Study On The Characteristics Of Renal-infiltrating T Cells In Spontaneous Murine Lupus

Background and ObjectiveSystemic lupus erythematosus (SLE) is a chronic, multisystem-involved typical autoimmune disease characterized by a diverse array of autoantibody production, complement activation and immune complex deposition, causing tissue and organ damage, such as serious glomerulonephritis. The etiology and pathogenetic mechanisms of SLE have not been clearly elucidated. At present, there is no cure for SLE and treatment is aimed at controlling symptoms.The study on SLE mouse model is very important for exploring the etiology, pathogenesis and new treatments of human SLE. Over the past30years, mouse models of SLE have provided valuable insights concerning the pathogenesis of lupus. Based on how they were generated, these models can be divided into three groups:(1) spontaneous mouse lupus models,(2) congenic mouse models, and (3) engineered mouse models of lupus. Spontaneous mouse lupus models occur in the natural condition, which is more similar to human lupus. The availability of several murine strains that spontaneously develop an autoimmune syndrome resembling human SLE, such as MRL/Mpr-lpr/lpr (MRL/lpr), NZB/NZW F1(NZB/W F1) and BXSB/Yaa mice. MRL/lpr mice were derived mainly from the LG/J strain with contributions from AKR/J, C3H/Di, and C57BL/6J mouse strains. The spontaneous autosomal recessive mutation lymphoproliferation (lpr) was first observed at the12th generation of inbreeding. The Fas defect, caused by lpr gene mutation, accelerates the autoimmunity of MRL/lpr mice, and MRL/lpr mice spontaneously develop glomerulonephritis, vasculitis, and arthritis, with characteristic production of rheumatoid factor, immune-complexes, and anti-double-stranded DNA autoantibody. The lupus-like syndrome is earlier in onset and more acute in females than in males. The NZB/W F1is the oldest classical model of lupus generated by the F1hybrid between the NZB and NZW strains. Both NZB and NZW display limited autoimmunity, while NZB/W F1hybrids develop severe lupus-like phenotypes comparable to that of lupus patients. The lupus-like syndrome is earlier in onset in females than in males. Female NZB/W F1mice spontaneously produce auto-antibodies starting at5-6months of age and eventually die from kidney failure at8-9months of age. BXSB/Yaa mice, a recombinant inbred strain, generated by the hybrid between the female C57BL/6and male SB/Le strains. Male BXSB/Yaa mice develop SLE faster than females, which is opposite of that observed in humans.The excessive activation and dysfunction of T lymphocyte and B lymphocyte cause the abnormal immune system response in SLE patients. T lymphocytes play a central role in cell-mediated immunity. T cells can be divided into several subsets with distinct functions, including the T helper cells (Th), Suppressor T cells (Ts), Effector T cells (Te), Cytotoxic T cells (Tc) and Delayed type hypersensitivity T cells (Td). Dysfunctions of subsets, distribution imbalance and activation of different subsets have been implicated to contribute to disease progression of SLE. Recent studies have found that Th cells and its cytokines play an important role in the pathogenesis of SLE and other autoimmune diseases. Th cells can regulate immune responses via induce or suppress proliferation, differentiation and activation of other immune cells. Mature Th cells express the surface protein CD4and are referred to as CD4+T cells. Th cells are not a single group of cells. Naive CD4+T cells may differentiate into several lineages of Th cells:Thl, Th2, regulatory T cells (Treg) and Th17, as defined by their pattern of cytokine production and function. The cytokines secreted by the four kind of Th cell and immune function each are not identical. Th1cells, which differentiate in the presence of IFN-y and IL-12, are largely responsible for cellular immunity against intracellular bacteria and viruses and are distinguished by their secretion of signature cytokine IFN-y. Thl cells tend to be good IL-2producers, and