Hironori Hamano

c‐myc, ras p21 and p53 Expression in pleomorphic adenoma and its malignant form of the human salivary glands

Using an immunohistochemical study and an immunoblot analysis, the expression of cellular oncogenes of the human salivary glands such as c‐myc, ras p21, and p53 tumorsuppressor gene in pleomorphic adenomas and its malignant form, carcinoma in pleomorphic adenomas was examined to evaluate a differential biological significance, in comparison with that in normal salivary gland tissues. Immunohistochemically, the c‐myc product was detected in 42% of the pleomorphic adenomas and in 56% of the carcinomas in pleomorphic adenoma. The ras p21 expression was observed in 24% of pleomorphic adenomas, and in 50% of carcinomas in pleomorphic adenoma. The p53 protein was detected in 18% of the pleomorphic adenomas and in 67% of the carcinomas in pleomorphic adenoma. Although there was no significant difference between the benign and malignant forms for the expression of c‐myc, a statistical significance in ras p21 and p53 expression was found between the pleomorphic adenoma and its malignant form (P < 0.05) and P< 0.001, respectively). An immunoblotting assay clearly demonstrated the expression of c‐myc and p53 gene products in both the benign and malignant forms of the pleomorphic adenoma, and that of ras p21 in the malignant form. These results indicate that activation of c‐myc and ras p21 proto‐oncogenes and the involvement of p53 mutation may play important roles in the malignant transformation of salivary gland pleomorphic adenoma.

Pathogenesis of Sjögren's syndrome‐like autoimmune lesions in MRL/Ipr mice

Sjögrens syndrome in humans is a chronic inflammatory disease with a presumed autoimmune etiology of the exocrine organs, involving in particular the salivary and lacrimal glands. The pathogenesis of this syndrome remains unclear, but the majority of infiltrating cells in the salivary glands are CD4+ T cells both in humans and rodents. Since many cytokines are involved in the development of T cell‐mediated autoimmunity, local cytokine gene expression was analyzed in vivo using an animal model for Sjögrens syndrome in MRL/Ipr mice. Overexpression of interleukin‐1 (IL‐1)β and tumour necrosis factor (TNF) was detected before the onset of inflammatory lesions in the salivary gland, and the up‐regulation of IL‐6 mRNA was also found in accordance with autoimmune sialadenitis in MRL/Ipr mice. The inflammatory cytokines such as IL‐1β, TNF, and IL‐6 have proved to play important roles as regulatory proteins inducing autoimmune phenomena. In addition, the expression of T cell antigen receptor β (TCR) β transcripts in the salivary gland tissues was analyzed. Transcript for Vβ8 was predominantly detected in the T cells infiltrating sialadenitis from the onset of the disease, suggesting that CD4+ T cells bearing TCR Vβ8 play an essential role in recognizing unknown autopeptide in the autoimmune sialadenitis of MRL/Ipr mice. Furthermore, Sjögrens syndrome‐like autoimmune lesions were successfully transferred into severe combined immunodeficiency (SCID) mice, and these lesions were prevented by administration of anti‐CD4, and anti‐Vβ8 monoclonal antibodies. This article will review recent observations of these pathogenetic analyses of autoimmune sialadenitis as it occurs in MRL/Ipr mice.

Cytokine Gene Expression and Autoantibody Production in Sjogren's Syndrome of MRL/lpr Mice

