11.4.4Peripheral Intracrine Synthesis of Sex Steroids

In addition to the synthesis in the gonads, primates are unique in synthesizing active sex steroids also locally in peripheral tissues. Unlike other mammals, the cortex of the adrenal glands of the primates secretes large amounts of prohormones. These precursors constitute a large reservoir of substrate and can be further processed into active sex steroid extragonadally. This allows tailor-making according to local tissue needs, enables similar function of ÒunisexÓ organs (organs, which work similarly in men and women in spite of the systemic differences in the sex steroid milieu), and provides a buffering system against changes over time in the systemic sex steroid levels. This local intracrine sex steroid production acts as a buffer with regards to circadian, menstrual, pregnancy, and lifelong chronobiological changes in systemic hormone levels. This Þeld of the study of the local synthesis of

active sex steroids from precursor hormones produced in adrenal glands is called intracrinology [138]. Sex steroids produced have usually but not always an intracrine mode of action [139, 132, 140]. Classical intracrine tissues include prostate, uterus, and mammary glands. However, many other tissues have been shown to produce sex steroids locally, adipose tissue and bone being among them [141].

Steroidogenic intracrine enzymes catalyze the peripheral synthesis of active androgens or estrogens from DHEA (Fig. 11.6) [142]. Approximately 50% of total androgens in the prostate of an adult man and 75% and nearly 100% of peripheral estrogens in premenopausal and post-menopausal women, respectively, are synthesized locally [138, 143]. It would be interesting to know the corresponding Þgures in exocrine glands. The higher the Þgure, the more dependent the exocrine glands are on their local intracrine enzymatic machinery.

11.4.5Intracrine Sex Steroids Production in pSS and sSS

In the periphery cytokines, e.g., TNF, IL-1, and IL-6, are generally considered to increase the activity of aromatase and to shift the production of active sex steroid toward estrogens, at the cost of androgens [144, 145]. The function of 17β-HSD is stimulated by epidermal growth factor (EGF), IFN-γ, TGF-α, and TGF-β and that of 5α-reductase by TGF-β1 and TGF-β2 [119]. Additionally, TNF-α has been shown to inhibit the conversion of DHEA-S to DHEA in rheumatoid arthritis synovial cells leading to decreased local concentrations of DHEA in the diseased joints [146].

The effect of sex steroids on the production of cytokines is double-faced: androgens tend to inhibit the synthesis of proinßammatory cytokines, whereas estrogens have an opposite role [147]. Testosterone decreases secretion of pro-inßammatory cytokines by macrophages and epithelial cells of the lacrimal glands. Androgens enhance production of antiinßammatory cytokines in the lacrimal gland [148, 149].

Intracrine conversion of DHEA to DHT is impaired in the exocrine glands in pSS [132, 140, 150]. If this is the primary underlying defect, then as long as there is enough prohormone input from the adrenal glands to the intracrine enzymatic machinery, the exocrine glands could produce enough DHT for their needs (for acinar cell remodeling). If such a SS-prone person happens to be genetically programmed to an early and/or deep adrenopause, this ÒtoleranceÓ or ÒstressÓ test would unmask the primary, peripheral intracrine defect and lead to a local DHT deÞciency in the glandular tissues. Adrenopause could explain the peak age of incidence of new SS cases. In this scenario pSS is associated with an inherent intracrine defect, which is aggravated by adrenopause. In sSS inßammation-associated changes impair adrenal gland function and when this has continued for 10Ð20 years such a systemic DHEA deÞciency might Þnally damage the glands and provoke an immune attack. Thus,

pSS would be mainly provoked by a primary intracrine (local) DHEA deÞciency, whereas sSS would be provoked by a secondary deÞciency caused solely by endocrine (systemic) DHEA failure.

11.4.6Sex Steroids in Female and Male Sjögren’s Syndrome

The above-mentioned paradigm has an additional advantage. It is not known why SS is so heavily dominated by women. The reason might be that the DHT demand in exocrine glands in women has to be totally satisÞed by the local intracrine conversion from DHEA-S. This requires coordinated function of at least four enzymes, which in addition can go astray due to the many crossroads in the intracrine metabolic pathways. Men have similar intracrine machinery, but are less vulnerable to its local malfunction because they also feed this system directly with testosterone, only one step away from DHT. This could secure men against SS and could explain the female dominance.

