Background
The etiology of autoimmune diseases remains largely unclear despite numerous research efforts, including clinical studies, epidemiological studies, and those involving experimental models. Hypothetical concepts speculate that autoimmunity results from a susceptible genetic background and the impact of specific environmental factors, including infectious agents and chemicals/xenobiotics [
1‐
4]. Therefore, autoimmune diseases seem to develop because of environmental triggers in combination with genetic and stochastic factors.
Induction of autoimmune disease by environmental agents, especially mercury, in susceptible rodent strains is a well-established and relevant model of systemic autoimmunity [
5,
6]. In Brown Norway (BN) rats, repeated administration of nontoxic doses of mercury chloride (HgCl
2) leads to T cell-dependent polyclonal activation of B cells characterized by lymphoproliferation, hypergammaglobulinemia, production of autoantibodies and immune complex (IC) deposits in the renal glomerular mesangium [
7,
8]. Target organs of systemic mercury-induced autoimmunity include lymphoid organs, kidneys, salivary glands, and mucocutaneous tissues [
7]. Among the mucocutaneous tissues, the oral mucosa is a target of mercury in BN rats [
7,
9,
10]. Histopathological findings of mercury-induced oral mucosal lesions were characterized by dense infiltration of mononuclear cells, including dendritic cells (DC) and macrophages, in the lamina propria beneath the surface epithelium of the mucosa. These changes seem to result from the establishment of mercury-induced autoimmunity in the oral mucosa. However, there are few research studies of mercury-induced disease that show evidence of direct interaction between local autoimmunity and cell infiltrates of immunocompetent cells in the oral mucosa.
Lupus erythematosus (LE) is a chronic inflammatory condition, considered the prototype of autoimmune human disease. Classically, LE has been subdivided into systemic and cutaneous forms. Whereas systemic LE is a multiorgan disease with variable prognoses, cutaneous LE is a more benign condition, limited to the skin and/or mucosal surfaces [
11]. The prevalence of oral mucosal involvement in LE patients is debatable. Some authors suggest that oral lesions are present in 9% - 45% patient with systemic LE and 3% -20% in those with cutaneous LE [
11]. These clinical data prompted us to examine whether systemic autoimmunity can lead to established lupus lesions in the oral mucosa. In the present study, we examined oral mucosal lesions in BN rats with systemic mercury-induced autoimmunity. First, we identified that immunohistopathological findings are characterized by dense infiltration of DC and macrophages in the basement membrane (BM) zone. Second, we determined that tissue expression of IL-4 mRNA is detected from early phase of oral lesions. Finally, the lupus band test is positive at junction between epithelium and lamina propria. Collectively, our data demonstrate that systemic mercury-induced autoimmunity induces LE-like lesions in the oral mucosa.
Methods
Rats
Inbred adult male BN rats (RTl
n) weighing 250–350 g were purchased from Kyudo Co. (Saga, Japan). All experiments were performed using rats matched for strain, age, and gender. Male rats were used because of their greater susceptibility to mercury-induced autoimmunity [
12]. The animal experimentation protocols were approved by the Animal Care and Use Committee of Fukuoka Dental College.
HgCl2 treatment
Groups of rats were injected with phosphate-buffered saline (PBS) or HgCl
2 (Sigma-Aldrich, St. Louis, MO, U.S.A.). Mercury-induced autoimmunity in the BN rats has been established by Aten et al. [
7]. HgCl
2, dissolved in distilled water (1 mg/ml), was injected at a dose of 1.0 mg of mercury per kilogram of body weight subcutaneously at days 0, 3, 5, and 7. At least five animals were used for each experimental point. PBS-treated rats were used as controls in all experiments.
Blood and tissue preparation
Blood samples were collected from tail veins of the mercury treated and control rats at various time points. The serum was separated and stored at −80°C for serum autoantibody determination. Kidneys and tongues were excised 2, 4, 6, 8, 10, 12, 14, and 21 days after injection from the mercury-treated group (n=5 each). Control tissues were collected from the control group at each time point (n=3 each). Half of the tissue specimens were fixed in 4% paraformaldehyde in PBS and embedded in paraffin. Paraffin sections (4-μm thickness) were then stained with hematoxylin and eosin (HE) to help visualize histopathological changes. The other specimens were immediately frozen in liquid nitrogen, and serial frozen sections were used for immunostaining, immunofluorescence (IF), and extraction of total RNA.
Immunohistochemistry
Monoclonal antibodies (mAb) used for immunohistochemistry are listed in Table
1. Acetone-fixed frozen sections were first incubated with normal rabbit serum to decrease nonspecific binding and then reacted with one of the mAbs. Sections were incubated with alkaline phosphatase-conjugated anti-mouse antibody (1:150 dilution; DakoCytomation, Tokyo, Japan). Immunohistochemical reactions were visualized using 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium chloride solution (BCIP/NBT solution; DakoCytomation). As a control, sections were treated with normal mouse IgG instead of the first set of antibodies. Infiltrating cells in the epithelial and subepithelial regions of the oral mucosa were counted in 25 randomly selected areas of 50 μm
2 each. Statistical analysis was performed with the two-tailed Student’s
t-test. Data are presented as the mean ± standard error and
P values of < 0.05 were considered statistically significant.
