self-tolerance [92, 93, 94]. It is proposed that such hidden epitopes are presented to lymphocytes in the secondary lymphatic organs and maybe also ectopically in the inßamed glands because tubuloacinar glandular HLA- DR-positive cells themselves may act as aberrant antigen-presenting cells [95, 96, 8, 6]. Cooperative T-cell and B-cell responses develop against such molecules, their nature being revealed in the speciÞcities of the autoantibodies produced, which in SS include ribonucleosomal bodies [97, 98, 99, 100], cell cytoskeleton [101], and muscarinic acetylcholine receptor [102].

VIP, although called a neuropeptide, is like many other Òneuropeptides,Ó also synthesized by non-neuronal cells, mainly Th2 CD4+ and type 2 CD8+ cells. VIP has been identiÞed as a potent anti-inßammatory factor [70, 33]. It participates in the regulation of DHEA and testosterone production [103], probably via gene transcription Þne-tuning steroidogenic enzymes. VIP activated directly androgen receptors via VIP receptor in an androgen-independent and protein kinase A- dependent manner [104]. On the other hand, low 17β-estradiol levels lead to an increased expression of VIP receptors [105]. Depletion of VIP from post-ganglionic, parasympathetic nerve Þbers could result from excessive stimulation in a dry mouth patient or muscarinic acetylcholine receptor autoantibodies [106], which might deprive the acinar cell from its acinotrophic support. VIP as a target for therapy has raised great interest, but attempts have been hampered by poor metabolic stability and poor penetration to targets [107]. These problems hint to a high potency of VIP. These difÞculties are in the human body circumvented by delivery from the protected environment of the nerve terminal.

11.4Sex Steroids

11.4.1Steroidogenesis in Adrenal Glands

In primates sex steroids are not only synthesized in gonads, but also locally from adrenal prohormones in peripheral tissues. Adrenal

gland-derived DHEA, DHEA-S, and androstenedione serve as substrates for the peripheral synthesis of active androgens and estrogens, like testosterone, dihydrotestosterone (DHT), and 17β-estradiol (17β-E2). Adrenal glands consist of two compartments which differ both structurally and functionally, medulla and cortex (constitutes 90% of the weight of the gland).

Catecholamines, epinephrine and norepinephrine, are produced in medulla, a specialized ganglion of the sympathetic nervous system. It is innervated by cholinergic post-ganglionic sympathetic nerves. Medullary neuroendocrine cells contain post-synaptic cholinergic receptors. Upon stimulation these chromafÞn cells secrete (nor)epinephrine, which is exocytosed directly into the bloodstream [108].

The cortex of the adrenal gland is organized into three morphologically distinct layers. The superÞcial glomerular zone represents 15% of the weight of the cortex, the intermediate fasciculate zone 65%, and the innermost reticular zone 20%. Their phenotypic differences reßect their specialized functions: glomerular zone produces mineralocorticoids (aldosterone), fasciculate zone glucocorticoids (such as cortisol), and reticular zone DHEA prohormones. Reticular zone is unique to primates and does not exist in non-primates, like in mice or rats often used as models for SS [109].

During their lifespan the cells of the cortex migrate from the outer parts of the cortex centripetally toward the inner parts, undergoing successive transformations from glomerular to fasciculate and Þnally to reticular zone cells, apparently guided by the changing composition of the basement membrane laminin chains [110]. Glomerular zone cells divide fast, whereas cells in the inner parts of the cortex in the reticular zone have a slow division rate. Conversely, reticular zone cells die more rapidly than in other cortical cells. Cells are forced from the superÞcial zone toward the deeper zones. These events regulate the cell turnover in the cortex [109].

Corticosteroids refer to hormones produced in the adrenal cortex. The metabolic pathway from cholesterol substrate to DHEA and DHEA- S involves three and four steps, respectively (Fig. 11.4) [111, 112]. DHEA-S concentration

