Lupus-like oral mucosal lesions in mercury-induced autoimmune response in Brown Norway rats

Abstract

Background: Administration of mercury at nontoxic doses induces systemic autoimmune disease in Brown Norway (BN) rats. The pathogenesis of lupus-like oral mucosal lesion by mercury-induced autoimmunity is still unclear, even though the oral mucosa is observed to be commonly affected in mercury-treated BN rats. In this study, we investigated the immunopathology of lupus-like oral mucosal lesions in a model of mercury-induced systemic autoimmunity.

Methods: Brown Norway male rats were injected subcutaneously with either phosphate-buffered saline (control) or mercury at a dose of 1.0 mg per kilogram of body weight on days 0, 3, 5, and 7. Blood, kidney, and tongue samples were taken at various timepoints for evaluation by immunohistochemistry, RT-PCR, and lupus band test (LBT).

Results: Oral mucosal lesions were classified according to three consecutive temporal phases on the basis of infiltration of immunocompetent cells as follows: (phase I) infiltration of MHC class II+ dendritic cells (DC) and macrophages; (phase II) addition of ED1+ macrophage infiltrates; and (phase III) focal infiltration of pan T cells following increased infiltration of DC and macrophages. Dense infiltration of DC and macrophages was observed in the basement membrane (BM) zone of the oral epithelium. Tissue expression of IL-4 mRNA was detected in early lesions (phase I), suggesting that locally produced IL-4 may be responsible for Th2-mediated immune response. A linear and continuous smooth pattern of fluorescence was observed in the oral epithelial BM in addition to renal glomeruli, indicating immune complex deposits.

Conclusions: Local autoimmune responses are involved in the pathogenesis of mercury-induced lupus-like lesions of the oral mucosa.

Figure 1

Immunohistochemical staging of infiltrating mononuclear cells in the tongue of the mercury-treated rats. A-D: The tongue specimens from the control rats show MHC class II⁺ cells distributed sparsely to both the lamina propria and surface epithelium (A). The number of MHC class II⁺ cells increases continuously as phase progress (B-D). E-H: A few ED1⁺ cells are in the lamina propria of normal tongue (E). During progression of phases, the number of ED1⁺ cells is increased in upper lamina propria (F-H). I-L: A small number of CD5⁺ T cells are distributed in the lamina propria of tongue specimens from phases I and II as well as the control rats (I-K). In phase III specimens, focal accumulations of CD5⁺ T cells are present in both the lamina propria and surface epithelium (L). M-P: No obvious changes are noted in hematoxylin-eosin (HE)-stained tongue sections taken from thec ontrol, phase I and phase II rats (M-O). Infiltrates of mononuclear cells are observed in the lamina propria of the oral mucosa in phase III specimens (P). Bar= 100μm.

Figure 2

Infiltration of MHC class II⁺, ED1⁺, and OX19⁺ mononuclear cells in the tongue from the control rats and the mercury-treated rats. Cells were counted from day 0 (d0) to day 21 (d21). The tongues from control rats are represented by black columns (n=3 per day), the tongues from mercury-treated rats are indicated by white columns (n=5 per day). Cell numbers/ mm² indicated as mean± standard error (SE). *, significantly different at p< 0.01 compared with control. **, significantly different at p< 0.005 compared with control. There are no significant differences in groups of white columns jointed to horizontal bars are at a p value of <0.05.

Figure 3

The number of MHC class II⁺, ED1⁺, and OX19⁺ mononuclear cells at various phases of oral mucosal lesions in the mercury-treated rats. Cell numbers/ mm² indicated as mean± standard error (SE). *, significantly different at p< 0.01. **, significantly different at p< 0.05.

Figure 4

Tissue expression of interleukin-4 (IL-4) and interferon-γ (IFN-γ) mRNA in the tongue of the mercury-treated rats by real-time reverse transcription-polymerase chain reaction (qRT-PCR). A and B: qRT-PCR analyses examine expression levels of mRNA encoding IL-4 (A) and IFN-γ (B) normalized by those of glyceraldehyde-3-phosphate dehydrogenase (G3PDH). Data represents mean ± standard error (SE) of pooled data derived from three to five independent experiments. *, significantly different at p< 0.05 compared with the control. **, significantly different at p<0.01 compared with the control. §, significantly different at p<0.05 compared with phases I and II. §§, significantly different at p<0.05 compared with control, phases I, and II. C: Agarose gel electrophoresis analysis of qRT-PCR. Ctr, control; I, phase I; II, phase II; III, phase III.

Figure 5

Antibody immunofluorescence (IF) analysis. A: Detection of antinuclear autoantibodies (ANA) by indirect IF using serum from the mercury-treated rats with phase III lesions, incubated on HEp-2 cells. Both nucleolar and nuclear cytoplasmic fluorescences are noted. Bar=50 μm. B: The reciprocal titer of IgG ANA in test serum samples from the rats in control and various phases. Horizontal bars denote median values. *, significantly different at p<0.05 compared with phase I. Controls show no titers. C: The reciprocal titer of autoantibodies bound to renal capillaries and basement membrane (BM) of the oral mucosa by indirect IF analysis. Horizontal bars denote median values. *, significantly different at p<0.05 compared with phases I. Controls show no titers. D: Indirect IF images of autoantibodies in the renal capillaries and basement membrane (BM) of the oral mucosa using serum from Brown Norway (BN) rats with phase III lesions. (A) IF reaction of the renal capillaries and mesangium is seen in the normal kidney. (B) IF labeling on the BM of the oral epithelium in normal tongue. Bars= 100 μm.

Figure 6

Lupus band test (LBT) with FITC-conjugated anti-rat IgG, Fcγ, antibody (1:300) incubated on frozen sections of the kidneys and tongues from mercury-treated rats. A-C: No immunofluorescence (IF) staining is seen in phase I (A). IF reaction on the glomerular mesangial regions during phases II (B) and III (C). D-F: A very weak or faint reaction is seen in the portion of the basement membrane (BM) of the oral epithelium of the tongue by phase I (D). Remarkable IF appears on the BM of the epithelium of the tongue in phases II (E) and III (F). Bar= 100 μm.