Norio Haneji

Estrogen Deficiency Accelerates Autoimmune Exocrinopathy in Murine Sjögren's Syndrome through Fas-Mediated Apoptosis

Estrogenic action has been suggested to be responsible for the strong female preponderance of autoimmune diseases, but the role of estrogens in the female has not been well characterized. We evaluated the effects of estrogen deficiency in a murine model for autoimmune exocrinopathy of Sjögrens syndrome (SS). Severe destructive autoimmune lesions developed in the salivary and lacrimal glands in estrogen-deficient mice, and these lesions were recovered by estrogen administration. We detected an intense estrogen receptor in splenic CD8(+) T cells compared with that in CD4(+) T cells, and concanavalin-A-stimulated blastogenesis of splenic CD8(+) T cells with estrogens was much higher than that of CD4(+) T cells. We found a significant increase in serum autoantibody production against the organ-specific autoantigen alpha-fodrin. Moreover, an increased proportion of TUNEL+ apoptotic epithelial duct cells was observed in estrogen-deficient mice. It was demonstrated that Fas-mediated apoptosis in cultured salivary gland cells was clearly inhibited by estrogens in vitro. These results indicate that dysfunction of regulatory T cells by estrogen deficiency may play a crucial role on acceleration of organ-specific autoimmune lesions, and estrogenic action further influences target epithelial cells through Fas-mediated apoptosis in a murine model for SS.

Anti‐120‐kDa α‐fodrin immune response with Th1‐cytokine profile in the NOD mouse model of Sjögren's syndrome

Our recent study suggested that the 120‐kDa α‐fodrin molecule may be an important autoantigen in the pathogenesis of Sjögrens syndrome, and anti‐120‐kDa α‐fodrin antibodies have been detected in patients with Sjögrens syndrome. Here we have analyzed anti‐120‐kDa α‐fodrin immune responses during development of spontaneous autoimmune sialadenitis in NOD mice as a model of Sjögrens syndrome. We found specific autoantibody production against 120‐kDa α‐fodrin, and its production correlated closely with autoimmune sialadenitis. A specific T cell response of splenocytes against the 120‐kDa α‐fodrin autoantigen was observed in NOD mice from the early onset of autoimmune sialadenitis. In addition, production in vitro by splenic T cells of cytokines such as interleukin‐2 (IL‐2) and interferon‐γ (IFN‐γ), but not IL‐4, was detected by enzyme‐linked immunosorbent assays. We found up‐regulation of local cytokine genes, including those of Th1 type (IL‐1β, TNF‐α, IL‐2, IFN‐γ, IL‐6), as well as IL‐10 and IL‐12(p40), in the tissue‐infiltrating cells during the course of autoimmune sialadenitis. These findings suggest that in spontaneous autoimmune sialadenitis in NOD mice, there may be a specific anti‐120‐kDa α‐fodrin immune response in the development of autoimmune lesions resembling human Sjögrens syndrome, and that the autoreactive Th1 cells possess an up‐regulated cytokine profile besides IL‐10 and IL‐12.

Severe Destructive Autoimmune Lesions with Aging in Murine Sjögren’s Syndrome through Fas-Mediated Apoptosis

When we evaluated the age-associated changes in autoimmune exocrinopathy in a NFS/sld murine model for primary Sjögrens syndrome (SS), severe destructive autoimmune lesions developed in the salivary and lacrimal glands in the aged mice, compared with those observed in the younger model. We detected a decreased secretion of saliva and tear flow in the aged group. A significant increase of TUNEL(+)-apoptotic epithelial duct cells in the salivary glands was detected in the aged SS animal model. A higher proportion of mouse salivary gland cells bearing Fas was found in the aged group, whereas no significant changes were seen on tissue-infiltrating CD4(+) T cells bearing FasL in the salivary glands from young and aged mice. We detected an increased cleavage product of organ-specific autoantigen, 120-kd alpha-fodrin, in the aged salivary gland tissues on immunoblotting, and an increase in serum autoantibody production against 120-kd alpha-fodrin by enzyme-linked immunosorbent assay. An increase in the proliferative response of splenic T cells against organ-specific autoantigen was observed, whereas nonspecific concanavalin A responsiveness was decreased in the aged mice. In addition, a decrease in Fas expression was found on splenic CD4(+) T cells in the aged mice, and anti-Fas mAb-stimulated apoptosis was down-regulated on CD4(+) T cells. These results indicate that age-associated dysregulation of CD4(+) T cells may play a crucial role on acceleration of organ-specific autoimmune lesions in a murine model for primary SS through Fas-mediated apoptosis.

