RESUMO
Sjögren's Syndrome (SjS) is a chronic, systemic autoimmune disease causing xerostomia, xerophthalmia, and systemic symptoms. The principal pathological finding in SjS is the accumulation of lymphocytes in exocrine glandular tissue and elsewhere, leading to secretory dysfunction and other abnormalities. A rational therapeutic approach might be to interfere with lymphocyte migration to the periphery from central lymphoid tissues. We thus examined in an animal model of SjS the effects of Fingolimod (FTY720, Gilenya™), which interferes with migration of lymphocytes to peripheral sites. Fingolimod induces sequestration of lymphocytes in lymphoid organs by altering lymphocyte expression of sphingosine-1-phosphate receptors. In the C57Bl/6. NOD.Aec1Aec2 (AEC) model of SjS, Fingolimod reduced circulating T and B cell numbers. Treatment of AEC mice with Fingolimod increased salivary output and decreased the size of salivary gland infiltrates. Oral Fingolimod thus merits further consideration in the management of SjS in humans.
Assuntos
Cloridrato de Fingolimode/administração & dosagem , Saliva/metabolismo , Glândulas Salivares/efeitos dos fármacos , Síndrome de Sjogren/tratamento farmacológico , Administração Oral , Animais , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Glândulas Salivares/imunologia , Glândulas Salivares/metabolismo , Glândulas Salivares/patologia , Síndrome de Sjogren/genética , Síndrome de Sjogren/imunologia , Síndrome de Sjogren/patologiaRESUMO
Autoantibodies in systemic autoimmunity are directed against only ~5% of the proteome. The purpose of this study was to assess whether the properties of assemblages (also known as Membraneless Organelles and Biological Condensates) and their protein constituents partly explain the immunological selectivity of autoimmunity. Assemblages arise from phase separation of their protein components, akin to partitioning of oil droplets in water. We obtained from a prediction algorithm (Vernon et al., elife7, 2018) the propensity scores (PScores), i.e., likelihood, for phase separation of autoantigens and non-autoantigens. We then compared autoantigens with the highest PScores to identify shared structural properties. The mean PScores for autoantigens (n = 1050) and the entire human proteome of non-autoantigens (n = 17,532) were 1.46 and 1.09 (p = 1.2E-08). To varying extents, the 25 autoantigens with the highest phase separation propensities shared additional features such as compositional bias, repeated domains, coiled coil regions, nucleic acid binding, and disorder. Most of these properties were present with greater frequencies than their frequencies in the non-autoantigens. We conclude that, on average, autoantigens have a higher predisposition to undergo phase separation, thus, they are more likely to exist in assemblages compared with the average non-autoantigen. We suggest that assemblage formation and the greater than average presence of certain structural features are key factors in selection of a portion of the autoimmune repertoire. Other properties of assemblage proteins, such as high concentration and tendency to form novel complexes with other proteins, may partially explain why assemblages are potent sources of autoantigens.
Assuntos
Autoanticorpos/metabolismo , Autoantígenos/metabolismo , Doenças Autoimunes/imunologia , Biologia Computacional/métodos , Príons/metabolismo , Autoanticorpos/genética , Autoantígenos/genética , Autoimunidade , Humanos , Transição de Fase , Conformação Proteica , Multimerização ProteicaRESUMO
Systemic lupus erythematosus (SLE) is a complex multisystem autoimmune disease, characterized by a spectrum of autoantibodies that target multiple cellular components. Glomerulonephritis is a major cause of morbidity in patients with SLE. Little is known about the pathogenesis of SLE renal damage and compromised renal function. Activation of both Stat1 and Stat3 has been reported in lupus and lupus nephritis. The reciprocal activation of these two transcription factors may have a major impact on renal inflammation. To study the role of Stat1 in a lupus model, we induced lupus-like chronic graft-versus-host disease (cGVHD) in Stat1-knockout (KO) and wild-type (WT) mice by i.p. injection of class II-disparate bm12 splenocytes. WT recipients of these alloreactive cells developed anti-dsDNA autoantibodies starting at week 2 as expected, with a decline after week 4. In contrast, Stat1-KO hosts exhibited a prolonged and significant increase of anti-dsDNA autoantibody responses compared with WT mice (week 4 to week 8). Increased autoantibody titers were accompanied by increased proteinuria and mortality in the cGVHD host mice lacking Stat1. Further analysis revealed expression and activation of Stat3 in the glomeruli of Stat1-KO host mice but not WT mice with cGVHD. Glomerular Stat3 activity in the Stat1-KO mice was associated with increased IL-6 and IFN-γ secretion and macrophage infiltration. Interactions between Stat1 and Stat3 thus appear to be crucial in determining the severity of lupus-like disease in the cGVHD model.
