Jason Brayer

Evidence for antimuscarinic acetylcholine receptor antibody–mediated secretory dysfunction in NOD mice

OBJECTIVE Antibodies directed against general and specific target-organ autoantigens are present in the sera of human patients and animal models with autoimmune disease. The relevance of these autoantibodies to the disease process remains ambiguous in most cases. In autoimmune exocrinopathy (Sjögrens syndrome), autoantibodies to the intracellular nuclear proteins SSA/Ro and SSB/La, as well as the cell surface muscarinic cholinergic receptor (M3) are observed. To evaluate the potential role of these factors in the loss of secretory function of exocrine tissues, a panel of monoclonal and polyclonal antibodies was developed for passive transfer into the NOD animal model. METHODS Monoclonal antibodies to mouse SSB/La, rat M3 receptor, and a rabbit polyclonal antiparotid secretory protein antibody were obtained for this study. These antibody reagents were subsequently infused into NOD-scid mice. Saliva flow rates were subsequently monitored over a 72-hour period. Submandibular gland lysates were examined by Western blotting for alteration of the distribution of the water channel protein aquaporin (AQP). RESULTS Evaluation of the secretory response indicated that only antibodies directed toward the extracellular domains of the M3 receptor were capable of mediating the exocrine dysfunction aspect of the clinical pathology of the autoimmune disease. In vitro stimulation with a muscarinic agonist of submandibular gland cells isolated from mice treated with anti-M3 antibody, but not saline or the isotype control, failed to translocate AQP to the plasma membrane. CONCLUSION These findings define a clear role for the humoral immune response and the targeting of the cell surface M3 signal transduction receptor as primary events in the development of clinical symptoms of autoimmune exocrinopathy. Furthermore, the anti-M3 receptor activity may negatively affect the secretory response through perturbation of normal signal transduction events, leading to translocation of the epithelial cell water channel.

A novel NOD-derived murine model of primary Sjögren's syndrome.

OBJECTIVE The appearance of autoimmune diabetes prior to autoimmune exocrinopathy in the NOD mouse suggests that it is an excellent model of secondary, but not primary, autoimmune sicca complications. Since the unique major histocompatibility complex (MHC) I-A(g7) expression in NOD mice is essential for the development of insulitis and diabetes in these animals, we investigated exocrine gland function in NOD.B10.H2b mice, which have an MHC congenic to NOD, as a potential model for primary Sjögrens syndrome (SS). METHODS Histopathologic manifestations of lymphocytic infiltrates into the pancreas and exocrine tissues were examined by light microscopy. Sera were evaluated for the presence of antinuclear antibodies. Saliva, tears, and gland lysates were evaluated for total volume and protein concentration, the aberrant expression and processing of parotid secretory protein, and cysteine protease activity. RESULTS NOD.B10.H2b mice exhibited the exocrine gland lymphocytic infiltration typical of the SS-like disease and dysfunction observed in NOD mice, but without the insulitis and diabetes. These mice additionally expressed elevated levels of cysteine protease activity (a measure of apoptotic activity) and abnormal expression and cleavage of parotid secretory protein in the submandibular tissues. CONCLUSION The results of this study suggest that the unique NOD MHC I-A(g7) is not essential for exocrine tissue autoimmunity. Furthermore, the findings indicate that sicca syndrome occurs independently of autoimmune diabetes and that the congenic NOD.B10.H2b mouse represents a novel murine model of primary SS.