many make TNF-a as well. Th2cells, which differentiation is induced by IL-4and IL-2, are recognized to be integrally involved in the humoral response to parasitic infections and are defined by their characteristic secretion of cytokines of IL-4, IL-5and IL-13. They also make TNF-a, and some produce IL-9.The imbalance of T-cell-derived cytokines have been implicated to contribute to SLE progression, of which, imbalance in Thl/Th2plays an import role in pathogenesis of SLE. But studies have shown that SLE cytokine secretion abnormalities are not consistent."Thl prominence" and "Th2prominence" were proved in animal model and lupus patients. Indeed, some investigators propose that a predominance toward Thl generation could play a significant role in driving lupus-like autoimmune disease in MRL/Ipr mice. Cytokine profiles of spleen cells in BXSB/Yaa mice showed mainly increases in the Thl cytokine IFN-y. IFN-y accelerated, while anti-IFN-y antibody (Ab) or soluble IFN-y receptor prevented (onset or progression) of SLE in NZB/W F1mice. The research indicated that Thl type cytokines IFN-γ may play an important role in development of mouse models of lupus disease. Interestingly, Th2-type responses have also been observed with the development of SLE, where the numbers of IL-4-producing cells have been found to be higher in some lupus-susceptible strains. Similarly, treatment with blocking anti-IL-4Ab or soluble IL-4R reduced autoantibody production and nephritis in NZB/W F1and MRL-Fas mice. Additionally, IL-13, also a Th2cytokine, has been found to play a critical role in immunoglobulin production and the induction of antibody class switch in response to rheumatoid factors in human SLE patients.Recently, a new population of effector CD4+T cells characterized by the secretion of IL-17, identified as Thl7cells, which also produce of IL-21and IL-22. Thl7cells have been implicated in host defense, inflammatory disease, tumorigenesis, autoimmune diseases, and transplant rejection. IL-17has also been implicated in the pathogenesis of autoimmune and inflammatory diseases including SLE. Herber’s research results suggest that blocking IL-21with IL-21R reduces disease progression in MRL/lpr mice. We previously reported that both IL-13and IL-17were produced in MRL/lpr mice, however little is known about these two cytokines in NZB/W F1and BXSB/Yaa mice. We have also reported that renal T cell infiltrates may consist of a mixture of Thl, Th2and Th17cells in lupus patients. As of present, the roles of individual Thl, Th2and Thl7cell subsets in SLE remain unclear.Laser microdissection (LMD) is a well-established method for isolating individual cells or subcellular structures from a heterogeneous cell population. It could be used to precisely harvest cells of interest from a tissue specimen in a rapid and practical manner. Cell-, DNA-, RNA-, and protein-based techniques have been used in combination with LMD to gather important information regarding the genome, transcriptome, and more recently, proteome of individual microdissected cells. Together with reverse transcription polymerase chain reaction (RT-PCR) techniques, LMD can be used to study genetic alterations, gene expression, and protein expression in defined cell populations from complex normal and diseased tissues.In the present study, we employed RT-PCR following LMD to analyze cytokine IL-10, IL-13, IL-17and IFN-y mRNA profiles as well as protein expression by immunohistochemistry to further define the role of T cells in the kidneys of murine lupus models, which determine the position of Thl, Th2and Th17cells in the pathogenesis of the murine model and provide the basis to find SLE new biological marker and therapeutic target.Methods1. Analysis of renal tissue and cellular characterization of murine lupus:Kidney samples of female MRL/lpr, female NZB/W F1mice (n=9,36-40weeks) and male BXSB/Yaa mice (n=10,20-30weeks) were fixed with10%formalin in0.01M phosphate buffer, pH7.2, and embedded in paraffin. Sections (5μm thick) were stained with hematoxylin and eosin (H&E) for