Many exocrine glands, the ocular surface, and the lacrimal gland are affected by sex hormones, principally the androgens. It has been shown that androgen receptor protein exists within the epithelial cell nuclei of the human exocrine
glands, meibomian gland, conjunctiva, and cornea, suggesting that androgens influence their structural organization and functional activity, and have an especially profound impact on the immunology, molecular biology, and secretory capacity of the lacrimal gland (9). Such proven androgen effects have led to the idea that androgen deficiency states such as SS, SLE, RAs, aging, and the use of antiaging medications may be very important in the pathogenesis of dry eyes (10). The androgen binding to the receptors in the acinar nuclei of the exocrine glands (the lacrimal gland in the context of xerophthalmia) leads to an altered expression of numerous cytokines and proto-oncogenes. The activity of androgens in the exocrine glands is thought to induce the accumulation of antiinflammatory cytokines such as transforming growth factor-β (TGF-β) (10). The reduction of the androgen level below a certain threshold may result in the release of proinflammatory cytokines such as interleukin-1β (IL-1ß), IL-2, interferon-γ, and tumor necrosis factor-α (TNF-α) by the lymphocytes entering the gland (11). Experimental evidence has shown that progressive CD4⁺ T-cell and B-cell infiltration occurs in lacrimal and salivary glands of patients with SS (12). Alterations of the nerve fibers among the glandular acini are superimposed on the inflammatory process in these patients. It is also possible that the central nervous system plays an important role in the cascade of events that occur in SS where the amount of tear flow is initially decreased through cellular infiltration of the tear gland. Increased friction between the lids dry cornea may cause exfoliation of the superficial corneal epithelium, resulting in constant repeated C-fiber nerve stimulation, which becomes dominant in time and acts in combination with central parasympathetic inhibition of tear flow. This could also explain the clinical and experimental discrepancies of the concept of infiltration and destruction of the lacrimal gland as a single cause of tear flow depression. In addition, central neural mechanisms may also explain those cases in which patients have subjective rather than objective improvement of complaints (13).

Under normal circumstances, lymphocytes entering the lacrimal or an exocrine gland are expected to undergo apoptosis. In the presence of inflammation, however, the apoptotic response is aborted, and the lymphocytes release proinflammatory cytokines and inflammatory cytokines such as IL-1β, IL-6, and IL-8 (14,15). The increased expression of human leukocyte antigen (HLA)-DR and intercellular adhesion molecule (ICAM) in the conjunctiva, for instance, has been reported to be associated with “homing” of more T cells and greater cytokine release within the lacrimal gland, increasing the level of inflammation (16).
Numerous investigators have attempted to analyze the association of primary SS with cytokine polymorphisms. Both human and animal studies indicate the involvement of IL-10 in the pathogenesis of primary SS, and mice transgenic for IL-10 develop a Fas-ligand mediated exocrinopathy that resembles SS (17,18). A recent study described an association between primary SS and IL-10 promoter polymorphisms in a cohort of Finnish individuals, and a specific haplotype was found to correlate with high plasma levels of IL-10 (19). Conversely, no association was found for IL-10 promoter polymorphism and primary SS or the presence of Ro autoantibodies in an Australian cohort of primary SS patients (20). The IL-1 receptor antagonist regulates IL-1 activity in inflammatory disorders by binding to the IL-1 receptor and thus, inhibiting its activity. The human IL-1 receptor antagonist gene (IL1RN) has a variable allelic polymorphism within intron 2. An increased frequency and carriage rate of the ILRN^*2 allele has been found in primary SS (21,22).

Solomon et al. (22a) reported that IL-1α and -1β, IL-6, IL-8, TGF-β1, and TNF-α were expressed at elevated levels in the conjunctival epithelia of patients with SS compared with dry eye controls. These investigators also demonstrated that the conjunctival epithelium of SS patients displayed increased numbers of S-phase cells; the authors formulated a model to explain the ocular surface pathology of SS: mechanical abrasion secondary to tear deficiency creates an inflammatory environment, with conjunctival epithelial cells and lymphocytes stimulated to produce and secrete various cytokines into the tear film. Elevated cytokine levels within the tear film, combined with reduced concentrations of essential lacrimal-gland derived factors such as epidermal growth factor (EGF) and retinol, create an environment in which terminal differentiation of the ocular surface epithelium is impaired. As a consequence, the epithelium becomes hyperplastic, displaying increased mitotic activity, and loses the ability to express mature surface protecting molecules, including membrane bound mucin, MUC-1. Indeed, conjunctival impression cytology supports this hypothesis, demonstrating “snake-like chromatin cells,” increased squamous metaplasia, decreased goblet cell density and inflammatory cells in SS patients (24, 25, 26). The ocular surface changes in SS dry eyes are frequently associated with corneal hypoesthesia; this perpetuates a vicious cycle. These would all imply that antiinflammatory medications that suppress the inflammatory component of this cascade may ameliorate the ocular surface disease and discomfort experienced by SS patients.