A Dual Role for Interferon-γ in the Pathogenesis of Sjögren's Syndrome-Like Autoimmune Exocrinopathy in the Nonobese Diabetic Mouse

Sjögrens syndrome‐like autoimmune exocrinopathy (AEC) in the nonobese diabetic (NOD) mouse progresses from a preimmune phase to an immune phase, resulting in dry mouth and/or dry eyes. In the present study, the impact of the prototypical T‐helper type 1 cytokine, interferon‐gamma (IFN‐γ), on the onset of AEC was investigated using both the IFN‐γ and the IFN‐γ receptor gene knockout mice, NOD.IFN‐γ–/– and NOD.IFN‐γR–/–, respectively. Neither the NOD.IFN‐γ–/– nor the NOD.IFN‐γR–/– mice exhibited increased acinar cell apoptosis and abnormal salivary protein expression, typically observed in parental NOD mice prior to disease. Without these preimmune phase abnormalities, NOD.IFN‐γ–/– and NOD.IFN‐γR–/– mice showed no subsequent autoimmune responses against the salivary glands at 20 weeks. Interestingly, real‐time polymerase chain reaction and electrophoretic gel mobility shift assays suggested that IFN‐γ and STAT1, as well as the transcriptional activity of STAT1 in NOD glands, were increased at birth. Unlike the neonatal submandibular glands of NOD or NOD‐scid mice that show abnormal glandular morphogenesis at birth, the submandibular glands of the newly constructed congenic strain, NOD‐scid.IFN‐γ–/–, were found to be normal. Taken together, IFN‐γ appears to play a critical role not only during the later immune phase of AEC, but also the early preimmune phase, independent of effector functions of immune cells. How exactly IFN‐γ functions during this period remains speculative.

An Alternative Perspective to the Immune Response in Autoimmune Exocrinopathy: Induction of Functional Quiescence Rather Than Destructive Autoaggression

Sjögrens syndrome is characterized by dryness of the eyes and the mouth due to mononuclear cell infiltration of the lacrimal and salivary glands. The aetiology is unknown but autoimmunity is considered to play a significant role in the pathogenesis. Recent studies have focused on the fact that tear and salivary flow involves an entire functional system that includes the mucosal surfaces with adnexes (the site of inflammation), efferent nerve signals sent to the midbrain (lacrimal and salivary response region), and afferent neural signals from the brain to the acinar/ductal epithelial structures in the gland. Mononuclear cell infiltration in exocrine glands can lead to glandular destruction, suggested to be mediated through apoptosis. However, the functional impairment of exocrine glands could be regulated by cytokines and/or antibodies against the muscarinic M3 receptor by inhibiting the neural stimulation of the residual glands. This review discusses the possibility that the pathogenesis of Sjögrens syndrome comprises aberrant immune‐mediated neuro‐hormonal events.

IL-4-Dependent Effector Phase in Autoimmune Exocrinopathy as Defined by the NOD.IL-4-Gene Knockout Mouse Model of Sjogren's Syndrome

NOD mice manifest many features of autoimmune exocrinopathy (Sjögrens syndrome), a disease generally characterized by a chronic, progressive immunological attack against the exocrine tissues of the salivary and lacrimal glands. Previous studies using the NOD congenic partner strain, NOD.Igμnull, defined an important role for B lymphocytes in the development of xerostomia, implicating autoantibodies reactive with the acetylcholine muscarinic receptor (M3R) as the possible effector mechanism. In the present study, we have examined the impact of the cytokine, interleukin (IL)‐4, on autoimmune exocrinopathy by using the IL‐4 gene knockout (KO) NOD mouse strain, NOD.IL‐4−/−. Despite manifesting the physiological aberrations and marked leukocytic infiltration of the salivary glands characteristic of autoimmune xerostomia in NOD mice, the NOD.IL‐4−/− mice do not develop xerostomia. However, NOD.IL‐4−/− mice that received adoptively transferred T lymphocytes derived from NOD.Igμ−/− mice progress to xerostomia, thereby reversing the defect. While progression or lack of progression to xerostomia correlated with the ability of the NOD.IL‐4−/− mice to express detectable anti‐M3R autoantibodies, the precise mechanism of how IL‐4 influences the development of autoimmune xerostomia remains speculative.