In an attempt to elucidate the mechanism of development of organ-specific autoimmune lesions resembling human Sjögrens syndrome of MRL/lpr mice, we have analyzed local cytokine gene expressions and organ-specific autoantibody production in vivo. We have demonstrated that a major proportion of T cells bearing CD4 and V(beta)8 molecules are essentially responsible for triggering the autoimmunity in the salivary glands of MRL/lpr mice. The local cytokine gene expressions including interferon(IFN)-gamma, IL-12(p40) mRNAs were observed during the course of murine Sjogrens syndrome in MRL/lpr autoimmune strain. In particular, a high level of local expressions of IL-12 mRNA was detected earlier in the proinflammatory stage of autoimmune lesions. A significant level of local expression of MHC class-II(I-Ak) mRNA was detected before the onset of inflammatory lesions in the salivary glands, and I-Ak-positive epithelial duct cells were frequently observed in the salivary glands of MRL/lpr mice. In addition, we found the salivary gland-specific autoantibody in sera from MRL/lpr mice with early phase of autoimmune lesions by immunoblot analysis. These results suggest that cytokine gene stimulation and autoantibody production are essentially involved in the development of organ-specific autoimmune lesions in Sjögrens syndrome of MRL/lpr mice.

Expression of HLA-DR and cytokine genes on interferon-gamma-stimulated human salivary gland cell line.

Stimulation of a cultured human salivary gland (HSG) cell line by interferon (IFN)-gamma leads to HLA-DR gene expression concomitant with inflammatory cytokine genes such as IL-1 beta, tumor necrosis factor (TNF)-alpha, and IL-6 in vitro. IFN-gamma-induced HLA-DR mRNA expression was clearly detected at 2 h after the stimulation, and thereafter its level of gene expression increased until day 7 on HSG cells by reverse transcriptase (RT)-PCR. Immunofluorescence analysis revealed that cytoplasmic HLA-DR immunoreactivity was detected for the first time at 2 days after the stimulation, and its immunoreactivity increased gradually until day 7, while no immunoreactivity with HLA-DP and HLA-DQ was observed at any of the days. In addition, the expression of IL-1 beta, TNF-alpha, and IL-6 on the IFN-gamma-stimulated HSG cells was detected by immunohistochemistry and RT-PCR analysis. These results indicate that human salivary gland cells can be induced to express HLA-DR mRNA by IFN-gamma concomitant with inflammatory cytokine gene expressions such as IL-1 beta, TNF-alpha, and IL-6.

Prevention of adoptive transfer of murine Sjögren's syndrome into severe combined immunodeficient (SCID) mice by antibodies against intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-1 (LFA-1)

We have analysed the role of ICAM‐1 and LFA‐1 during development of autoimmune sialadenitis in MRL/1pr mice by direct analysis of RNA obtained from the salivary gland tissues, and the therapeutic effects with antibody administration on adoptive transfer system into SCID mice. The expression of cell adhesion molecules was assessed by using reverse transcriptase polymerase chain reaction (RT‐PCR) and Southern blot analysis, and immunohistochemical analysis. Up‐regulated expression of ICAM‐1 mRNA was observed before the onset of inflammatory lesions in the salivary glands at 1 month and 2 months old, and thereafter LFA‐1 mRNA was expressed within the typical inflammatory lesions, resembling human Sjögrens syndrome in MRL/1pr mice. Immunohistochemically, ICAM‐1 was localized exclusively in the endothelial cells of varying sized blood vessels before the onset of disease, and LFA‐1 expressing inflammatory ceils were found within these lesions. When the therapeutic effects in vivo were examined, antibodies to ICAM‐1 in combination with anti‐LFA‐1 prevented adoptive transfer of Sjögrens syndrome in MRL/1pr mice into SCID mice, while no significant effect was found when treated with either antibody. These findings ìndìcate that in Sjögrens syndrome‐like autoimmune lesions in MRL/1pr mice the ICAM‐1/LFA‐1 pathway may play a crucial role in the initiation and subsequent progression of T cell‐mediated autoimmunity in the salivary and lacrimal glands of MRL/1pr mice.

Immunopathological Analysis of Mucosal Melanocyte Distribution in the Human Lower Lip of the Elderly

Mucosal melanocyte distribution in the human lower lip was analyzed immunopathologically using a wide selection of 195 surgical specimens. An age-related increase of mucosal melanocyte distribution was observed in the human lower lip by Fontana-Masson argentaffin stain and S-100 protein immunoreactivity. The mean number of mucosal melanocytes increased gradually with advancing age. A large number of them was found at or beyond the seventh decade of life in both sexes, which was statistically significant compared with those in all other decades (p < 0.01 or < 0.05). In addition, the sex difference in mucosal melanocyte density was statistically significant (p < 0.001) in each decade (men > women). These results suggest that physiological changes in mucosal melanocyte distribution are dependent upon aging and sex difference, and may play an important role in development of melanocytic lesions such as senile lentigo, pigmented nevi, and also intraepithelial malignant melanomas.

