to younger women, and that the medical consequences of omission, especially frequent omission, may be severe. Although a strong association between omission and disordered eating was observed, these data suggest that this link may be complicated by important diabetes-specific factors. Patients preoccupied with eating and weight concerns may also become emotionally overwhelmed by diabetes and/or fearful of normoglycemia (and the associated weight-related consequences), thus reinforcing the desire to omit insulin and maintain elevated blood glucose levels.