Fig. 11.4 Endocrine metabolism of cholesterol to dehydroepiandrosterone sulfate in the reticular zone of the adrenal cortex. Cholesterol is Þrst converted to pregnenolone from cholesterol catalyzed by mitochondrial cholesterol side chain cleaving cytochrome P450 (P450ssc). This step consists of three separate reactions: 20-hydroxylation, 22-hydroxylation, and, Þnally, breakage of the 20,22 CÐC bond. Subsequently, 17α- hydroxylase activity of P450c17 hydroxylates pregnenolone to 17-OH-pregnenolone, which is in the next

step converted to DHEA by the 17,20-lyase activity of the same enzyme. DHEA sulfotransferase (in the adrenal gland SULT2A1 isoform) sulfates DHEA yielding DHEA-S, the most plentiful product of the adrenal glands and the most common steroid in the circulation in humans. DHEA-S can be desulfated by steroid sulfatase (STS). Instead of conversion to DHEA-S, DHEA can alternatively be converted to androstenedione by 3β- steroid dehydrogenase and [4, 5]-isomerase (3β-SDH)

is approximately 300-fold higher than that of DHEA. Actually, adrenal glands are almost solely responsible for the production of DHEA and DHEA-S so that 90% of DHEA and 98% of DHEA-S in the human body is produced in the cortex.

The synthesis of mineralocorticosteroids continues from pregnenolone to progesterone and then to deoxycorticosterone and corticosterone in reactions catalyzed by 3β-SDH, P450c21,

and P450c11, respectively. Reaction catalyzed by aldosterone synthase Þnally yields aldosterone. The synthesis of glucocorticosteroids from pregnenolone involves 3β-SDH, P450c21, and P450c11 following the route 17-OH- pregnenolone → 17-OH-progesterone →17-OH- deoxycortisol → cortisol.

Conversion of cholesterol to pregnenolone by P450scc is the rate-limiting step in corticosteroid synthesis. In contrast, P450c17 determines

the direction of the synthesis. When this enzyme is not present (glomerular zone), C-21 17-deoxysteroids, like aldosterone, are produced. On the other hand, when the 17α- hydroxylase activity of P450c17 is present, C-21 17-hydroxysteroids, like cortisol, are produced. Finally, in the presence of both 17α-hydroxylase and 17,20-lyase C-19 steroid DHEA(-S) is synthesized [112].

11.4.2Regulation of the Adrenal Steroidogenesis

Corticosteroid synthesis in the cortex is regulated by the hypothalamusÐpituitaryÐadrenal (HPA) axis. Corticotropin-releasing hormone (CRH) and corticotropin (adrenocorticotrophic hormone, ACTH) regulate synthesis of all corticosteroids. CRH released by hypothalamic neuroendocrine cells binds to its CRH-R1 receptor in the pituitary gland, which stimulates secretion of pro-opiomelanocortin (POMC). POMC is a precursor polypeptide, which is processed in a tissue-speciÞc manner to various hormonal peptides, in the pituitary gland by pituitary prohormone convertases to a large extent to ACTH, which is released into circulation. In adrenal cortex ACTH stimulates secretion of glucocorticosteroids, aldosterone, and sex steroid precursors [111]. Cortisol exerts negative feedback on the hypothalamus (CRH secretion) and pituitary gland (ACTH secretion).

Although in part regulated by ACTH, the primary regulators of aldosterone synthesis are angiotensin II and extracellular potassium. Adipocyte-derived factors, adrenaline, serotonin, and VIP, also increase production of corticosteroids, especially aldosterone [113, 114, 115]. Synthesis of cortisol is stimulated by high concentrations of potassium, T-cell-derived glucosteroid response-modifying factor (GRMF), and IL-1. Other systemic factors regulating DHEA(-S) synthesis include estrogens, insulin, and growth hormone [116, 117, 118].

Apart from systemic factors, intra-adrenal factors regulate corticosteroids independently from ACTH so that serum DHEA(-S) levels are low in

chronic inßammation in spite of normal ACTH levels [119]. Both immune and the adrenal cells themselves are potential sources [120]. Macrophages inÞltrate adrenal glands, especially the reticular zone, in healthy people [121]. These cells secrete cytokines, e.g., IL-1, IL-6, and tumor necrosis factor-α (TNF-α), which inßuence the function of the adrenal cortex. Lymphocyte inÞltrates are present in the adrenal glands, in particular in the reticular zone of elderly people, making lymphocyte-derived ACTH a potential regulator of DHEA(-S) synthesis [122].

Adrenocortical cells can themselves secrete cytokines. Glomerular zone cells secrete IL-6 and TNF-α, fasciculate zone cells TNF-α and IL-18, and reticular zone cells IL-1, IL-6, and IL-18 [120]. The effects of these cytokines on the adrenal glands are variable. IL-1 enhances glucocorticosteroids and inhibits aldosterone production. IL-6 stimulates release of cortisol and DHEA from the fasciculate and reticular zones, respectively. TNF-α induces a shift from cortisol to DHEA production in humans. Furthermore, corticosteroid-producing cells are regulated by nerve endings in contact with them [123].