Autoimmune diseases are mostly characterized by tissue destruction and functional decline due to autoreactive T cells that escape self-tolerance. Although the specificity of cytotoxic T-lymphocyte function has been an important issue of organ-specific autoimmune responses, the mechanisms responsible for tissue destruction in SS remain to be elucidated. The histopathologic changes seen in minor salivary gland biopsies are characterized by focal and/or diffuse lymphoid cell infiltrates and parenchymal destruction. The majority of cells in glandular biopsy specimens are CD4⁺ T cells, with a small proportion of CD8⁺ T cells (27). These T cells express the αβ receptor and cell surface antigens associated with mature memory T cells. When the repertoire of T-cell receptor (TCR) Vβ genes transcribed and expressed within the inflammatory infiltrates was analyzed in the animal model of primary SS, a preferential utilization of TCR Vβ genes was detected from the onset of the disease. A 120-kD organ-specific autoantigen was previously described from the salivary gland tissues of the animal model of SS [NFS/sld mutant mouse thymectomized 3 days after birth (3d-TX)]. The sequence of the first 20 NH₂-terminal residues is identical to that of the cytoskeletal protein human α-fodrin (27). And sera from patients with SS reacted positively with the purified 120-kD antigen, and induced a proliferative response of peripheral blood lymphocytes. Purified antigen was detected from SS patients, but not from SLE or RA patients and healthy controls. These results indicate that the anti-120-kD α-fodrin immune response may play a role in the development of primary SS. Recent reports have demonstrated evidence that caspase-3 is required for α-fodrin cleavage during apoptosis (27). It has been speculated that an increase in activity of apoptotic proteases is involved in the development of α-fodrin proteolysis during the development of SS. Fodrin cleavage by caspases can lead to cytoskeletal alterations. It is of interest to remember that α-fodrin binds to ankylin, which contains a cell death domain. It has been shown that cleavage products of α-fodrin inhibit adenosine triphosphate (ATP)-dependent glutamate and γ-aminobutyric acid accumulation into synaptic vesicles where a cleavage product can be a novel component of an unknown immunoregulatory network such as cytolinker proteins (28). It is now also clear that the interaction of Fas with FasL regulates a large number of pathophysiologic processes via apoptosis. Immunohistologic studies revealed that the majority of T cells infiltrating salivary glands bear FasL in the SS model, and epithelial duct cells express Fas antigen on their cell surface (29). CD4⁺ T cells isolated from the affected glands bear a large proportion of FasL compared to CD8⁺ cells (flow cytometry). Tsubota and associates (30) previously showed the presence of apoptosis of acinar cells, FasL expression in lacrimal glands (Fig. 29-1), and that the FasL expression highly correlated with glandular function, especially in those patients without glandular enlargement. FasL expression of infiltrating lymphocytes was low in the lacrimal glands of patients with SS with enlarged exocrine glands where the lacrimal gland function was well preserved even with massive lymphocyte invasion
(30). In other words, the infiltration of lymphocytes alone did not cause glandular dysfunction. Apoptosis of acinar cells may explain the differences.

Transgenic mice containing the human T-cell lymphotropic virus type-1 (HTLV-1) ax gene under the control of viral long terminal repeat develop an exocrinopathy that involves the salivary and lacrimal glands, resembling the pathology of SS (31). It has been suggested that the HTLV-1 infection might represent a primary event in the development of exocrinopathy by virally induced proliferation and perturbation of the function of the ductal epithelium. Sialadenitis and inflammation in lacrimal glands histologically resembling SS have been reported to be associated with hepatitis C viral infection (32). Epstein-Barr virus (EBV) has also long been believed to play some role in the development of SS. Increased levels of EBV DNA in salivary glands of SS patients have been demonstrated by polymerase chain reaction technology, suggesting that viral reactivation or homing of EBV-infected circulating B cells may produce chronic autoimmune destruction of the salivary gland (33). Pflugfelder and associates (34) detected EBV DNA sequences in 80% of lacrimal glands from SS patients. SS has also been shown to develop after infectious mononucleosis, a condition caused by EBV. Crouse et al. (35) reported that EBV DNA was detected more in lacrimal glands compared to salivary glands. It has indeed been reported that, in any given individual with SS, lymphocytic infiltration of the lacrimal gland was far greater than that of the salivary gland (36). These findings corroborate the clinical evidence that dry eyes are more severe than dry mouth in SS. Pflugfelder et al. (37) have suggested that the conjunctival squamous metaplasia seen in SS may be primarily due to a dysfunctional immune system; the detection of EBV DNA in the conjunctival epithelium in their study was believed to support this hypothesis. It has been reported that 1% to 2% of the conjunctival cells from SS patients are T or B lymphocytes, with the latter serving as a possible source of EBV (38). It is not yet clear whether these EBV-infected B cells come from the lacrimal gland or subepithelial connective tissue. Like the lacrimal gland epithelial cells, conjunctival epithelium may have EBV receptors. Although further study is necessary, it seems likely that the conjunctiva is also primarily affected in SS.