11.4.7Putative Mechanism of Action of the Intracrine Processing Defect

The acinotrophic VIP may play an important role in the maintenance of acini. Acinar remodeling also means that apoptotic and/or necrotic (lost) acinar cells have to be replaced. Progenitor cells residing in the intercalated ducts have the capacity to undergo asymmetric cellular divisions. This way they retain their stemness, but one of the two daughter cells can trans-differentiate to an acinar cell, to replace the lost one. This progenitor capacity has been conÞrmed by cellular cloning [151, 152].

It is not known what initiates the asymmetric division of the progenitor cell(s), but loss of conßuence of the cells on the tubuloalveolar basement membrane upon death and detachment of one or more acinar cells might trigger it. The intercalated duct progenitor cell destined to

become an acinar cell has Þrst to transmigrate from the duct to the acinar space. Second, it needs to receive a site-speciÞc or acinus-speciÞc differentiation signal. It seems that these two functions are fulÞlled by the extracellular basement membrane matrix read by cellular integrin receptors. Indeed, similar to the cloned intercalated duct cells, human submandibular gland HSG intercalated duct cell line [153], when cultured on laminin α1 (laminin-α1β1γ1 or simply laminin-111) containing basement membrane (Matrigel), trans-differentiate into acinar cells and start to produce acinar cell markers, such as salivary amylase and cystatin C [154, 155]. They form acinus-like clusters, where cells in the middle, not in contact with laminin-111, undergo apoptosis creating an acinar lumenlike structure. Matrigel membranes used in such experiments are growth and differentiation factors depleted to avoid confounding factors. It seems that laminin-111 is necessary for the trans-differentiation.

Progenitor cells migrate on basement membrane using laminin-111-binding integrin receptors. The acinus-speciÞc differentiation signal responsible for the outsideÐinside signaling and trans-differentiation is also read using these α1β1 and α2β1 integrins [156]. Their level of expression is greatly increased by DHEA treatment, 5.6-fold and 11.7-fold in ductal cells and 3-fold and 5-fold in acinar cells [157]. Thus, in DHEA deÞciency the progenitor cells have probably a much decreased capability to migrate to the acinus and, in particular, to trans-differentiate there to a secretory acinar cell. SS is characterized by low levels or loss of acinar-speciÞc (not found in the salivary ducts) laminin-111 [158] and of the corresponding epithelial cell integrins α1β1 and α2β1 [156]. In advanced SS acini disappear, which is associated with a reciprocal ductal cell hyperplasia [159], as if the ductal cells had Þlled the acinar spaces without being able to trans- differentiate into secretory acinar cells, probably due to deprivation of the laminin-111/Int α1β1 and α2β1 signaling.

11.5Inflammatory Cells and Cytokines

11.5.1 General Histopathology

Impairment of the quantity and/or quality of the secretory acinar cells not only leads to a compensatory increased stimulation of acetylcholine secretion, but also to a depletion of the acinotrophic VIP. In this speculative scenario immune attack would be secondarily activated against altered self and further impair the structure and function of the exocrine glands.

At early stages of involvement of the adaptive immune system, lymphocytes start to accumulate in exocrine glands via tethering, rolling, adhesion, and migration through the high endothelial cell venules (homing). If some recirculating lymphocytes happen to get into contact with altered self, they become activated. The process continues to be facilitated by ampliÞcation and chemokines. Apoptosis of the inÞltrating cells is inhibited by cytokines IL-2, IL-10, TNF-α, and others.

This leads to the characteristic histopathological Þnding, formation of focal, periductal lymphoplasmacytoid inÞltrates, which is the single best criterion of the salivary gland involvement and syndrome.

The inÞltrating cells are not only predominantly mature T lymphocytes (80%), mostly CD4+ cells, but also CD8+ cells, with the remaining 20% being composed of B lymphocytes and plasma cells. InÞltrates are Þrst dominated by T lymphocytes, but the proportion of B lymphocytes and in particular plasma cells increases over time. Monocytes or NK cells are rare, but macrophages and dendritic cells appear when germinal centers are formed [160].