Assuntos
Doença Enxerto-Hospedeiro/imunologia , Nefrite Lúpica/etiologia , Fator de Transcrição STAT1/fisiologia , Fator de Transcrição STAT3/fisiologia , Animais , Autoanticorpos/biossíntese , Linfócitos B/imunologia , Linfócitos B/metabolismo , Doença Crônica , Interferon gama/fisiologia , Interleucina-6/metabolismo , Nefrite Lúpica/imunologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Chronic inflammation is increased in patients with chronic kidney disease (CKD) and contributes to cardiovascular morbidity and mortality. Specific immune mechanisms and pathways that drive and maintain chronic inflammation in CKD are not well described. The TAM ligands (Gas6 and protein S) and receptors (Axl and Mer) have been recently recognized as playing a prominent role in immune regulation. The receptors exist in both soluble and cell-bound forms; the soluble receptors (sAxl and sMer) are believed to compete with the bound receptors and thus inhibit their function. In this study, we determined the expression of cell-bound and soluble TAM proteins in patients with CKD. CKD patients had significantly lower expression of Mer in monocytes, yet increased expression of soluble TAM receptors sAxl and sMer in plasma compared to controls. The metalloproteinase ADAM 17, responsible for cleavage of Mer to its soluble form, was increased in patient monocytes. Elevated levels of soluble TAM receptors were more evident in patients with progressive renal failure. These observations suggest that functional deficiency of TAM receptor-mediated regulation of inflammation may contribute to chronic inflammation in patients with CKD.
Assuntos
Regulação da Expressão Gênica/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteína S/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Insuficiência Renal Crônica/metabolismo , Humanos , Inflamação , Peptídeos e Proteínas de Sinalização Intercelular/genética , Monócitos/metabolismo , Proteína S/genética , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Insuficiência Renal Crônica/imunologia , c-Mer Tirosina Quinase , Receptor Tirosina Quinase AxlRESUMO
Exposure of human monocytes/macrophages to anti-inflammatory agents, such as IL-10 or glucocorticoids, can lead to two separate fates: either Fas/CD95-mediated apoptosis or differentiation into regulatory and efferocytic M2c (CD14(bright)CD16(+)CD163(+)Mer tyrosine kinase(+)) macrophages. We found that the prevalent effect depends on the type of Th cytokine environment and on the stage of monocyte-to-macrophage differentiation. In particular, the presence of IFN-γ (Th1 inflammation) or the prolonged exposure to IL-4 (chronic Th2 inflammation) promotes apoptosis of monocytes/macrophages and causes resistance to M2c differentiation, thus provoking impaired clearance of apoptotic neutrophils, uncontrolled accumulation of apoptotic cells, and persistent inflammation. In contrast, the presence of IL-17 (Th17 environment) prevents monocyte/macrophage apoptosis and elicits intense M2c differentiation, thus ensuring efficient clearance of apoptotic neutrophils and restoration of anti-inflammatory conditions. Additionally, the Th environment affects the expression of two distinct Mer tyrosine kinase isoforms: IL-4 downregulates the membrane isoform but induces an intracellular and Gas6-dependent isoform, whereas IFN-γ downregulates both and IL-17 upregulates both. Our data support an unexpected role for IL-17 in orchestrating resolution of innate inflammation, whereas IFN-γ and IL-4 emerge as major determinants of IL-10 and glucocorticoid resistance.
