Pathogenic effects of maternal antinuclear antibodies during pregnancy in women with lupus

Lupus is an autoimmune disease that primarily affects young women of childbearing age. Fertility rates in lupus patients depend on various factors, including disease activity, nephritis, and the presence of antiphospholipid antibodies; however, after lupus patients become pregnant, different factors may affect the course of pregnancy, such as the production of autoantibodies, pre-existing renal disease, and eclampsia, among others. The placenta is a temporary hemochorial organ that prevents immunological conflict due to exposure to alloantigens at the maternal-fetal interface; placental regulatory T cells play a major role in maternal-fetal tolerance. Typically, significant amounts of maternal IgG class antibodies cross the placenta and enter the fetal circulation. This transition depends on the distribution of Fc receptors along the syncytiotrophoblast. The production of antinuclear antibodies (ANA) is a hallmark of lupus, and these autoantibodies can form immune complexes that are typically trapped in the placenta during gestation. However, the entry of ANA into the fetal circulation depends on the IgG-ANA concentration and the FcR placental density. Maternal antinuclear antibodies with anti-Ro or anti-La specificity might be pathogenic to the fetus if transfused by the placental pathway and could induce neonatal pathologies, such as neonatal lupus and congenital heart block. Here, we review the experimental and clinical data supporting a pathogenic role for maternal autoantibodies transmitted to the fetus

Apoptosis and cell proliferation: the paradox of salivary glands in sjögren’s disease

Este estudo avalia a apoptose e a proliferação nas glândulas salivares dos doentes com Síndroma de Sjögren primária. Métodos: A apoptose foi estudada por imunohistoquímica utilizando anticorpos monoclonais anti- -Fas, FasL e Caspase 3 e as características apoptóticas por TUNEL. Os estudos foram executados em vinte e quatro glândulas salivares minor de doentes com Síndroma de Sjögren primária e num igual número de controlos. A proliferação foi avaliada com anticorpos monoclonais anti-PCNA e anti-Ki67. Resultados:Todas as glândulas salivares dos doentes com Sjögren apresentavam moléculas apoptoticas no epitélio dos ductos salivares, e menos no tecido acinar, consequentemente a presença do caspase 3, Fas/FasL eram concordantes com a expressão da apoptose por TUNEL. Os marcadores de proliferação foram encontrados nas células inflamatórias presentes, mas não no epitélio ductal nem nos acinos. A expressão de marcadores de apoptose ou de proliferação nos tecidos das biopsias dos controlos foi escassa. Conclusão: Os dados actuais sugerem que as células do epitélio ductal e dos acinos das glândulas salivares dos doentes com doença de Sjögren têm aumento da apoptose. A proliferação foi observada principalmente no infiltrado celular linfóide. Em conjunto, estes eventos constituem um paradoxo biológico relacionado com o processo inflamatório das glândulas salivares na Síndrome de Sjögren.Aim: To assess apoptosis and proliferation in salivary glands of patients with primary Sjögren’s syndrome. Methods: Studies were performed in twenty four minor salivary glands from patients with primary Sjögren’s syndrome and an equal number of controls. Apoptosis was studied by immunohistochemistry using monoclonal antibodies anti-Fas, FasL and Caspase 3 and apoptotic features by TUNEL. Proliferation was assessed with monoclonal anti-PCNA and anti-Ki67 antibodies. Results: All salivary glands from Sjögren’s display apoptotic molecules along the epithelia of salivary ducts, and in a smaller amount in acinar tissue. The presence of Caspase 3, Fas/FasL was concordant with the expression of apoptosis by TUNEL. Proliferation markers were encountered in inflammatory emigrant cells, but not in ductal epithelia nor in acini. Control biopsies poorly expressed apoptotic or proliferation markers. Conclusion: Present data suggests that the ductal epithelial and acinar cells of salivary glands from Sjögren’s disease patients exhibit increased apoptosis. Proliferation was mainly observed in infiltrating lymphoid cells. Both events constitute a biological paradox related to the inflammatory process of salivary glands in Sjögren’s disease

Camptothecin induces the transit of fASl trimers to the cell surface in apoptotic heP-2 cells

