Syndrome in the Salivary

Glands

Primarily for diagnostic purposes, a salivary gland biopsy is evaluated for the presence and frequency of focal cellular aggregates, deÞned as clusters of at least 50 mononuclear cells [1]. Inßammatory foci tend to form around the ductal epithelium and inÞltrates seem to subsequently expand and occupy at a later-stage acinar epithelium as well [2]. Analyses of gene expression proÞles of salivary gland tissue from patients with SS conÞrmed the presence of many aspects of chronic inßammation [3Ð5]. The inÞltrates in the salivary glands consist of T cells, B cells [6, 7], plasmacytoid dendritic cells (pDCs) [5], follicular DCs (FDC) [8], and macrophages [9]. Recent studies in rather large patient groups indicated that ultrasonography (US) and magnetic resonance imaging might be promising alternatives to the conventional invasive sampling of a lower lip biopsy [10, 11]. Whereas US takes the advantage as a diagnostic tool for routine examinations, both methods appear to correlate well with results obtained using histopathological evaluations [10, 11]. Such non-invasive imaging technologies have, however, not yet been applied to Þelds such as disease progression or prognosis and follow-up on treatment interventions in SS. These areas of study would most certainly beneÞt from a non-invasive method to access salivary gland pathology.

15.1.1 Endothelial Cells

Endothelial cells lining the lumina of blood vessels are involved in leukocyte extravasation

movement of cells in particular directions to speciÞc locations [12] (Fig. 15.1). Leukocytes thereby migrate in response to, and along concentration gradients of chemotactic factors such as chemokines [13]. Despite their uncontested potential as candidates for therapeutic intervention, and their involvement in angiogenesis, Þbrosis, and malignancy [14], chemokines remain a rather poorly examined Þeld of study in SS [15Ð19]. The growth of blood vessels from pre-existing vasculature, termed angiogenesis or neovascularization, contributes signiÞcantly to inßammatory cell migration and depends critically on the balance or imbalance between angiogenic mediators and inhibitors [20, 21]. Neovascularization is recognized as a pathogenic process in rheumatoid arthritis (RA) and key molecules in the molecular pathway of angiogenesis have been explored as targets for therapeutic intervention [22]. In the Þeld of SS, conclusions derived from experimental studies argue for an interrelationship between neovascularization and impaired secretory function [19]. Additional research initiatives, however, need to clarify the exact role of neovascularization in the formation and perpetuation of the salivary gland pathology characteristic for SS.

Lymphocyte migration into the target tissue in SS is partly mediated by vascular cell adhesion molecule (VCAM)-1 and peripheral node addressin (PNAd) expressed on vascular endothelium (Fig. 15.1). Inhibition of these two molecules or their ligands (α4-integrin, L-selectin, and lymphocyte function-associated antigen-1 [LFA-1]) showed a nearly complete block of lymphocyte migration into the lacrimal glands of non-obese diabetic (NOD) mice [23], a strain accepted as a model of SS [24, 25].

Fig. 15.1 Mechanisms involved in lymphocyte migration. An important feature in regulation of lymphocyte recirculation is the ability of lymphocytes to recognize and bind to the surface of endothelial cells before migrating through the vessel into surrounding tissue.

(1) Circulating lymphocytes enter high endothelial venules. (2) Initial transient tethering and rolling: most lymphocyte adhesion molecules such as L-selectin are found on the tips of the lymphocytes microvilli where they can easily contact the endothelium by binding glycosylation-dependent cell adhesion molecule

(GlyCAM)-1 or CD34. (3) Appropriate activating factors: chemokines such as CXCL13 encountered in the local environment render possible a Þrst lymphocyte activation step which facilitates Þrm adhesion. Activated integrins, LFA-1 and VLA-4, on lymphocytes interact with ICAMs and VCAMs. (4) Lymphocyte diapedesis: migration through endothelium, from circulation to tissue, a process probably also directed by chemokines. (5) Migrated lymphocytes contribute to the local immune response. Figure adapted from Delaleu et al. [229]

In contrast, mucosal addressin cell adhesion molecule-1, E-selectin, or P-selectin did not seem to mediate monocyte migration into the target organs [23].

