Jau-Ling Suen

Tamoxifen Decreases Renal Inflammation and Alleviates Disease Severity in Autoimmune NZB/W F1 Mice

It has been documented that sex hormone may play a role in the pathogenesis of murine lupus. To determine the effect of tamoxifen (TAM) on NZB/W F1 female mice, a total dose of 800 μg (22 mg/kg body weight) of TAM was administered subcutaneously every 2 weeks. The control mice were injected with peanut oil only. After treatment with TAM for 5 months, the mice were killed and immunological parameters were evaluated. The results suggest that NZB/W F1 mice treated with TAM had less severe proteinuria and increased survival rate compared to controls. Flow cytometric analysis of splenocytes revealed a significantly lower percentage of B cells and CD5+ B cells in the TAM‐treated group. There was a significantly lower serum level of soluble tumour necrosis factor (TNF) receptor I and II molecules in the TAM‐treated mice. Immunohistological study showed that control mice had severe immune complex deposition in the kidney. In contrast, TAM‐treated mice had much less pathological change. In summary, this study demonstrated that TAM treatment might be able to alleviate the symptoms of lupus nephritis, influence B‐cell count, modulate the expression of cytokine receptors and thereby subsequently affect immune function. Further studies to determine the cellular mechanisms in lupus nephritis may increase our understanding of this complex disease and provide additional targets for therapeutic intervention.

Altered homeostasis of CD4(+) FoxP3(+) regulatory T-cell subpopulations in systemic lupus erythematosus.

The role of naturally occurring regulatory T cells (Treg), known to be phenotypically heterogeneous, in controlling the expression of systemic lupus erythematosus (SLE) is incompletely defined. Therefore, different subpopulations of CD4+ FoxP3+ Tregs in patients with active or inactive SLE were investigated and compared with those of healthy subjects and patients with ankylosing spondylitis (AS). Characterization of different subsets of circulating CD4+ FoxP3+ Tregs was examined using flow cytometry. CD4+ CD25high T cells were sorted and examined for suppressive activity in vitro. The results showed first that a significant decrease in the frequency of CD4+ CD25high FoxP3+ T cells was present in patients with active SLE (n = 58), compared with healthy controls (n = 36) and AS patients (n = 23). In contrast, the frequencies of CD25low FoxP3+ and CD25− FoxP3+ CD4+ T cells were significantly increased in patients with active SLE by comparison with the control subjects. The elevation of these two putative Treg subpopulations was associated with lower plasma levels of complement C3 and C4 in patients with SLE. In addition, the ratios of the three subsets of CD4+ FoxP3+ Tregs versus effector T cells (CD4+ CD25+ FoxP3−) were inversely correlated with the titer of anti‐double‐stranded DNA IgG in patients with inactive, but not active, SLE. These results suggest that the pathogenesis of SLE may be associated with a defect in the homeostatic control of different Treg subsets.

The role of CD4+CD25+ T cells in autoantibody production in murine lupus

Systemic lupus erythematosus (SLE) is a chronic, systemic autoimmune disease characterized by the loss of tolerance to self‐antigen. Because it is currently not known if regulatory T (Treg) cells are involved in the pathogenesis, we determined the frequency of CD4+CD25+ T cells and assayed the related gene expression levels in CD4+CD25+ T cells isolated from both lupus mice (NZB/NZW F1) and normal control mice (DBA2/NZW F1). The results showed that the frequency of CD4+CD25+ T cells in lupus mice was lower than that of normal mice. Except for the high expression level of interleukin (IL)‐10 mRNA, CD4+CD25+ T cells from lupus mice expressed normal forkhead box P3 (Foxp3) and transforming growth factor (TGF)‐β mRNA, and exerted suppressive functions. Furthermore, we depleted CD25+ Treg cells of non‐autoimmune mice with anti‐CD25 antibody and broke their tolerance with apoptotic cell‐pulsed dendritic cells for the follow‐up of autoantibody levels. The mice in the CD25+ cell‐depleted group had higher titres of anti‐double‐strand/single‐strand DNA antibodies than those of the isotype control antibody‐treated group. These findings indicated that CD4+CD25+ T cells might be involved in the regulatory mechanism of autoantibody production.