Fas ligand (L) is a membrane protein from the tumor necrosis factor (TNF) family. It induces apoptosis upon contact with its Fas/CD95/APO1 receptor. Trimerization of FasL on the surface of effector cells is essential in the binding of the Fas trimer of the target cells. The receptor then recruits an adaptor and caspase-like proteins which lead apoptosis. This paper reports on the fate of FasL in HEp-2 cells committed to apoptosis by induction with campthotecin. Our main results demonstrated that in non-apoptotic cells, FasL aggregates in the cytoplasm forming trimers of 120 kDa. Apoptosis increases the trimeric FasL species, but also induces its dissociation into monomers of 35 kDa. In conclusion, camptothecin appears to perturb the Fas and FasL segregation in the cytoplasm by promoting the transit of FasL to the cell surface, thus fostering a process of autocrine or paracrine apoptosis. FasL is trimerized prior to Fas/FasL complex formation, and after apoptosis, FasL undergoes an intense turnover

shRNA targeting caspase-3 inhibits apoptosis and cell detachment induced by Pemphigus Vulgaris autoantibodies

Pemphigus is an organ-specific autoimmune disease that affects the skin and mucous membranes. It is induced by the deposition of pemphigus IgG autoantibodies, which mainly target Dsg1 and 3 and cause a loss of cell adhesion in a phenomenon known as acantholysis, and clinically is reflected as intraepidermal blistering. The present work assessed the effect of pemphigus vulgaris IgG (PV-IgG) on cell adhesion and caspase 3- dependent apoptosis in HaCaT cells. The expression of caspase-3 induced by PV-IgG was silenced in cells pre-treated with caspase 3-shRNA. PV-IgG induced cell detachment and apoptotic changes as demonstrated by the annexin-FITC assays. Treatment of cell cultures with normal IgG (control; N-IgG) did not have relevant effects on the aforementioned parameters. Then, the effect of PV-IgG on cells previously treated with shRNA was tested. The results demonstrated that shRNA reduced apoptotic features and the relative expression of caspase-3 measured by qRT-PCR, which showed a decrease of 96%. In conclusion shRNA prevented cell detachment and apoptosis of HaCaT cells induced by PV-IgG. The presented results further our understanding of the molecular pathophysiologic mechanisms involved in pemphigus diseases.Pemphigus is an organ-specific autoimmune disease that affects the skin and mucous membranes. It is induced by the deposition of pemphigus IgG autoantibodies, which mainly target Dsg1 and 3 and cause a loss of cell adhesion in a phenomenon known as acantholysis, and clinically is reflected as intraepidermal blistering. The present work assessed the effect of pemphigus vulgaris IgG (PV-IgG) on cell adhesion and caspase 3- dependent apoptosis in HaCaT cells. The expression of caspase-3 induced by PV-IgG was silenced in cells pre-treated with caspase 3-shRNA. PV-IgG induced cell detachment and apoptotic changes as demonstrated by the annexin-FITC assays. Treatment of cell cultures with normal IgG (control; N-IgG) did not have relevant effects on the aforementioned parameters. Then, the effect of PV-IgG on cells previously treated with shRNA was tested. The results demonstrated that shRNA reduced apoptotic features and the relative expression of caspase-3 measured by qRT-PCR, which showed a decrease of 96%. In conclusion shRNA prevented cell detachment and apoptosis of HaCaT cells induced by PV-IgG. The presented results further our understanding of the molecular pathophysiologic mechanisms involved in pemphigus diseases

Activation of Peptidylarginine Deiminase in the Salivary Glands of Balb/c Mice Drives the Citrullination of Ro and La Ribonucleoproteins

The goal of the present study was to determine whether peptidylarginine deiminase PAD2 and PAD4 enzymes are present in Balb/c mouse salivary glands and whether they are able to citrullinate Ro and La ribonucleoproteins. Salivary glands from Balb/c mice were cultured in DMEM and supplemented with one of the following stimulants: ATP, LPS, TNF, IFNγ, or IL-6. A control group without stimulant was also evaluated. PAD2, PAD4, citrullinated peptides, Ro60, and La were detected by immunohistochemistry and double immunofluorescence. PAD2 and PAD4 mRNAs and protein expression were detected by qPCR and Western blot analysis. PAD activity was assessed using an antigen capture enzyme-linked immunosorbent assay. LPS, ATP, and TNF triggered PAD2 and PAD4 expression; in contrast, no expression was detected in the control group (p < 0 001). PAD transcription slightly increased in response to stimulation. Additionally, PAD2/4 activity modified the arginine residues of a reporter protein (fibrinogen) in vitro. PADs citrullinated Ro60 and La ribonucleoproteins in vivo. Molecular stimulants induced apoptosis in ductal cells and the externalization of Ro60 and La ribonucleoproteins onto apoptotic membranes. PAD enzymes citrullinate Ro and La ribonucleoproteins, and this experimental approach may facilitate our understanding of the role of posttranslational modifications in the pathophysiology of Sjögren’s syndrome