Table 1
Monoclonal anti-rat monoclonal antibodies used for immunohistochemical analysis
OX6 | RT1B | MHC class II antigens |
OX19 | CD5 | Pan T cells |
ED1 | CD68 | Macrophages, monocytes |
Real-time reverse transcription-polymerase chain reaction (qRT-PCR)
Total RNA was isolated from serial frozen sections by acid guanidiniumthiocyanate-phenol-chloroform extraction using an ISOGEN Kit (Nippon Gene, Toyama, Japan). One microgram of total RNA was transcribed into cDNA using random primers, oligo (dT) primers, and 10 μl of reverse transcriptase (ReverTra® Ace qPCR RT Kit; Toyobo Co., Ltd., Osaka, Japan). The reverse transcription was performed at 37°C for 15 min and then at 98°C for 5 min. Resulting templates were subjected to a LightCycler Nano real-time PCR system according to the manufacturer’s procedure (Roche Diagnostics, Tokyo, Japan). Predesigned primers and probe reagents for rat interleukin-4 (IL-4), interferon-γ (IFN-γ), and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) were commercially obtained from Roche Diagnostics. G3PDH was used as an internal control. The relative quantification of mRNA expression was calculated as a ratio of IL-4 and IFN-γ genes to G3PDH. Sequences of the primers and TaqMan probe were as follows: IL-4 forward primer, 5′-CATCGGCATTTTGAACGAG-3′; reverse primer, 5′-CGAGCTCACTCTCTGTGGTG-3′; Universal ProbeLibrary probe no. 2; IFN-γ forward primer, 5′-TCAAAAGAGTTCCTTATGTGCCTA-3′; reverse primer, 5′-TACGAGGACGGAGAGCTGTT-3′; Universal ProbeLibrary probe no. 69; G3PDH forward primer, 5′-AATGATCCGTTGTGGATCTGA-3′; reverse primer, 5′-GCTTCACCACCTTCTTGATGT-3′; Universal ProbeLibrary probe no. 80. Furthermore, 1.8% agarose gels were run to confirm that clean products of the expected length had been obtained.
Detection of autoantibodies and lupus band test (LBT) by IF
Serum samples from the mercury-treated or control rats were tested for the presence of autoantibodies by indirect IF. Detection of antinuclear autoantibodies (ANA) was used for HEp-2 cells as substrate [
13]. Briefly, serum samples were diluted 1:50–1:1000 in PBS and were incubated on slides with monolayer HEp-2 cells (GA Generic Assay, Dahlewitz, Germany), followed by Alexa Flour 488-conjugated goat anti-rat IgG Ab (Molecular Probes, Eugene, OR, USA) diluted 1:100. Titers were expressed as the reciprocal value of the highest serum dilution that gave a clear positive reaction. No staining at a serum dilution of 1:50 was considered as a negative result.
In situ binding of anti-BM autoantibodies from serum samples was tested by indirect IF using frozen sections of the kidneys and tongues from the control rats as substrates [
14]. Serum samples diluted 1:50 up to 1:1000 were incubated on frozen sections of kidneys and tongues. Binding sites of serum samples were detected by Alexa Flour 488-conjugated goat anti-rat IgG (Molecular Probes). The titers were expressed as the reciprocal value of the highest serum dilution that gave a clear positive reaction.
LBT was performed by the direct IF method. Frozen sections of the kidneys and tongues from the mercury-treated or control rats were incubated with FITC-conjugated mouse anti-rat IgG, Fcγ (Jackson ImmunoResearch Laboratories, West Grove, PA, USA), diluted 1:10 up to 1:500.
Discussion
In this study we used BN rats treated subcutaneously with mercury to elucidate whether oral mucosal lesions develop as a result of local events associated with immune responses, which were similar to those in systemic LE. Although the oral mucosa has been proposed as one of the targets in systemic autoimmune disorders of rats administered mercury, uncertainty remains as to whether the pathology underlying oral mucosal lesions include local immunological phenomena [
7,
9,
10,
23,
24]. We present three lines of evidence to support the conclusion that local immunological events play a role in the elicitation of lupus-like oral mucosal lesions an accompanying symptom of systemic mercury-induced autoimmunity. First, immunohistochemical approaches confirmed that an infiltration of both DC and macrophages occurred early during lesion development. Second, RT-PCR results indicated that local IL-4 release was found in the early lesions. Third, IgG deposition was detected at the junction between the surface epithelium and lamina propria of the oral mucosa by LBT.