Tamoxifen alleviates disease severity and decreases double negative T cells in autoimmune MRL-lpr/lpr mice

Previous study suggested that MRL‐lpr/lpr mice treated with tamoxifen (TAM) had less severe proteinuria, reduced serum titre of anti‐dsDNA autoantibodies and an increased survival rate. To investigate further the regulatory mechanisms of TAM on MRL‐lpr/lpr female mice, a total dose of 200 µg per mice (5·5 mg/kg) was given every 2 weeks subcutaneously, while the control mice were injected with oil only. After being treated with TAM four times, the mice were killed and cellular functions were evaluated. The TAM‐treated groups had smaller sized spleen and lymph nodes. Flow cytometric analysis of splenocytes had a significantly lower percentage of cell number of T cells and double negative T cells (CD4– CD8– T cells). There was no difference in cytokine production (interleukin (IL)‐2, IL‐4, IL‐5, IL‐10 and interferon‐γ (IFN‐γ)) from splenocytes stimulated with concanavalin A (Con A) or cytokines (IL‐6) secreted by peritoneal exudate cells when stimulated with lipopolysaccharide (LPS). However, IL‐2 from lymph node cells was significantly higher on TAM‐treated mice. Finally, splenocytes or purified T cells stimulated with anti‐CD3 antibody plus cross‐linking immunoglobulin G (IgG) of the TAM‐treated group had higher 3H‐incorporation of proliferation assay compared with that of control groups. In vitro study further demonstrated that IL‐2‐activated proliferation of lymph node double negative (DN) T cells can be inhibited by TAM treatment in a dose‐dependent manner. Our finding demonstrated that TAM may potentially influence T cells and modulate the immune function, which offers a novel approach to explore the feasibility of hormone therapy for autoimmune diseases.

Characterization of self-T-cell response and antigenic determinants of U1A protein with bone marrow-derived dendritic cells in NZB × NZW F1 mice

Systemic lupus erythematosus (SLE) is characterized by the existence of a heterogeneous group of autoantibodies directed against nuclear intact structures, such as nucleosomes and small nuclear ribonucleoproteins (snRNPs). Autoantibodies against snRNPs are of special interest because they are detectable in the majority of SLE patients. Although the B‐cell antigenic determinants have been well characterized, very limited data have been reported in regard to the T‐cell epitopes of snRNPs. Furthermore, several studies have demonstrated that determination of the auto‐T‐cell epitopes recognized by freshly isolated T cells is difficult from unprimed lupus mice when self‐antigen‐pulsed B cells or macrophages are used as antigen‐presenting cells (APCs) in vitro. In the present study, we showed a novel approach for determining the auto‐T‐cell epitopes, using bone marrow‐derived dendritic cells (BMDCs) pulsed with the murine U1A protein – an immunodominant antigen of the U1 snRNPs – which is capable of activating freshly isolated T cells from unprimed (NZB × NZW) F1 (BWF1) mice in vitro. The T‐cell epitope area was found to be located at the C‐terminus of U1A, overlapping the T‐cell epitope of human U1A that has been reported in human SLE. Identification of the autoreactive T‐cell epitope(s) in snRNPs will help to elucidate how reciprocal T–B determinant spreading of snRNPs emerges in lupus. The results presented here also indicate that it is feasible to use this approach to further explore strategies to design immunotherapy for patients with lupus.

In vivo tolerance breakdown with dendritic cells pulsed with U1A protein in non-autoimmune mice: the induction of a high level of autoantibodies but not renal pathological changes

One of the hallmarks of systemic autoimmune diseases, such as systemic lupus erythematosus (SLE), is the immune response to nuclear autoantigens. Several studies have proposed that dendritic cells may acquire the nuclear autoantigens from the apoptotic cells to initiate the systemic autoimmune responses. To examine the immune response to a nuclear autoantigen induced by dendritic cells, bone marrow‐derived dendritic cells (BMDCs) pulsed with U1 small nuclear ribonucleoprotein (snRNP)‐A protein (U1A) were intravenously injected into non‐autoimmune mice. The results showed that BMDCs pulsed with U1A proteins by intravenous injection into BALB/c (H‐2d) and DBA‐2×NZW F1 (H‐2d/u) mice were capable of activating the autoreactive T cells and inducing a high titre of immunoglobulin G (IgG) anti‐U1A antibodies. Both groups of mice with a high anti‐U1A autoantibody titre also transiently developed IgG against double‐stranded (ds) DNA. However, unlike NZB×NZW F1 (BWF1) (H‐2d/u) mice, no obviously histopathological changes to the glomeruli were noted in the mice treated either with BMDCs or with U1A‐pulsed BMDCs. Several months after immunization, all mice treated with U1A‐pulsed BMDCs did develop IgG, but not the complement C3 deposit in the glomeruli. The cytokine profile produced by the U1A‐specific T cells of primed DBA‐2×NZW F1 mice was skewed toward the T helper type 1 phenotype compared with that of BWF1 mice. The model we describe here adds to the further understanding of the pathogenic mechanisms, such as self‐antigen shifting, and the mechanisms that account for the different responses to self‐antigens when in a normal or an autoimmune state.