Soluble Fas and the −670 Polymorphism of Fas in Lupus Nephritis

This study was performed to clarify the role of soluble Fas (sFas) in lupus nephritis (LN) and establish a potential relationship between LN and the −670 polymorphism of Fas in 67 patients with systemic lupus erythematosus (SLE), including a subset of 24 LN patients with proteinuria. Additionally, a group of 54 healthy subjects (HS) was included. The allelic frequency of the −670 polymorphism of Fas was determined using PCR-RFLP analysis, and sFas levels were assessed by ELISA. Additionally, the WT-1 protein level in urine was measured. The Fas receptor was determined in biopsies by immunohistochemistry (IHC) and in situ hybridization (FISH) and apoptotic features by TUNEL. Results. The −670 Fas polymorphism showed that the G allele was associated with increased SLE susceptibility, with an odds ratio (OR) of 1.86. The sFas was significantly higher in LN patients with the G/G genotype, and this subgroup exhibited correlations between the sFas level and proteinuria and increased urinary WT-1 levels. LN group shows increased expression of Fas and apoptotic features. In conclusion, our results indicate that the G allele of the −670 polymorphism of Fas is associated with genetic susceptibility in SLE patients with elevated levels of sFas in LN with proteinuria

An Animal Model Using Metallic Ions to Produce Autoimmune Nephritis

Autoimmune nephritis triggered by metallic ions was assessed in a Long-Evans rat model. The parameters evaluated included antinuclear autoantibody production, kidney damage mediated by immune complexes detected by immunofluorescence, and renal function tested by retention of nitrogen waste products and proteinuria. To accomplish our goal, the animals were treated with the following ionic metals: HgCl2, CuSO4, AgNO3, and Pb(NO3)2. A group without ionic metals was used as the control. The results of the present investigation demonstrated that metallic ions triggered antinuclear antibody production in 60% of animals, some of them with anti-DNA specificity. Furthermore, all animals treated with heavy metals developed toxic glomerulonephritis with immune complex deposition along the mesangium and membranes. These phenomena were accompanied by proteinuria and increased concentrations of urea. Based on these results, we conclude that metallic ions may induce experimental autoimmune nephritis

Apoptosis in chronic cutaneous lupus erythematosus, discoid lupus, and lupus profundus

: Introduction: Lupus erythematosus is a multisystemic disease that is characterized by autoantibody production and immune complex deposition in such tissues as the mucosa, joints, the central nervous system, and skin. Cutaneous lupus erythematosus is categorized as acute, subacute, and chronic. Chronic cutaneous lupus erythematosus comprises discoid lupus erythematosus (DLE) and lupus profundus (LP). Aim: To analyze the expression of proapoptotic molecules in patients with lupus erythematosus discoid and lupus profundus. Material and methods: Descriptive study, the study groups comprised 10 cases of LP and 10 cases of DLE, and a control. Skin samples of cases and controls were processed for immunohistochemistry and by TUNEL technique. The database and statistical analysis was performed (statistical test X2) SPSS (Chicago, IL, USA). Results: Apoptotic features were broadly distributed along the skin biopsies in epidermal keratinocytes as well as at dermis. By immunohistochemistry the expression of Fas receptor and Fas-L was higher in the skin of lupus patients compared with controls. We also noted differences in Fas-L, -Fas, and -Bax proteins expression intensity in discoid lupus erythematosus patients in the epidermis, and hair follicles. Conclusions: Fas and Fas-L are expressed similarly in LP and DLE

Apoptosis and redistribution of the Ro autoantigen in Balb/c mouse like in subacute cutaneous lupus erythematosus

In subacute cutaneous lupus eryhematosus (SCLE) the cutaneous antigens constitute the main source of Ro and La autoantigens. The aim of this investigation was to demonstrate if UV light increases the availability of Ro autoantigen in the skin, also the blocking effect of Ac-DEVD-CMK a caspase inhibitor was assessed. For this purpose newborn Balb/c mice were UVB irradiated (5–30 mJ/cm2) equivalent to a moderate to severe sunburn. Animals were injected with monoclonal anti-Roantibodies from SCLE patients. Apoptosis was also induced by anti-Fas antibody injection. Skin samples were examined by direct immunofluoresence, by TUNEL, and the expression of caspase 3 by RT-PCR. Major findings of present studies were: 1. UVB irradiation and anti-Fas induced apoptosis of keratinocytes. 2. Apoptosis redistribute the Ro antigen on cell surface and is better triggered by Ro antibody. 3. The caspase 3 inhibitor Ac-DEVD-CMK decreases the availability of Ro autoantigen in epidermis and prevents deposition of anti-Ro. In conclusion, the caspase pathway would be blocked to avoid anti-Ro deposition along skin; this finding would be a prospect in the treatment of SCLE patients