Aging affects DHEA(-S) production (Fig. 11.5) [124]. The fetal adrenal cortex consists of two layers, the fetal zone, which produces high concentrations of DHEA(-S), and neocortex. Glomerular zone and fasciculate zone later develop from the neocortex, whereas the fetal zone atrophies. The focal development of the reticular zone begins Þrst around the age of 3 and a continuous reticular zone has developed by Þlled 6 years. In addition to these morphological zonation changes, adrenarche involves changes in the expression of steroidogenic enzymes. Cytochrome b5 (CYB5) and steroid sulfotransferase (which are regulators of the 17,20-lyase activity of P450c17 and of the enzyme converting DHEA to DHEA-S, respectively) increase in the enlarging reticular zone [125] concomitantly with a decrease of 3β-SDH, which competes with P450c17 for substrate [126].

After adrenarche, circulating concentrations of DHEA(-S) increase and reach their adult peak levels at 20Ð30 years. Thereafter, their concentrations start to decline due to adrenopause

Fig. 11.5 Effect of aging on DHEA-S concentration. Fetal adrenal glands produce great quantities of DHEA-S so that its concentration is very high in newborns. DHEA-S concentrations fall rapidly after birth and stay low for a few years upon regeression of the fetal zone. Between the ages of 6 and 8 the adrenal glands undergo changes in the expression of steroidogenic enzymes and morphology in a process called adrenarche, which leads to formation of the reticular zone and to a rise in the systemic concentration of DHEA-S to high levels between the ages of 20 and 30. Thereafter, DHEA-S concentrations start to decline due to adrenopause and regression of the reticular zone and by the ages of 70Ð80 the DHEA-S levels are only 20 and 30% of the peak concentrations in men and women, respectively

[111, 123]. Reticular zone decreases in thickness and zonal irregularity develops [109]. The exact mechanisms behind these changes are still obscure. Cell deaths caused by infarcts, telomere shortening, or mutations could disrupt the architecture of the adrenal cortex. Cytokines and enzymes related to senescence could contribute [109]. A decrease in the 17,20-lyase activity could explain diminished DHEA(-S) production [127]. Finally, a decrease in the DHEA response of the adrenal cortex to CRH has been observed in aging people, with no change in ACTH or cortisol [128].

Adrenal synthesis of so-called prohormones DHEA(-S) plays a remarkable role in primates for the peripheral synthesis of sex steroids.

11.4.3Adrenal Function in Sjögren’s Syndrome

During long-lasting inßammatory diseases secretion of the adrenal prohormones decreases

[129], probably as the result of the abovementioned neuroimmunoendocrine dysregulation. Accordingly, patients with both pSS and sSS have decreased DHEA-S concentrations [130, 131, 132], probably due to decreased production in the adrenal glands. However, the ultimate reason behind this diminished production might differ in pSS and sSS.

Low systemic levels of DHEA in pSS may ensue from premature and deeper-than-usual adrenopause [109] as a result of primary adrenal gland failure. There are no differences in the concentrations of ACTH or cortisol between patients with pSS and healthy controls [130], suggesting changes in the reticular zone.

Patients with sSS have an underlying autoimmune rheumatic disease, which precedes its development. This underlying disease causes alterations in the entire cytokine and cellular environment of the patientÕs body [133, 134]. In the short term, inßammatory cytokines acutely stimulate CRH and ACTH secretion and thus stimulate production of corticosteroid ÒstressÓ hormones. However, during chronic inßammation the responsiveness of the HPA axis becomes blunted. This is especially noticeable for DHEA(-S), which is decreased and remains decreased even after stimulation with ACTH. Chronic inßammation leads to a secondary adrenal gland failure. There are contradictory Þndings about the levels of cortisol secreted in chronic inßammation, but cortisol levels seem inappropriately low considering the diseaserelated stress.

Cytokines, especially IL-1β, IL-2, IL-3, IL-6, and TNF-α, directly decrease DHEA(-S) synthesis [119], causing a shift from production of DHEA toward cortisol [135, 136]. Primary SS is characterized by lower concentrations of cortisol than sSS [137]. Thus, production of DHEA is perhaps decreased for a different reason in pSS and sSS. Naturally, the cytokine-mediated down-regulation also develops in pSS, in addition to the primary failure of the adrenal glands in pSS. We will later see how this diminution of the endocrine DHEA(-S) production can unmask the peripheral intracrine DHEA-to-DHT conversion defect in the exocrine glands.