Abnormal organogenesis in salivary gland development may initiate adult onset of autoimmune exocrinopathy

Objectives: Salivary gland organogenesis was evaluated in NOD mice, an animal model for autoimmune exocrinopathy, to determine when disease onset is first present in the target tissues. Methods: Submandibular glands were removed for histological, immunohistochemical and biochemical evaluation from neonatal NOD and congenic strains as well as healthy control C57BL/6 mice. Results: Histomorphological analyses of neonatal submandibular glands, the primary target for autoimmune exocrinopathy at 1 day postpartum, revealed delayed morphological differentiation during organogenesis in autoimmune-susceptible NOD mice when compared to nonsusceptible C57BL/6 mice. Acinar cell proliferation was reduced, while expression of Fas, FasL and bcl-2 were increased. Acinar cell proliferation was reduced, while expression, of Fas, FasL and bcl-2 were increased. Throughout the preweaning period (21 days) submandibular glands from NOD and NOD congenic strains aberrantly expressed an increased matrix metalloproteinase (MMP)-2 and MMP-9 activity. Substitution of two susceptibility alleles (Idd3 and Idd5) in NOD mice resulted in an hierarchical and additive reversal of delayed organogenesis, elevated MMP-9 activity, and aberrant expression of parotid secretory protein. Discussion: NOD-derived mice whose submandibular glands showed normal organogenesis did not progress to develop autoimmune exocrinopathy. Altered organogenesis of target tissue may therefore provide a cellular microenvironment capable of activating autoimmunity.

Differential absorption and distribution of epidermal growth factor and insulin-like growth factor in diabetic NOD mice

Previous studies have shown that absorption of growth factors occurs through the gastrointestinal tract and the oral cavity. The non-obese diabetic (NOD) mouse, a model for spontaneous development of type 1 insulin-dependent diabetes (IDDM), was evaluated for the absorption and systemic distribution of growth factors. Radiolabeled epidermal growth factor (EGF) and insulin-like growth factor, type I (IGF-I), were administered by gavage into the stomach or by lozenge into the sublingual vasculature of either diabetic or nondiabetic mice. After a time-dependent uptake, the levels of absorption and distribution through the tissues were measured. A similar time course of EGF absorption following gavage administration was determined for NOD and C57BL/6 mice, with a maximum tissue distribution by 30-min post infusion. Diabetic NOD mice showed similar levels of IGF uptake and tissue distribution compared with nondiabetic NOD and normal healthy C57BL/6 mice, whether administered by gavage or sublingual lozenge. On the other hand, gavage uptake and tissue distribution of EGF was significantly higher in diabetic mice when compared to sublingual administration in nondiabetic NOD or C57BL/6 healthy control mice. These findings suggest that the overall potential uptake and distribution of saliva-derived growth factors in systemic wound-healing processes is retained with diabetes onset, and may offer a new avenue to treating this complication of diabetes.

Gastrointestinal absorption of insulin-like growth factor in the mouse in the absence of salivary insulin-like growth factor binding protein

Based on previous observations of the presence of both insulin-like growth factors I and II (IGF-I and IGF-II) in murine saliva (kerr et al., Biochem Pharmacol 49: 1521-1531, 1995), the saliva from BALB/c and Non-obese diabetic (NOD) mice was examined for the presence of insulin-like growth factor binding proteins (IGFBPs). Using a western-blot type ligand binding assay with 125I-labeled IGF-I, a series of binding proteins with molecular masses (M), between 25 and 45 kDa were detected in the sera, but not saliva, from both BALB/c and diabetic NOD mice. In the diabetic NOD mice, there were detectable changes in the concentrations of several of the IGFBPs relative to BALB/c mice. Using specific antibody to the binding proteins, one of these was identified as IGFBP-2. Gavage administration of [125I]IGFI indicated substantial uptake from the gastrointestinal tract and significant tissue distribution. There was an increase in serum concentrations of radiolabeled IGF-I in diabetic NOD mice over that in BALB/c mice but less recovered from most of the tissues. Intact 125I-labeled IGF-I was extracted and purified from various tissues, following gavage, and shown to retain biological activity. Thus, the uptake of biologically active IGFs from saliva would appear to take place independently of specific binding proteins.