histologic examination by light microscopy. Immunohistochemical staining of L3T4, Ly-2, B220and Thy-1was performed by avidin-biotin complex.2. Analysis of renal cytokine gene expression in murine lupus:Cells infiltrating the glomeruli and perivascular areas in MRL/lpr, NZB/W F1and BXSB/Yaa mice were captured by LMD. Samples were subjected to nested RT-PCR with primers specific for β-actin, T-cell receptor β chain (TCR-Cβ), IL-10, IL-13, IL-17, and IFN-y.Results 1. Histopathologic Examinations:MRL/lpr mice (10females) and NZB/W F1mice (9females) with developing glomerulonephritis and vasculitis, as well as BXSB/Yaa mice (10males) with developing glomerulonephritis were used. Unlike MRL/lpr and NZB/W F1, almost no infiltrating cells were detected at the perivascular areas of BXSB/Yaa mice.2. Immunohistologic Examinations:Thy-1, B220, CD4, and CD8staining were observed in glomeruli from all three strains, especially in the glomerular circumference of MRL/lpr mice. Thy-1, B220, CD4, and CD8staining were also observed in perivascular areas of MRL/lpr and NZB/W F1mice. Areas positive for Thy-1were larger than those positive for B220, CD4, and CD8in both the glomeruli and perivascular areas.3. Analyses of gene expression by LMD and nested RT-PCR:Single glomeruli and perivascular areas of similar sizes as the glomeruli were captured by LMD. Samples from ten MRL/lpr mice (292pooled samples positive for both β-actin-and TCR-C-by RT-PCR), nine NZB/W F1mice (315pooled samples positive for both β-actin-and TCR-C-by RT-PCR), and ten BXSB/Yaa mice (188pooled samples positive for both β-actin-and TCR-C-by RT-PCR) were used.T cells infiltrating the glomeruli and perivascular areas prominently produced IFN-y, IL-13, and IL-17in MRL/lpr, NZB/W F1, and BXSB/Yaa mice. However, the amount of IL-17expressing cells in the glomeruli of BXSB/Yaa mice was lower than that of MRL/lpr and NZB/W F1mice. In the glomerular lesions, the percentage of positive IFN-y samples was48.6%in T cells of the NZB/W F1mice, which is significantly lower than in the BXSB/Yaa mice (66.4%, P<0.05). IFN-expression of almost the same percentage (63.1%) was found in the MRL/lpr mice. The percentages of positive IL-13samples were68.0%,63.3%, and63.5%in MRL/lpr mice, NZB/W F1mice, and BXSB/Yaa mice, respectively. The percentages of positive IL-17samples were50.7%and60.8%in the MRL/lpr and NZB/W Fl mice, both of which were significantly greater than that from BXSB/Yaa mice (29.7%). The expression level of IL-10was not detectable in the glomeruli of all three mouse models. Similar to glomerular infiltrating T cells, the perivascular infiltrating T cells also expressed high levels of IFN-γ, IL-13, and IL-17in MRL/lpr and NZB/W F1mice. Additionally, IL-10expression was detected at the same strength in the perivascular infiltrates from these two strains. The percentages of positive IFN-γ and IL-13samples were higher than60%, with IFN-at66.9%and60.2%, and IL-13present at72.5%and61.6%in the MRL/lpr and NZB/W F1mice, respectively. The percentages of samples positive for IL-17were41.7%, and56.6%in MRL/lpr and NZB/W F1mice, respectively. The percentages samples positive for IL-10were64.4%in the MRL/lpr mice and49.7%in the NZB/W F1mice.Conclusion1. Female MRL/lpr and female NZB/W F1mice develop glomerulonephritis and vasculitis, male BXSB/Yaa mice develop only glomerulonephritis.2. T cells infiltrating of three lupus model mice were observed mainly in glomeruli and T cell of glomeruli produce IFN-γ, IL-13and IL-17. The perivascular infiltrating T cells in MRL/lpr and NZB/W F1mice also were observed and produce IFN-γ, IL-13, IL-17and IL-10.3. Cytokine balance in murine SLE is complex and cannot be attributed simply to the balance between Th1and Th2cells. Th17cells may play a critical role in disease pathology, possibly with greater contribution toward disease progression in MRL/lpr and NZB/W F1mice than in BXSB/Yaa mice. Furthermore, these findings lend support to the concept that different molecular mechanisms underlie glomerulonephritis as compared to vasculitis.