Besides guiding speciÞc inßammatory cell populations to migrate through the vessel wall into surrounding tissues, endothelial cells are also crucial in the physiologic process of saliva secretion [26]. Upon parasympathetic nerve stimulation, vasodilatation and increased capillary blood pressure lead to increased Þltration of ßuid from glandular capillaries into the interstitial space of the salivary glands. Through this process acinar cells are provided with the ßuid required for the secretion of primary saliva [27]. Reduced blood ßow responses to parasympathetic stimuli have been reported in patients with SS and may contribute to the reduced salivary gland secretion [27, 28].

15.1.2 Epithelial Cells

Although being at the center of SS pathology and primary targets of the pathogenic autoimmune reaction, acinar and ductal epithelial cells in the past often took a back seat compared to cells of the immune system with respect to the extent of research conducted on different cell types [29] (Fig. 15.2).

The important role of the epithelial cells in the pathogenesis of SS is suggested by the occurrence of lesions in various epithelial tissues (described as autoimmune epithelitis) as well as the increased epithelial expression of several inßammatory proteins in the histopathological lesions of patients [29]. Although professional antigen-presenting cells (APCs), such as DCs, macrophages, and B cells, are present in states of chronic inßammation such as SS

Fig. 15.2 Events related to etiology and pathogenesis of SS. Immune reactions to speciÞc non-self or selfstructures are initially determined by their nature. If recognized, the immune systemÕs appropriate decision on quality and extent of the reaction meets the potential threat posed by the antigen. At the same time, it must consider tissue-speciÞc requirements, spare healthy tissue, and subsequently ensure proper instauration of tissue homeostasis. Processes involved in mounting and shaping immune reactions are manifold and require co-operation of multiple immune-cell subsets. Such interactions also appear to expand the immune systemÕs cognition from ÒselfÓ and Ònon-selfÓ to Òhow foreignÓ and probably also

Òhow dangerousÓ the recognized entity is. In addition, speciÞc immune-cell subsets appear to adequately regulate the ongoing effector phase of every immune reaction. Although, many of the processes described above are not entirely understood, different lines of evidence indicate that deÞciencies in one or several of these complex processes may trigger or contribute to the pathogenesis manifested as SS. The exocrine glands in SS are characterized by distinct inßammatory foci, mainly consisting of T and B cells. Their distinct effector functions appear to speciÞcally contribute to the pathogenesis of SS characterized by distinct exocrine gland inßammation and severe impairment of the affected glandsÕ secretory capacity

[9], interestingly, epithelial cells have been proposed to contribute to the process of antigen presentation. Central for the testing of such a possibility was the establishment of methodology for the long-term cultivation of non-neoplastic

salivary gland epithelial cell (SGEC) lines (of ductal type). These techniques have permitted the analysis of various aspects of phenotypic and functional properties of SGEC [30]. Two major concepts have emerged from these studies,

namely, the functional participation of SGEC in immune responses and the occurrence of Òintrinsic activationÓ of salivary gland epithelial cells in SS patients [29].

The notion of epithelial cells acting as nonprofessional antigen-presenting cells in the salivary glands of SS is underpinned by the presence in vivo and functional analyses in vitro of several molecules representing the APCÕs side required for the formation of an immunological synapse [29] (Fig. 15.2). Major histocompatibility complex (MHC) class II molecules, HLA-DR/DP/DQ [31], on the epithelial cellÕs surface, provided the presence of immunogenic peptides, could enable the delivery of the primary activation signal to T cells. Furthermore, β-2 microglobulin, a principal component of the MHC class I molecule, has been localized on salivary gland epithelium [31].

Toll-like receptors (TLRs), whose signals are pivotal in either supressing or activating APCs, are expressed on epithelial cells from the salivary glands [32, 33]. Upon binding of pathogen-associated molecular patterns (PAMPs) [34] or speciÞc endogenous factors [35], TLR ligation leads to signiÞcant cytokine production and upregulation of co-stimulatory and adhesion molecules [34, 35]. Indeed, co-stimulatory molecules such as cluster of differentiation (CD)80 (B7.1), CD86 (B7.2) [36], and CD40 [37], which mediate the second crucial signal for induction of an immune response, are expressed on epithelial cells and appear to be functional when tested in vitro [38]. Similar studies also suggested that CD86 on epithelial cells from patients might be more responsive to activation signals mediated via CD28 ligation compared to inhibitory signals provided by binding of cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) [36]. The increased presence of adhesion molecules, VCAM-1, endothelialleukocyte adhesion molecule (ELAM)-1, and more modestly of intercellular adhesion molecule (ICAM)-1, might further impact the outcome of non-professional APC/T-cell interactions [36, 37, 39].

