Laboratory of Molecular Medicine, Department of Veterinary Clinical Sciences and 1 Laboratory of Anatomy, Department of Biomedical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, 060-0818, Japan.
Correspondence to: M. Inaba, Laboratory of Molecular Medicine, Department of Veterinary Clinical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, N18W9, Sapporo, Hokkaido 060-0818, Japan. E-mail: inazo{at}vetmed.hokudai.ac.jp
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Abstract |
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Methods. Systemic histopathological analysis was performed on IQI/Jic mice at various ages. Phenotypes of infiltrated lymphocytes were determined using immunohistochemical techniques.
Results. Inflammatory lesions were observed not only in the lacrimal and salivary glands, but also in multiple organs, including the lung, pancreas and kidney at advanced ages, and were mainly composed of CD4+ T cells and B cells. The incidence and severity of the inflammatory lesions increased with age in all these organs. The histological appearance and spreading of lesions were similar to those in human primary SS.
Conclusions. IQI/Jic mice spontaneously develop inflammatory cellular infiltrates in multiple exocrine and non-exocrine organs. This characteristic distinguishes IQI/Jic mice from other murine models, making them favourable for studies on the pathogenesis of systemic involvement in primary SS.
KEY WORDS: IQI/Jic mouse, Primary Sjögren's syndrome, Autoimmune disease
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Introduction |
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Several animal models of SS have been proposed based on the histopathological findings of focal lymphocytic infiltrates in the lacrimal and salivary glands. They include autoimmune-prone mice that develop SS-like pathology associated with other autoimmune conditions, such as systemic lupus erythematosus, rheumatic arthritis and insulitis [58], and other rodent strains requiring experimental manipulations, such as antigen sensitization and neonatal thymectomy, before they develop inflammatory lesions [911]. However, few of these animal models bear a resemblance to primary SS, in which multiple organs, including exocrine and non-exocrine organs, are affected simultaneously.
IQI/Jic mice have been described previously as a novel model of primary SS [1214]. They spontaneously develop autoimmune infiltration of lymphocytes into the lacrimal and salivary glands, leading to dacryoadenitis and sialoadenitis. The incidence of the disease is higher in females than in males [12]. Sialoadenitis in female mice can be detected from 2 months of age onwards [14], and significant progress of the lesions is observed after 9 months of age [12]. Inflammatory lesions occurring at young ages mainly consist of CD4+ T cells with lesser abundance of CD8+ T cells, B cells and macrophages, and the proportions of B cells and plasma cells are elevated in accordance with increasing magnification of the lesions. Production of antinuclear antibodies, one of the prominent pathophysiological features in patients with SS, is also observed in old IQI/Jic mice [13].
Interestingly, during our histopathological studies on IQI/Jic mice [14], we found that focal accumulations of mononuclear cells frequently occurred also in other organs at advanced ages. In the present study, we followed the development of inflammatory lesions in various organs of IQI/Jic mice histologically and immunohistochemically. We report that IQI/Jic mice develop inflammatory lesions in the lung, pancreas and kidney in addition to the lacrimal and salivary glands that are very similar to those in patients with primary SS, suggesting that the IQI/Jic strain is an animal model suitable for the investigation of the pathogenesis of SS, with progression from oral and ocular diseases to systemic disorder.
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Methods |
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Histological analysis
Various organs removed from the mice at each age were fixed in 10% formaldehyde and embedded in paraffin. Sections were stained with haematoxylin and eosin, and examined under a microscope. The severity of the inflammatory lesions in the lacrimal and salivary glands and the pancreas was classified into five grades (04) according to modified criteria [15] as follows: 0, no visible change; 1, mild accumulation of mononuclear cells within the interstitium; 2, focal accumulation of mononuclear cells without any parenchymal destruction; 3, focal accumulation of mononuclear cells with parenchymal destruction; 4, extensive infiltration of mononuclear cells with severe tissue damage. The lesions in the lung were similarly distinguished into grades 03: 0, no visible change; 1, mild accumulation of mononuclear cells surrounding blood vessels and bronchi; 2, moderate accumulation of mononuclear cells surrounding blood vessels and bronchi; 3, extensive infiltration of the interstitium with mononuclear cells. Grading of renal tissues was: 0, no visible change; 1, infiltration of the interstitium with mononuclear cells without destruction of the urinary tubules; 2, infiltration of the interstitium with mononuclear cells with the destruction of urinary tubules.
Immunohistochemistry
Immunohistochemical staining was performed on cryostat sections of various organs from 10-month-old mice using the biotinavidin immunoperoxidase method. Briefly, the sections were fixed in acetone for 10 min, treated with 3.0% H2O2 in methanol for 15 min, and then blocked with 1.0% bovine serum albumin in phosphate-buffered saline (pH 7.2) for 20 min. Thereafter, they were incubated with biotinylated rat anti-mouse CD4, CD8 or B220 monoclonal antibodies (BD Pharmingen, San Diego, CA, USA) for 2 h, and then with streptavidin-conjugated peroxidase (Nichirei, Osaka, Japan). The sections were reacted with a mixture of 0.05% 3,3'-diaminobenzidine and 0.005% H2O2 in 50 mM TrisHCl (pH 7.6) and 150 mM NaCl, followed by counterstaining with Mayer's haematoxylin. All control samples were incubated with normal rat serum (Nichirei) instead of the monoclonal antibodies, and showed no non-specific staining.