A genetic predisposition to SS appears to exist, and several families involving two or more cases of SS have been described. However, the extent of genetic contribution remains unknown (39, 40, 41, 42). Large twin studies in SS are lacking, and the twin concordance rate cannot be estimated (43). A few available case reports reveal a very similar phenotype, with almost identical clinical presentation, including dry eyes and dry mouth, similar serologic data, with identical fine specificity in their immune responses to 60-kD Ro/SSA, and identical labial salivary gland focus scores (43). It is also common for an SS proband to have relatives with other autoimmune diseases (approximately 30% to 35%) (44,45). The major histocompatibility complex (MHC) genes are the best-documented genetic risk factors for the development of autoimmune disease (46, 47, 48). In European and American Caucasians, SS was found to be associated with the haplotypes B8, DRw52, and DR3 (49). The increased frequency of HLA-B8 was presumably due to an association (linkage disequilibrium) with the HLA class II allele HLA-DRB1^*03 (50). However, a novel association of HLA class I alleles (HLA-A24) to susceptibility in primary SS was also recently described. An association with DR2 has been found in Scandinavians and with DR5 in Greeks (51). All of the haplotypes are in strong linkage
disequilibrium, resulting in difficulties in establishing which of the genes contains the locus that confers the risk (52). It has been claimed that SS patients with DQ1/DQ2 alleles have a more severe autoimmune disease than do patients with any other allelic combination at HLA-DQ, and the DR3-DQ2 haplotype has been indicated as a possible marker for a more active immune response in Finnish patients with SS (53). Autoantibodies to Ro/SSA and La/SSB have been found to be associated with DR3, DQA, and DQB alleles (54, 55, 56). In Japan, HLA class II allele association has been reported to differ among anti-Ro/SSA (+) individuals according to the presence or absence of coexisting anti-La/SSB (8). The contribution of Ro/SSA and La/SSB in SS is not fully understood. It is not known how tolerance breakdown and autoantibody response to Ro/SSA and La/SSB is generated. The ribonucleoproteins are endogenous proteins that are normally hidden from the immune system and should not give rise to abnormal B-cell responses. However, stress, such as ultraviolet radiation, viral infections, and apoptosis have been suggested to lead to cell surface exposure of previously hidden autoantigens to the immune system (57). Genes that encode transporters associated with antigen processing (TAP genes) have also been associated with susceptibility to SS (58). Others have indicated a putative role for the cysteine-rich secretory protein 3 (CRISP-3) gene as an early response gene that may participate in the pathophysiology of the autoimmune disease in SS (59).

Aquaporins (AQPs) are water-specific membrane channel proteins that provide the molecular pathway for water permeability in many tissues (60, 61, 62). Among them, aquaporin 5 (AQP5) is normally expressed in the apical membranes of acinar cells in lacrimal and salivary glands, as well as the corneal epithelium and type-1 pneumocytes of the lung (60). At least five AQPs are expressed in the eye: AQP0 (MIP) in lens fiber; AQP1 in cornea endothelium, ciliary and lens epithelia, and trabecular meshwork; AQP3 in conjunctiva; AQP4 in ciliary epithelium and retinal Müller cells; and AQP5 in corneal and lacrimal gland epithelia. This cell-specific expression pattern suggests involvement of AQPs in corneal and lens transparency, intraocular pressure (IOP) regulation, retinal signal transduction, and tear secretion. Indeed, humans with mutant AQP0 develop cataracts. Mice lacking AQP1 have reduced IOP and impaired corneal transparency after swelling, and mice lacking AQP4 have reduced light-evoked potentials by electroretinography. There is evidence of impaired cellular processing of AQP5 in lacrimal glands of humans with SS (61). We carried out immunolabeling for AQP5 in biopsy samples from normal controls and patients with SS or non-SS type of dry eyes. An apical membrane-staining pattern was noted in normal subjects and in non-SS and Mikulicz’s syndrome patients. In contrast, diffuse cytoplasmic staining was observed in the lacrimal gland biopsy samples of patients with SS, with almost no labeling at the apical membranes (63) (Fig. 29-2). Quantification of AQP5 by enzyme-linked immunosorbent assay (ELISA) in these three groups did not reveal significant differences of AQP5 expression, suggesting a defect in protein trafficking rather than synthesis. The cause of the defect remains unknown (Fig. 29-3). Additional immunohistochemical analyses showed that the sodium-potassium adenosine triphosphatase (ATPase) channels in the basolateral membranes and sodium channels in
apical membranes were normally distributed in all patients (Table 29-3). In a recent study, we found that AQP5 increased in the tears of patients with severe SS compared with control subjects (64). This finding corresponds to a previous report that leakage of AQP5 in the tear was related to the lacrimal gland damage in experimental dacryoadenitis models. Although significant correlations exist between tear lactoferrin and EGF and clinical indices such as tear function index, Schirmer test and rose-Bengal staining scores, no correlation between AQP5 levels and clinical indices was observed. These differences suggest that lactoferrin and EGF produced by the lacrimal gland directly preserve ocular surface function, whereas AQP5 has not a direct but rather a secondary influence on the ocular surface disorder, caused by a decrease in the number of lacrimal gland cells (64).