Epithelial cells were also shown to produce several pro-inßammatory cytokines, e.g.,

interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF)-α [40], and possibly contribute to the cellular composition and organization of glandular inßammation in SS through production of chemokines, such as C-X-C motif ligand (CXCL)13, CXCL12, C-C motif ligand (CCL)21, CCL19, and CCL18 [16, 41]. In NOD mice, however, injection of anti-CXCL12 antibodies was effective in preventing diabetes and insulitis but did not affect the SS-like disease in this strain [42]. By and large, these data strongly suggest the operation of intrinsic activation mechanisms in the epithelial cells of SS patients and provide support to the notion of active participation of epithelial cells in the pathogenesis of this inßammatory condition [29]. However, whether epithelial cell activation is an event closely linked to the initiation of SS or has to be considered as a secondary phenomenon of the ongoing immune response triggered and shaped by other cell types requires further investigation.

Epithelial cells express Fas and FasL together with B-cell lymphoma-2-associated X protein (Bax) [43, 44]. These and similar results led to the proposition of increased apoptosis as a major mechanism responsible for acinar cell destruction. However, despite aberrant expression of pro-apoptotic molecules apoptosis appears to be a rare event among acinar and ductal cells of patients with SS [45]. Researchers also suggested that increased matrix metalloproteinases (MMP) activity, e.g. MMP-2, MMP-3, and MMP-9, could be related to the dramatic changes in the structural organization in the basal lamina and apical surface of acini observed in patients with SS [46, 47].

15.1.3 T cells

The mononuclear cell inÞltrate consists of up to 80% T cells representing a CD4+ to CD8+ T-cell ratio of more than 2 [6]. However, the proportion of different cell types within individual foci appears to vary signiÞcantly, possibly in association with the degree of lymphoid organization and disease progression [48]. Most CD4+ T cells are of a primed memory phenotype (CD45R0+)

and large shares express CD40L [49]. In context of T-cell activation, IL-2R has been found on a large fraction of inÞltrating lymphocytes [31, 50]. Interestingly, circulating T cells showed only weak proliferation after exposition to antiCD2 and anti-CD3 antibodies [51]. Furthermore, T-cell responses to stimulation with recombinant Ro and La in vitro seemed rather weak [52].

Analyses of T-cell receptors (TCRs) revealed that most T cells bear α/β TCRs [53] and the usage of the Vβ family repertoire was found to be relatively restricted. In addition, complementarity-determining region (CDR)3 analyses showed some conserved amino acid motifs suggesting a relatively limited number of recognized antigens [54]. Some T cells in the inßamed lesions expand clonally indicating an antigen-driven inßammation [55].

One form of how T cells and natural killer (NK) cells might contribute to the pathogenesis of SS is by epithelial cell death, induced by CD8+ T cells, natural killer T cells, and NK cells [56] (Figs. 15.2 and 15.3). The different lymphocyte

subsets involved in cytotoxic effector responses differ in the recognition process through which they identify infected or distressed somatic cells [12]. Cytotoxic lymphocytes use several pathways to induce target cell apoptosis, including Fas ligation and granule exocytosis of enzymes such as perforin and granzyme B [12].

High proportions of T cells within the salivary glands express Fas (CD95) and/or members of the B-cell lymphoma (Bcl) family [57]. Nevertheless, despite abundant expression of these pro-apoptotic molecules, neither Fas/FasLinduced apoptosis nor apoptosis implicating the Bax/Bcl-2 pathway seems to occur more often among inÞltrating mononuclear cells in SS compared to control individuals [45, 58]. High expression of Bcl-2 compared to Bax in these cells may explain their resistance to apoptosis, a situation which might result in a prolonged production of pro-inßammatory mediators [58].