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Results |
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The pulmonary lesions appeared much later in life, after 7 months of age, and perivascular and peribronchial cell infiltrations were obvious (Fig. 1C). Progressive accumulation of mononuclear cells was observed in the pulmonary interstitium in some aged mice (Fig. 1D).
In the pancreas, histological features were essentially similar to those found in the lacrimal and salivary glands. Foci of mononuclear cell infiltration were constitutively located in periductal and perivascular areas, resembling those in the lacrimal and salivary glands (Fig. 1E). Destruction of pancreatic acini was recognized in the magnifying processes of the foci. A few mice showed marked damage, and partial replacement with fibrils and adipose tissue (Fig. 1F). In contrast, the pancreatic islets were spared from the infiltrations in all age groups.
In the renal lesions, mononuclear cells mainly infiltrated into the interstitium, in contact with the large- and medium-sized blood vessels (Fig. 1G), and they were observed in mice older than 7 months of age (Table 1). Although mild destruction of urinary tubules was occasionally observed (Fig. 1H), the histological grade remained low throughout life (Table 1), being consistent with the absence of periodic paralysis, the typical sign of hypokalaemia due to renal tubular acidosis, in all mice examined.
Immunohistochemical analysis for infiltrating cells
The results of histological analysis were processed to examine the phenotypes of infiltrating mononuclear cells in the lacrimal glands, salivary glands, lungs, pancreas and kidneys of 10-month-old IQI/Jic mice by immunohistostaining. As shown in Fig. 2, the major populations of mononuclear cells were CD4+ T cells and B cells bearing B220 antigen in all organs examined, whereas CD8+ T cells were absent or scattered as extremely minor components of the infiltrates.
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Discussion |
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Many studies have demonstrated the spontaneous development of periductal foci of lymphocytic infiltration in the salivary and lacrimal glands in several murine strains, including NOD, MRL/lpr, NZB/W F1 and their backcrosses [58, 17]. Nevertheless, there is a paucity of animal models exhibiting histological characteristics of systemic autoimmune epithelitis in SS, like those found in IQI/Jic mice. Senile C57BL/B6 mice develop inflammatory lesions in multiple organs, including the salivary glands, lungs, pancreas and kidneys [18]. However, they do not show lesions in the lacrimal glands, and pancreatic lesions in C57BL/B6 mice are insulitis in contrast to the exocrine pancreatitis found in IQI/Jic mice, where the islets are completely exempted from damage. Mice possessing homozygous mutation for alymphoplasia (aly/aly) also develop mononuclear cell infiltrations in the periductal areas of exocrine glands, such as the lacrimal and salivary glands and pancreas, as well as in the areas surrounding pulmonary veins [15]. Although the affected organs and the locations of foci in each organ are essentially similar in aly/aly and IQI/Jic mice, there are several differences. In aly/aly mice, lymphocytic foci appear in the pancreas at the relatively young age of 14 weeks, when those in the lacrimal and salivary glands develop, and progress to marked tissue damage at between 20 and 29 weeks. On the other hand, in IQI/Jic mice, pancreatic lesions appeared much later than those in the lacrimal and salivary glands, and severe destructive lesions were rarely seen in the pancreas. These characteristics, specific to strains, may imply differences in the pathogeneses of their diseases, including the origin of the autoimmune reaction and the mechanism underlying the development of the autoimmune process.
It was reported previously that the proportion of B cells in infiltrates was drastically elevated with progress of the lesions in the lacrimal and salivary glands in IQI/Jic mice [12, 14]. The present study demonstrated that a similar phenomenon also occurred in the pancreas, but not in the lungs and kidneys (Fig. 2). This is unique to IQI/Jic mice, as CD4+ T cells are the predominant population infiltrating the lacrimal glands, salivary glands and pancreas throughout life in other murine models of SS, including NOD, NZB/W F1, MRL/lpr and aly/aly mice [13]. SS is characterized by the production of a wide range of autoantibodies, which illustrates that B cells are activated against various autoantigens in several restricted tissues or those with ubiquitous tissue distribution [19]. B-cell activation in SS often leads to monoclonal gammmopathies, pseudolymphomas and malignant lymphomas with increased frequency [20]. It has been suggested that B-cell malignancies mostly originate from infiltrates within glandular organs [21], and a sequential shift from T cells to B cells in proliferation preceding the monoclonal B-cell proliferation has been observed [22]. Therefore, investigations on IQI/Jic mice, particularly of the exact nature of infiltrating B cells, including clonality and phenotypes, could also be exploited to unravel the mechanism of B-cell lymphomagenesis in primary SS.
In conclusion, we demonstrated that IQI/Jic mice spontaneously developed lymphocyte infiltrates not only in the lacrimal and salivary glands, but also in the lungs, pancreas and kidneys, as they aged. These histopathological findings, unique to IQI/Jic mice, and their similarities with the observations reported in patients with SS suggest that this model may facilitate study of the aetiology of the progressive involvement of multiple exocrine organs and non-exocrine organs in primary SS.
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Acknowledgments |
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The authors have declared no conflicts of interest.
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References |
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