Fas-ligand-expressing adenovirus-transfected dendritic cells decrease allergen-specific T cells and airway inflammation in a murine model of asthma

T cells expressing a type-2 T helper profile of cytokines (Th2 cells) have been demonstrated to play an important role in the initiation and progression of allergic asthma, and it is well known that Fas ligand (FasL) induces apoptosis when bound to its receptor, Fas. In the present study, we examined the possibility of modulating asthma manifestations by dendritic cells (DCs) genetically engineered to express FasL (DC-FasL), which could deliver a death signal to T cells in an antigen-specific manner. The delivery of DC-FasL into ovalbumin (OVA)-immunized allergic mice decreased the airway hyper-responsiveness (AHR). Moreover, we established a mouse model of airway inflammation by using an adoptive transfer of Th2 cells derived from ovalbumin T cell receptor transgenic mice to study the effect of DC-FasL on airway reactivity. The administration of DC-FasL in Th2-cell-induced allergic mice had significantly decreased AHR, airway inflammation, and IL-4, IL-5 and IL-13 production. Furthermore, the numbers of OVA-specific T cells were decreased in the lung of mice receiving DC-FasL. These results demonstrate that FasL-expressing dendritic cells might be applied for the modulation of allergic responses.

CD4(+)FoxP3(+) regulatory T-cells in human systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by a loss of immune tolerance to self antigens and by the persistent production of pathogenic autoantibodies. Recent studies have suggested a dysregulation of regulatory T-cells (Tregs), particularly CD4(+)CD25(high)FoxP3(+) (forkhead box P3) Tregs, as one of the major factors conferring the risk for expression of human autoimmune diseases, including SLE. However, detailed studies of CD4(+)FoxP3(+) T-cells in patients with SLE remain limited. We attempt here to integrate the current experimental evidence to delineate the role of CD4(+)CD25(high) and other subsets of CD4(+)FoxP3(+) T-cells in human SLE.

Morin Promotes the Production of Th2 Cytokine by Modulating Bone Marrow-Derived Dendritic Cells

Our previous studies had reported that morin decreased the interleukin-12 (IL-12) and tumor necrosis factor-alpha (TNF-alpha) production in lipopolysaccharide (LPS)-activated macrophages, suggesting that morin may promote helper T type 2 (Th2) response in vivo. Dendritic cells (DCs) are the most potent antigen presenting cells and known to play a major role in the differentiation of helper T type 1 (Th1) and Th2 responses. This study aimed to reveal whether morin is able to control the Th differentiation through modulating the maturation and functions of DCs. Bone marrow-derived dendritic cells (BM-DCs) were incubated with various concentrations of morin and their characteristics were studied. The results indicated that morin significantly affects the phenotype and cytokine expression of BM-DCs. Morin reduced the production of IL-12 and TNF-alpha in BM-DCs, in response to LPS stimulation. In addition, the proliferative response of stimulated alloreactive T cells was significantly decreased by morin in BM-DCs. Furthermore, allogeneic T cells secreted higher IL-4 and lower IFN-gamma in response to morin in BM-DCs. In conclusion, these results suggested that morin favors Th2 cell differentiation through modulating the maturation and function of BM-DCs.

Identification of T‐cell epitopes on U1A protein in MRL/lpr mice: double‐negative T cells are the major responsive cells

Systemic lupus erythematosus (SLE) is characterized by the existence of a heterogeneous group of autoantibodies such as anti‐DNA, chromatin, histone, and ribonucleoprotein antibodies (Abs). Although the B‐cell antigenic determinants have been well characterized, very limited data about the T‐cell epitopes of self‐antigen (Ag) have been reported. In the present study, we analysed auto‐T‐cell epitopes using bone marrow‐derived dendritic cells (BM‐DCs) pulsed with murine U1A (mU1A) protein capable of activating autoreactive T cells from unprimed MRL/lpr mice in vitro. The data suggested that there are at least four T‐cell epitopes on the U1A protein, U1A31−50, U1A61−80, U1A201−220 and U1A271−287, and U1A31−50 had the most significant T‐cell proliferative response. In addition, the main responsive T cells are the CD4− CD8− double‐negative subgroup of T cells. Furthermore, we also demonstrated that the activation of double‐negative T cells is major histocompatibility complex class II restricted. The study here provides information on T‐cell epitope analysis of the U1A antigen using BM‐DCs as the effective antigen‐presenting cells.