Accelerated onset of age-related autoimmune lesions in MRL/+ mice by ovariectomy

MRL/Mp +/+ (MRL/+) mice, not bearing the lpr gene, are known to have age-related autoimmune lesions in several organs such as pancreas, salivary and lacrimal glands at 30-weeks-old or more. In this study, MRL/+ mice were ovariectomized at 4-weeks-old, and their natural histories were analysed. Ovariectomy (Ovx) of MRL/+ mice led to marked acceleration of organ-specific autoimmune lesions exclusively in the salivary and lacrimal glands at 8-weeks-old or more, whereas no significant inflammatory change was observed in the pancreas. In the vast majority of inflammatory infiltrates, CD3+ CD4+ T cells were predominant in both the salivary and lacrimal glands of Ovx-MRL/+ mice. Up-regulated expression of cytokine genes including IL-1 beta, TNF-alpha, IL-2, interferon (IFN)-gamma, and IL-6 was detected in the salivary gland of Ovx-MRL/+ mice by reverse transcriptase (RT)-PCR analysis. FACS analysis of spleen cells of Ovx-mice revealed increase in I-Ak expression on B220+ cells, and autoantibody production against the salivary gland-specific antigen in sera from Ovx-MRL/+ mice, but not in control mice. These results suggest that age-related autoimmunity in the salivary and lacrimal glands were accelerated in Ovx-MRL/+ mice, and that autoreactive Th1 cells were activated associated with organ-specific autoantibody production.

Immunopathology of Phenotypic Change on Human Parotid Gland Adenocarcinoma

Immunopathological analysis was made of phenotypic change in a recurrent parotid gland adenocarcinoma occurring in a patient with a long clinical course of 30 years or more. At the first and second operations, in 1959 and 1978, the resected parotid gland tumors were diagnosed histopathologically as acinic cell carcinoma. However, 11 years after the second operation, in 1989, the resected recurrent tumor showed a microscopically phenotypic change towards adenocarcinoma with typical tubular arrangement. At the last operation in 1991, histopathological examination of the tumor revealed adenocarcinoma with diffuse oncocytic change in association with cervical lymph node metastasis. These findings suggest that phenotypic change may occur in vivo among human neoplasms during a long period, which may be related to the cytodifferentiation in the salivary gland tumor.

Interstitial renal lesions as an autoimmune phenomenon in the aged

Spontaneously occurring, organ-specific autoimmune lesions develop in aged C57BL/6 mice of both sexes, especially in 24-month-old senescent mice. Inflammatory lesions of autoimmune nature associated with advance of age were found in many organs such as renal interstitium, salivary gland, pancreatic islet, lung and liver. The incidence and severity of spontaneously occurring organ-specific autoimmune lesions in this strain of non-autoimmune mice increase with age. In humans, autopsy material of elderly people, examined histopathologically and immunopathologically, showed infiltrating lymphocytes in several organs such as the salivary glands, thyroid, adrenal corteces. Recently, we found spontaneously occurring inflammatory lesions in the renal interstitium, probably indicating an autoimmune phenomenon. Extensively infiltrating foci in the renal interstitium were frequently found in persons over 70 years of age. The infiltrating mononuclear cells were predominently CD4+ cells, whereas CD22+ B cells were fewer in number. Moreover, a considerable proportion of T cells was activated as judged by IL-2 receptor expression. Based on these findings, we propose that the development of interstitial renal lesions in the elderly is an autoimmune phenomenon involving the cellular immune system, and may be related to an age-related disturbance in regulatory T-cell function.