Currently, no effective treatment for malignant pheochromocytoma exists. The aim of our study was to investigate the role of chromogranin A (CgA) as a specific target molecule for immunotherapy in a murine model for pheochromocytoma. Six amino acid-modified and non-modified CgA peptides were used for dendritic cell vaccination. Altogether, 50 mice received two different CgA vaccination protocols; another 20 animals served as controls. In vitro tetramer analyses revealed large increases of CgA-specific cytotoxic T cells (CTL) in CgA-treated mice. Tumors of exogenous applied pheochromocytoma cells showed an extensive infiltration by CD8+ T cells. In vitro, CTL of CgA-treated mice exhibited strong MHC I restricted lysis capacities towards pheochromocytoma cells. Importantly, these mice showed strongly diminished outgrowth of liver tumors of applied pheochromocytoma cells. Our data clearly demonstrate that CgA peptide-based immunotherapy induces a cytotoxic immune response in experimental pheochromocytoma, indicating potential for therapeutic applications in patients with malignant pheochromocytoma.

During sepsis, an intact adrenal gland glucocorticoid stress response is critical for survival. Recently, we have shown that Toll-like receptors, particularly TLR2 and TLR4, are crucial in HPA axis regulation following inflammation, establishing a direct link between bacterial and viral ligands and the endocrine stress response. However, the exact role which TLRs play in adrenal homeostasis and malfunction is not yet sufficiently known. Using quantitative real-time PCR, confocal microscopy and the NF-kappaB reporter gene assay, we aimed to analyse both, expression and function of all relevant TLRs in the human adrenocortical cell line-NCI-H295R and adrenal cells in primary culture. Our results demonstrate a differential expression pattern of TLR1-9 in human adrenocortical cells as compared to immune cells and adrenocortical cancer cells. Consequently, activation of these cells by bacterial ligands leads to differential induction of cytokines including IL6, IL8 and TNF-alpha. Therefore, Toll-like receptors expression and function is a novel feature of the adrenal stress system contributing to adrenal tissue homeostasis, regeneration and tumorigenesis.

Aldosterone synthesis is primarily regulated by angiotensin II and potassium ions. In addition, endothelial cell-secreted factors have been shown to regulate mineralocorticoid release. We analyzed the pathways that mediate endothelial cell-factor-induced aldosterone release from adrenocortical cells, NCI-H295R using endothelial cell-conditioned medium (ECM). The cAMP antagonist Rp-cAMP caused a 44% decrease in the ECM-induced aldosterone release but inhibition of cAMP-dependent PKA had no effect on aldosterone release. Interestingly, inhibition of cAMP-regulated guanine nucleotide exchange factor Epac with brefeldin-A decreased the ECM-induced aldosterone release by 45%. Similarly, inhibition of p38 MAP-kinase; PI-3-kinase and PKB significantly reduced the ECM-induced aldosterone release whereas inhibition of ERK1/2 and PKC did not decrease aldosterone release. These results provide evidence for the existence of a cAMP-dependent but PKA-independent pathway in mediating the ECM-induced aldosterone release and the significant influence of more than one signaling mechanism.

Endothelial cells play an important role in the development and functioning of endocrine tissue and endothelial cell-derived factors have been shown to regulate mineralocorticoid release in bovine adrenal cells. In the present study, we analysed the role of human endothelial cells in the synthesis and release of aldosterone from adrenocortical cells (NCI-H295R). Endothelial cell-induced aldosterone release was rapid and lasted as a long-term effect over a period of 48 h. This stimulant effect was influenced by the duration of endothelial cell conditioning and decreased linearly with increasing dilutions of the conditioned medium. At the molecular level, an increase in the mRNA transcripts of aldosterone synthase and StAR could be observed. Cellular interaction with endothelial cell-factors enhanced the activation of CRE, and the promoter activity of both StAR and SF-1 reporter genes. In conclusion, human endothelial cells are important intra-adrenal regulators of human aldosterone synthesis and release.

The A-ZIP/F-1 transgenic mouse is a model of lipoatrophic diabetes with severe insulin resistance, hyperglycemia and hyperlipidemia. Recently, a regulatory role of adipose tissue on adrenal gland function and blood pressure has been suggested. To further explore the importance of adipose tissue in the regulation of adrenal function and blood pressure, we studied this mouse model of lipodystrophy. A-ZIP/F-1 mice exhibit significantly elevated systolic and diastolic blood pressure values despite lack of white adipose tissue and its hormones. Furthermore, A-ZIP/F-1 lipoatrophic mice have a significant reduction of adrenal zona glomerulosa, while plasma aldosterone levels and aldosterone synthase mRNA expression remain unchanged. On the other hand, lipoatrophic mice present elevated corticosterone levels but no adrenocortical hyperplasia. Ultrastructural analysis of adrenal gland show significant alterations in adrenocortical cells, with conformational changes of mitochondrial internal membranes and high amounts of liposomes. In conclusion, lipodystrophy in A-ZIP/F-1 mice is associated with hypertension, possibly due to hypercorticosteronemia and/or others metabolic-vascular changes.