Blockade of lymphotoxin-beta receptor signaling reduces aspects of Sjögren's syndrome in salivary glands of non-obese diabetic mice

Sjögren syndrome (SS) is a chronic autoimmune disorder characterized by lymphocytic infiltrates of the exocrine glands. Patients present clinical symptoms of dry eyes and dry mouth. The exocrinopathy may occur alone (primary SS) or in association with another autoimmune disorder (secondary SS) such as rheumatoid arthritis or systemic lupus erythematosus [1]. Inflammatory reactions in the lacrimal and salivary glands with lymphoid neogenesis, and specifically germinal center formation, are reported in SS patients [2‐5].

The lymphotoxins (LTα and LTβ) and LIGHT (a cytokine homologous to LTs that shows inducible expression and competes with herpes simplex virus glycoprotein D for herpes virus mediator [HVEM], a receptor induced on T cells) and their receptors are part of the tumor necrosis factor (TNF) superfamily. LTα exists in both secreted and membrane-bound forms. The membrane-bound form is a complex of LTα and LTβ [6], forming in humans a predominant LTα₁β₂ heteromer [7]. This ligand binds the LTβ receptor (LTβR) [8] along with LIGHT [9]. For a detailed review of LT and LIGHT signaling, see [10].

The LTβR signaling pathway is important in the development of organized lymphoid structures [10‐18]. In the differentiated tissue, the LTβR signaling pathway is involved in the control of expression of chemokines and adhesion molecules that aid in the movement of lymphocytes and in their compartmentalization into T- and B-cell zones, high endothelial venule (HEV) development, and follicular dendritic cell (FDC) network formation and in the positioning and numbers of dendritic cells [19]. This signaling pathway is also crucial in lymphoid neogenesis at sites of inflammation [20]. Lymphoid neogenesis is reported in human chronic inflammatory diseases (including SS) and associated mouse models (reviewed in [21, 22]).

The efficacy of LTβR pathway blockade (that is, the blocking of activation of LTβR by either of its ligands via the pharmacological inhibitor LTβR-immunoglobulin fusion protein [LTβR-Ig]) has been studied in many murine disease models (reviewed in [19]), and a human version was used in rheumatoid arthritis clinical trials [13].

In non-obese diabetic (NOD) mice, treatment with LTβR-Ig prevented insulin-dependent diabetes mellitus (IDDM) and reversed insulitis [23, 24]. In addition, soluble LTβR-Ig transgene expression on the NOD background blocked diabetes development [25]. In addition to spontaneously developing IDDM, the NOD mouse spontaneously develops lymphocytic infiltrates in the exocrine glands, with associated glandular dysfunction [26]. Although the Idd3 and Idd5 susceptibility loci for diabetes have a role in the development of sialadenitis, the IDDM and sialadenitis are two independent autoimmune events [27‐29].

The aim of the present study was to analyze the effects of LT/LIGHT axis blockade by the pharmacological inhibitor LTβR-Ig on SS-like salivary gland disease in female NOD mice, introducing the inhibitor prior to the development of the SS-like disease. The study confirmed that blocking the LTβR pathway results in ablation of the lymphoid organization in the NOD salivary glands, leading to an improvement in salivary gland function.

The results disclosed that blocking the LTβR pathway in NOD mice not only has an effect on the blood glucose levels, as reported previously [23, 25], but also has profound effects on the salivary gland lesion, a hallmark of SS-like disease in NOD mice. The loss of FDC networks on blocking the LTβR pathway in adult mice has been described previously [19, 34], but the present study is the first to report that LTβR-Ig treatment results in FDC network loss in the salivary glands. FDC networks constitutively release a substantial amount of CXCL13 [35], although there are other sources of CXCL13, such as macrophages, dendritic cells, and primed follicular helper T cells [35]. CXCL13 recruits B cells and follicular helper T cells to the B-cell zones. Immunohistochemistry disclosed that both LTβR-Ig-treated mice and controls expressed CXCL13 at the protein level in the salivary glands. There was, however, a depletion of B-cell zones by 19 weeks (LT19 group). This was probably attributable to the reduction in the amount of CXCL13, as indicated by the reduction in CXCL13 mRNA expression levels in the LT19 group compared with controls. The complete loss of FDC networks following LTβR-Ig treatment may also have contributed to the lack of B-cell zones in the LT19 group.

There was also an all-out loss of HEVs in the salivary glands in the LT16 and LT19 groups. HEVs exhibit specific address signals that are recognized by circulating lymphocytes. PNAd is one such signal (antigen) that was first defined by reactivity with monoclonal antibody MECA-79 [36]. L-selectin on the circulating lymphocytes binds carbohydrate epitopes of PNAd, facilitating homing of these naïve lymphocytes [37]. Previous studies have reported a prominent role for the LTβR pathway in maintaining the HEV structures in a functional state in lymph nodes of adult mice [13, 33] and in sites of lymphoid neogenesis [38]. The LTβR pathway is also important in de novo lymphangiogenesis in sites of lymphoid neogenesis [39].