High incidence of autoimmune dacryoadenitis in male non‐obese diabetic (NOD) mice depending on sex steroid

The NOD mouse develops spontaneous autoimmune lesions in the lacrimal and salivary glands, besides a well characterized T cell‐mediated autoimmune pancreatic β cell lesion. We report unique pathological findings developed in the lacrimal glands as an autoimmune dacryoadenitis of NOD mice in contrast to those found in the salivary glands and pancreas. A high incidence of autoimmune lesions in the lacrimal glands was observed exclusively in male NOD mice at any age. Histology of autoimmune dacryoadenitis in male NOD mice showed severe destructive changes compared with those observed previously as an autoimmune lesion in the lacrimal glands. Castration in male NOD mice significantly decreased the incidence of autoimmune dacryoadenitis, and testosterone treatment with castration also increased the incidence of autoimmune lesions. Oestrogen treatment with castration did not increase the incidence, but tamoxifen treatment without castration significantly increased the incidence of autoimmune dacryoadenitis in NOD mice. In addition, we detected up‐regulation of local cytokine genes (IL‐1β, tumour necrosis factor‐alpha (TNF‐α), IL‐2, interferon‐gamma (IFN‐γ), IL‐6, IL‐10, and IL‐12 p40) during the course of autoimmune dacryoadenitis. These data suggest that in spontaneous autoimmune dacryoadenitis of male NOD mice there may be an intimate relationship with sex steroids, particularly testosterone, in the development and progression of autoimmune lesions, and autoreactive Th1 cells secrete up‐regulated cytokine genes, including IL‐10 and IL‐12.

Treatment with anti-CD86 costimulatory molecule prevents the autoimmune lesions in murine Sjögren's syndrome (SS) through up-regulated Th2 response.

Intraperitoneal administration with anti‐CD86 (B7.2) MoAb into the murine model for primary SS in NFS/sld mutant mice resulted in dramatically inhibitory effects on the development of autoimmune lesions, while no significant effects were observed when the mice were administered with anti‐CD80 (B7.1) MoAb. We found that spleen cells in the murine SS model treated with anti‐CD86 MoAb showed a significant impairment of autoantigen‐specific T cell proliferation. T cell activation markers (CD44high, CD45RBlow, Mel‐14low) were significantly down‐regulated in the spleen cells gated on CD4 in anti‐CD86‐treated mice. We detected a higher level of cytokine production of IL‐4 from splenic T cells in anti‐CD86‐treated mice, but not of IL‐2, and interferon‐gamma (IFN‐γ), compared with those in the anti‐CD80‐ and PBS‐treated SS model. Moreover, serum autoantibody production against α‐fodrin autoantigen was almost entirely suppressed in anti‐CD86‐treated mice. These data provide strong evidence that in autoimmune exocrinopathy resembling SS in NFS/sld mutant mice, the CD86 costimulatory molecule plays a crucial role in the initiation and subsequent progression of Th1‐mediated autoimmunity in the salivary and lacrimal glands.

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.

Requirement for splenic CD4+ T cells in the immune privilege of the anterior chamber of the eye

Injection of antigen into the anterior chamber of the eye induces suppression of antigen‐specific DTH, called anterior chamber‐associated immune deviation (ACAID). It has been shown that the spleen is required for the induction of ACAID and detecting the ACAID‐inducing signal from the eye. To examine the in vivo role of spleen cells, fractions of spleen cells were adoptively transferred into splenectomized mice. The present study showed that DTH was not suppressed in splenectomized mice, but was inhibited in splenectomized mice transferred with a primed CD4+ T cell‐containing fraction of spleen cells. This indicates that the splenic CD4+ T cells comprise the regulatory T cells for the DTH response. When we examined the cytokine profile of the infiltrating T cells in the eye of primed mice by reverse transcriptase‐polymerase chain reaction (RT‐PCR), we found that they expressed IL‐4, IL‐10 mRNA (Th2 type), but not IL‐2 and interferon‐gamma (IFN‐γ) mRNA (Th1 type). By contrast, T cells which can elicit normal DTH response expressed IL‐2 and IFN‐γ mRNA. These results suggest that splenic CD4+ T cells comprising the regulatory phenotype are required for the induction of ACAID, and that a DTH response to the antigen may be prevented by Th2‐dominant CD4+ T cells.

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.

Autoantigen-Specific CD4+CD28low T Cell Subset Prevents Autoimmune Exocrinopathy in Murine Sjögren’s Syndrome

Organ-specific autoimmune exocrinopathy resembling Sjögren’s syndrome (SS) that spontaneously develops in NFS/sld mutant mice thymectomized 3 day after birth is dependent on Th1-type CD4+ T cells. We previously reported that a cleavage product of 120-kDa α-fodrin may be an important autoantigen in the pathogenesis of SS in both an animal model and the patients. We demonstrate that in an animal model of SS with overt exocrinopathy, a unique CD4+ T cell subset expressing CD28low is dramatically increased in spleen cells before the disease onset, but that the CD4+ T cells of diseased mice were virtually all CD28high. We found that the spleen cells in these mice before the disease onset showed a significant increase in autoantigen-specific T cell proliferation. Analysis of in vitro cytokine production by spleen cells indicated, before the disease onset, severely impaired production of IL-2 and IFN-γ in the animal model, whereas high levels of IL-4 were observed. Expression of cytokine genes, including IL-4, IL-10, and TGF-β, was detected in FACS-sorted CD4+CD28low T cells by RT-PCR analysis. Transfer of CD4+CD28low T cells into the animal model actually prevented the development of autoimmune lesions including autoantibody production. These results suggest that a CD4+CD28low T cell subset that is continuously activated by an organ-specific autoantigen may play a regulatory role in the development of organ-specific autoimmune disease in an animal model of SS.