CD4+ T cells, so-called T helper (TH) cells, are involved in activating and directing other immune cells to meet the challenge posed by

Fig. 15.3 Proposed mechanisms mediating glandular destruction and dysfunction in SS. (1) Altered apoptosis of glandular epithelial cells. (2) Antibodies targeting the M3R may directly be related to the impairment of the salivary glands by inhibiting neuronal innervation of acinar cells. (3 and 4) AQPs are suggested to play a

major role in water transport through cell membranes. (3) AQP-1 expression was decreased in myoepithelial cells from patients with SS. (4) AQP-5 expression may be decreased in acinar cells from patients with SS. Figure adapted from Delaleu et al. [6]

antigens of different nature while, at the same time, considering tissue-speciÞc requirements [59Ð62]. TH cells are essential in determining B- cell antibody class switching, in the activation and growth of cytotoxic T cells, and in maximizing bactericidal activity of phagocytes such as macrophages. Based on the cytokines secreted, TH cells are currently subdivided into three major and distinct functional effector subsets, termed TH1, TH2, and TH17 [59Ð62].

These T cells differentiate from not yet committed TH0 cells in response to exposure to speciÞc cytokines [59Ð62]. Such stimuli are followed by the induction of independent patterns of gene transcription. TH1 cells tend to be involved in host defense against intracellular pathogens and thought to perpetuate autoimmune responses [59, 61, 62]. TH1 cell differentiation from TH0 cells is dependent on interferon (IFN)-γ and IL12. In their turn, TH1 cells produce IL-2 and IFN-γ. TH2 cells on the other hand tend to engage in immune responses, which depend on a substantial humoral component for the elimination of the pathogen [59, 61]. Key molecules of a TH2-dominated immune response are IL-4, IL-5, and IL-13 [59, 61]. TH1 and TH2 cells counteract each other, mainly because their respective cytokines can exert inhibitory effects on the opposite TH cell subset [59, 61].

An optimal scenario, before the emergence of TH17 and regulatory T-cell (Treg) subsets, seemed to consist of a well-balanced TH1 and TH2 responses, tailored in accordance with the encountered immunological challenge [59]. For example, protection from insulin-dependent diabetes mellitus (IDDM) in NOD mice has been associated with a shift from a TH1 to a TH2 cytokine expression proÞle in β-cell-speciÞc

or genetic modiÞcation [63]. Subsequent studies indicated, however, that compartmentalization into disease-promoting TH1 and protective TH2 cytokines may represent an oversimpliÞcation, which cannot always be applied to the pathogenesis of IDDM or other autoimmune diseases [64]. Considering the limited knowledge about cytokines at the time the TH1/TH2 model was

developed [59] and despite its ßaws, the TH1/TH2 paradigm showed remarkable durability and still conserved some of its validity [65].

Cytokine proÞles have been studied in blood and salivary gland tissues from patients with SS [17, 40, 66] and in mouse models for SS [18, 67]. However, because cytokines are characterized by redundancy, pleiotropism, synergism, and antagonism, the interpretation of results on cytokines is often challenging.

Applying the by-now expanded concept of TH1/TH2 cytokines and autoimmunity, it has been postulated that TH2 cytokines may be predominant in an early phase of SS, while TH1 cytokines would be associated with a later stage of the disease [68]. In opposition stands the proposed principle that the decrease in salivary ßow, which is thought to follow the emergence of glandular inßammation [25, 69], might be associated with TH2 cytokines [70Ð72]. The latter model is largely based on murine studies, in which crucial molecules involved in either TH1 or TH2 responses have been deleted. NOD Ifn-γ −/− and NOD Ifn-γ R−/− mice did not develop inßammation in the salivary glands and retained normal salivary secretion capacity, while, however, the inßammation within the lacrimal glands persisted [73]. In contrast, IL-4- [70, 71], alike signal transduction and activators of transcription (STAT)6-deÞcient [72] NOD or NOD-related strains retained salivary secretion rates similar to BALB/c mice. These genetically modiÞed strains concomitantly failed to produce anti-muscarinic M3 receptor (M3R) antibodies of the IgG1 isotype, despite the development of sialadenitis [70, 72].

The revision of the TH1/TH2 paradigm was caused by the emergence of a T-helper cell subset termed TH17, producing IL-17 (IL-17A), IL-17F, and IL-22 [60]. A major role of IL-17 has been described in various models of immunemediated tissue injury and autoimmune diseases, e.g., RA [60]. Recent results suggest that the TH17/IL-23 system is activated in SS patients and C57BL/6.NOD-Aec1Aec2 mice during the overt disease state. Functional associations between IL-17/IL-23 levels and speciÞc clinical manifestations have, however, not yet been identiÞed [18, 74].