Although the mRNA expression levels of CCL19 were not significantly reduced in the LTβR-Ig-treated mice, there was a complete loss of CCL19 protein in the salivary gland infiltrates of LTβR-Ig-treated mice. CCL19 and CCL21 are necessary for the transmigration of naïve T cells and central memory T cells across the HEVs and in the formation of T-cell zones [40]. Therefore, the combination of ablation of FDC networks, loss of HEVs, and reduced chemokine expression would ultimately result in a reduced influx of B and T cells to the salivary glands. This may explain the reduced B- and T-cell zones in the LTβR-Ig-treated mice.

Another interesting observation was the different response to LTβR-Ig treatment by the salivary gland lesion and the draining lymph nodes. There is no consensus as to which of these sites drives the immune response or whether there is a contribution from both [13]. Our findings indicate that the LTβR-Ig treatment had a more profound effect on the microarchitecture of the salivary gland lymphoid tissue than on that of the lymph node.

Our findings also suggest that the salivary gland lesion has a prominent role in SS-like disease, as partial saliva flow restoration occurs in association with the phenotypic changes in the salivary gland. The ectopic lymphoid tissue has a prominent role in several diseases/conditions [13]. In the NOD mouse, pancreatic lymphoid neogenesis could initiate and sustain the progression of IDDM in the absence of draining lymph nodes [41]. Interestingly, it has been proposed that LTβR-Ig treatment would result in a quiescent state of the lymph nodes, draining the sites of inflammation [13]. This is partly supported by the present study, showing microarchitectural differences between LTβR-Ig-treated mice and control lymph nodes. However, the lymph node microarchitectural changes in our experimental setup are unlike the grossly disturbed primary or secondary organ development observed in mice deficient in components of the LTβR pathway.

The present study was based on the hypothesis that LTβR-Ig treatment would preserve the integrity of the salivary gland by preventing development and progression of inflammation and lymphoid neogenesis, thus preventing the occurrence of salivary gland dysfunction. Our results, however, show that LTβR-Ig treatment may reverse pathological events that have already occurred in the salivary gland. This possibility is underscored by the finding of HEVs in the salivary glands of 12-week-old mice given three LTβR-Ig injections (although in only one mouse) but loss of HEVs in 16-week-old and 19-week-old mice treated with LTβR-Ig. Earlier analyses in mice suggested that a period of at least 2 weeks of LTβR-Ig treatment was required to downmodulate HEVs and therefore any early HEVs could still be present at week 12 [33]. Also, the saliva flow rate appeared to fall (comparing LT12 and LT16 groups) but recovered in the LT19 group, indicating that the salivary gland dysfunction may have been prevented or reversed.

The dichotomous distribution of saliva flow rates in NOD mice has been described previously [42]. The same study also used saliva proteins to describe the efficacy of intervention agents in SS-like disease of NOD mice. In our study, the striking similarity in the cytokine profiles of the pre-disease NT9 group and the LT19 group may indicate a restoration of the pre-disease quality of saliva in the LT19 group. However, analysis of pro- and anti-inflammatory cytokines and chemokines in saliva from untreated mice across different ages disclosed no obvious trend indicative of progressive disease status. However, 16 weeks may mark a time of potentially heightened cytokine production and activity in the NOD mouse.

Accelerated clearance of immunoglobulin-based agents is commonly observed in many rodent autoimmune disease models, and our finding of a reduction in the serum levels of LTβR-Ig as the mice age also points to a possible increased clearance of the fusion protein in these older NOD mice. It is postulated that, had the high levels of LTβR-Ig observed in 12-week-old mice been sustained throughout the experiment, the protective/reversal effects of LTβR-Ig would have been more pronounced.

There are severe technical limitations to finding proper controls for fusion proteins when faced with the need to use high-level dosing in autoimmune disease models to maintain coverage. A protective effect of polyclonal human IgG used as a control protein in experiments to determine the efficacy of LTβR-Ig in collagen-induced arthritis was reported [34]. The mechanism behind the MOPC 21 effect may involve non-specific binding of MOPC 21 to targets in the pathways involved in the disease process, resulting in a protective effect.

The immune dysregulation occurring in the female NOD background leads to autoreactivity and inflammation of the salivary glands. This inflammation is accompanied by the formation of lymphoid tissues in the salivary glands with HEVs and FDC networks. Although the role of these tertiary lymphoid tissues in the pathogenesis remains unclear, the present study clearly demonstrates that LTβR inhibition, whether via its effects on trafficking or lymphoid architecture, results in a less destructive environment in the target organ.

This study shows that blocking the LTβR pathway in NOD mice ablates lymphoid neogenesis in the salivary glands and this is accompanied by an improvement in salivary gland function. To our knowledge, the effect of interfering with the LTβR axis has not previously been explored in SS-like disease. It is hoped that our results will contribute to the development of appropriate methods for treatment of human SS.