The immunohistochemical results presented here indicate that infiltration of DCs and macrophages occurs in the early stage of oral lesion formation related to systemic mercury-induced autoimmunity. These findings are supported by previous reports [
9,
10,
24]. Because infiltration of DCs and macrophages precedes that of T cells, we speculate that development of oral lesions is mediated by DCs and macrophages. Generally, increased MHC class II antigen is most often associated with immunological stimuli [
25]. The earliest events in which MHC class II
+ cells infiltrate around the BM zones suggest that local immune responses related to systemic mercury-induced autoimmunity may be established in the oral mucosa. Following increased infiltration of MHC class II
+ cells, numerous ED1
+ macrophages migrate to the BM zone, indicating that the BM zone is the target tissue of oral mucosal lesions in systemic mercury-induced autoimmunity. Furthermore, accumulation of macrophages can indicate phagocytosis of injured fragments around the BM zone. Although increased infiltration of DCs and macrophages is not a primary event in mercury-induced autoimmunity, it may play an important secondary role in the pathogenesis of this syndrome. In contrast to the predominance of DC/macrophage-infiltration, participation of T cells seem to be less important in oral lesions. These findings suggest that DCs and macrophages may accumulate to react against local immune responses around the BM zone.
RT-PCR showed that tissue expression of IL-4 mRNA was observed from the early phase of oral lesions, suggesting that a local Th2-mediated immune response is responsible for the development of mercury-induced oral mucosal lesions. Previous RT-PCR analyses of mercury-treated BN rats show upregulation of IL-4 mRNA [
26,
27]. These reports speculate that IL-4 may serve multiple roles in the development of lupus. Phase I specimens were found to express IL-4 mRNA by RT-PCR, along with immunohistochemically detected infiltration of DC and macrophages in the absence of T cells. These findings suggest that cells other than T cells may be associated with IL-4 synthesis, even though T cells are known to participate in the secretion of this cytokine [
28,
29]. Although our immunohistochemical results reported here do not identify the type (s) of cells expressing IL-4 mRNA, a previous study demonstrated that mercury induced upregulation of IL-4 mRNA expression in mast cells of BN rats [
27]. Future work will address the origin of cells bearing IL-4 mRNA in mercury-induced oral mucosal lesions.
The observation of upregulated expression of IFN-γ mRNA in phase III specimens was unexpected. IFN-γ is generally responsible for the development of Th1-mediated immune responses, such as graft-versus-host disease. Our previous report examined IFN-γ induced epithelial expression of intercellular adhesion-1 (ICAM-1) in the early phase of oral mucosal graft-versus-host disease [
30]. It is difficult to explain why IFN-γ expression is upregulated in the late stage of mercury-induced oral mucosal lesions. Mercury-induced autoimmunity in BN rats is generally characterized by its weak symptoms, most of which resolve by 20 days after onset [
31]. A speculative possibility is that a Th2-mediated immune response responsible for the development of mercury-induced oral lesions may be decreased by the predominant Th1-mediated immune response in the late stage.
IF analyses indicate that the oral mucosa is one of the targets in mercury-induced systemic autoimmunity in BN rats. According to autoantibody production results and a positive LBT reaction in renal glomeruli, we support the mercury-treated BN rats as a model for the development of a systemic lupus-like syndrome. These findings are in concordance with data from other research groups, which showed that the features of mercury-induced autoimmunity in rats and mice, including lymphadenopathy, hypergammaglobulinemia, humoral autoimmunity, and IC deposits, are consistent with the autoimmunity observed in systemic LE [
21,
32‐
34]. In this study, serum samples from the mercury-treated BN rats contained autoantibodies, such as ANA and anti-BM antibody. It is known that ANAs could signal the body to begin attacking itself, which can lead to autoimmune diseases, including lupus, scleroderma, Sjögren’s syndrome, drug-induced lupus, and autoimmune hepatitis [
35,
36]. Indirect IF, using serum samples from the mercury-treated rats, showed a linear reaction in the renal glomeruli. Similarly, LBT of the kidney in the mercury-treated rats revealed a linear IF reaction in the glomeruli. LBT can be used to confirm IC-mediated reactions. Both indirect and direct IF studies demonstrated the deposition of glomerular IC in mercury-treated rat kidneys. Glomerular IC deposits are a hallmark of lupus-like autoimmune disease [
22]. In the tongue of the mercury-treated rats, LBT, as well as indirect IF assays, showed the linear deposition of IC in the BM zone, similar to the findings in the kidney. This suggests that the oral mucosa is affected by the lupus-like autoimmune disease. On the basis of these results, we suggest that the lupus-like oral mucosal lesions from mercury-induced systemic autoimmunity, are initiated by the deposition of IC in BM, followed by infiltration of DCs and macrophages into the IC deposition.
In summary, these results provide additional support for the characterization of oral mucosal lesions, in mercury-induced autoimmune disease. The IC deposits in BM are undoubtedly a key step in the pathogenesis of lupus-like oral mucosal lesions.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
KS and JO planned the study, performed the experiments and data analysis, and wrote the manuscript. NO carried out the immunostaining and helped to draft the manuscript. TH and KT supervised manuscript writing. All authors read and approved the final manuscript.