Some salivary ducts become slightly dilated. With increasing severity of disease, there is an increase in the number of lymphocyte foci and a progressive loss of acini. At the end stage, ducts undergo hyperplasia and only a few acini remain in a conßuent mass of lymphocytoid cells [6].

11.5.2 T Lymphocytes

The majority of the inÞltrating CD4+ and some CD8+ cells secrete IL-17 and are actually Th17 cells [161]. TGF-β, IL-6, and IL-18 induce IL-17 production [162]. IL-17, in co-operation with IL-18, stimulates secretion of IL-6 and IL-8 by salivary epithelial cells, which may be important for the recruitment of immigrant inßammatory cells. The majority of the CD4+ cells are CD45RO+ with αβ TCR (T-cell receptor), with perhaps a low frequency of T cells expressing TCRs with Vβ 7.2 in SS patients producing autoantibodies [163]. Adhesion and costimulatory molecules may lead to perpetuation and they secrete IFN-γ, which induces ectopic HLA-DR expression of the ductal epithelial cells.

CD8+ T cells expressing αEβ7 (CD103) integrin localize around acinar epithelial cells, which express E-cadherin. However, the number of apoptotic acinar cells is not much increased in

SS[164].

11.5.3B Lymphocytes

surrounded by a mantle zone containing small B cells.

Primary SS is characterized by a disturbed B- cell maturation with abnormal selection, defects in editing Ig receptors, and abnormal mutational targeting. Disturbances in the B-cell maturation during early B-cell development and in germinal centers might play a role in the autoimmune/lymphoproliferative syndrome [165]. B cell may play a much more important role in SS than has been previously appreciated and, accordingly, B-cell ablative treatment seems to work quite well in SS [166].

IL-12 or IL-18 might play a role in lymphoma development in pSS and excessive BAFF might result in inappropriate maturation of B- cell subsets, paired with an increased survival of mature cells. Constitutive activation of activationinduced cytidine deaminase (AID) promoting illegitimate DNA recombinations and somatic mutations could result in neoplastic transformations [167].

11.5.4 Chemokines

Altered proportions of Bm1Ð5 subpopulations with a relatively high percentage of circulating activated B cells have been observed in SS. A diminished percentage of peripheral memory B cells, with accumulation of such cells and plasma cells in the parotid glands, suggest disturbances in B-cell trafÞcking.

IgG is the predominant isotope expressed in the inÞltrating B cells, whereas IgA dominates in normal glands [164]. A substantial number of the B cells inÞltrating salivary glands are potentially self-reactive CD5+ B-1 plasma cells. Ectopic germinal center-like structures are frequently found in pSS, reßecting antigen-driven, T-cell- dependent, B-cell-mediated immune responses. Histologically they in their fully developed stage contain follicular dendritic cells and comprise a central (dark) zone of proliferating B-cells (centroblasts) surrounded by a light zone of B cells (centrocytes) now undergoing selection for high afÞnity surface antibody expression to generate memory cells or plasma cells. Germinal center is

The role of chemokines attracting lymphocytes to exocrine glands in SS has generated a lot of interest. CXCL13 (B-cell attracting chemokine 1), CXCL12 (stromal cell-derived factor-1), CCL19 (EBV-induced molecule 1 ligand chemokine), and CCL21 (secondary lymphoid tissue chemokine) all contribute to lymphoid cell homing and to the persistence of chronic inßammation in pSS. Regarding B lymphocytes, ectopic expression of CXCL13 was found on endothelial cells and germinal center-like structures and CXCL12 was strongly expressed by ductal epithelial cells. CXCL9 (monokine induced by IFN-γ) and CXCL10 (IFN-γ inducible protein-10) and impaired function of their epithelial receptor CXCR3 or Òchemokine scavengerÓ are involved in the accumulation of T lymphocytes [168]. The CXCL13 receptor CXCR5 on inÞltrating mononuclear cells contributes to recruitment of B cells and activated T cells. B lymphocyte attracting chemokines contributes to local