Assuntos
Diferenciação Celular , Interferon gama/imunologia , Interleucina-17/metabolismo , Macrófagos/citologia , Macrófagos/imunologia , Neutrófilos/imunologia , Fagocitose , Anti-Inflamatórios/farmacologia , Apoptose , Células Cultivadas , Glucocorticoides/farmacologia , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Interleucina-10/metabolismo , Interleucina-10/farmacologia , Interleucina-17/imunologia , Interleucina-17/farmacologia , Interleucina-4/imunologia , Macrófagos/metabolismo , Monócitos/metabolismo , Neutrófilos/citologia , Neutrófilos/metabolismo , Isoformas de Proteínas , Receptor fas/metabolismoRESUMO
OBJECTIVES: Hyperplasia of synovial fibroblasts, infiltration with lymphocytes and tissue hypoxia are major characteristics of rheumatoid arthritis (RA). Extensive data support a key role for toll-like receptors (TLRs) in RA. Little is known regarding the impact of hypoxia on TLR-induced inflammation in RA. The aim of this study was to reveal the effects of hypoxia and its regulator, hypoxia-inducible factor-1α (HIF-1α), on the inflammatory response of RA synovial fibroblasts (RASF) to TLR ligands. METHODS: Hypoxia was induced in RASF by incubation with Na2S2O4. TLR3 ligand polyIC, TLR2 ligand peptidoglycan, TLR4 ligand LPS and TLR9 ligand CpG were used to stimulate the cells. Effects of hypoxia on TLR-induced inflammatory mediators were determined by RT-PCR, qPCR and ELISA. Overexpression of HIF-1α as well as knocking-down its expression was used to reveal its fundamental role. RASF-induced inflammatory T cell expansion was determined by flow cytometry analysis of T helper (Th)1/Th17 cells, and IFN-γ/IL-17 production by ELISA after RASF/T cell coculture. RESULTS: Hypoxia potentiated the expression of inflammatory cytokines, metalloproteinases and VEGF in RASF stimulated by different TLR ligands, especially polyIC, a synthetic mimic of dsRNA from viruses or apoptotic cells. HIF-1α played a fundamental role in this synergy. Moreover, HIF-1α overexpression enhanced RASF-mediated expansion of inflammatory Th1 and Th17 cells, leading to proinflammatory IFN-γ and IL-17 production. CONCLUSIONS: Our findings suggest that hypoxia and HIF-1α may function in conjunction with TLR-stimulated innate immune responses to drive inflammation in RA. This pathway may serve as a therapeutic target for the disease.
Assuntos
Artrite Reumatoide/metabolismo , Fibroblastos/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Inflamação/metabolismo , Transdução de Sinais , Receptores Toll-Like/metabolismo , Artrite Reumatoide/imunologia , Hipóxia Celular , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Imunidade Inata/imunologia , Inflamação/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/fisiologia , Membrana Sinovial/metabolismoRESUMO
The Mertk receptor tyrosine kinase facilitates macrophage and DC apoptotic-cell clearance and regulates immune tolerance. Mertk may also contribute to B-cell activation, because Mertk-KO mice fail to develop autoantibodies when allo-activated by T cells. We investigated this possibility with a well-characterized model in which injection of mice with goat anti-IgD antibody causes membrane IgD cross-linking that induces T-independent B cell activation and antigen presentation to T cells. Goat anti-mouse IgD antibody-injected C57BL/6 Mertk-KO mice had normal initial B cell activation and proliferation, but significantly lower T cell activation and proliferation, as well as lower IgE and IgG anti-goat IgG responses, as compared to C57BL/6 WT controls. B cell antigen processing, analyzed by evaluating B cell fluorescence following injection of monoclonal anti-IgD antibody labeled with biotin or FITC, was comparable between Mertk-KO mice and WT mice. IgD Ab primed B cells from Mertk-KO mice exhibited significantly lower ability in activating memory T cells isolated from WT mice injected with the same antigen 10 days before. These observations suggest that Mertk expression is required for optimal B-cell antigen presentation, which is, in turn, required in this model for optimal T cell activation and subsequent T cell-dependent B cell differentiation.
Assuntos
Linfócitos B/imunologia , Comunicação Celular/imunologia , Imunoglobulina D/imunologia , Proteínas Proto-Oncogênicas/imunologia , Receptores Proteína Tirosina Quinases/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Linfócitos T/imunologia , Animais , Linfócitos B/citologia , Comunicação Celular/genética , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Regulação Enzimológica da Expressão Gênica/genética , Regulação Enzimológica da Expressão Gênica/imunologia , Imunoglobulina D/genética , Capeamento Imunológico/genética , Capeamento Imunológico/imunologia , Memória Imunológica/genética , Ativação Linfocitária/genética , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Receptores de Antígenos de Linfócitos B/genética , Linfócitos T/citologia , c-Mer Tirosina QuinaseRESUMO
Mer tyrosine kinase (MerTK) is a major macrophage apoptotic cell (AC) receptor. Its functional impairment promotes autoimmunity and atherosclerosis, whereas overexpression correlates with poor prognosis in cancer. However, little is known about mechanisms regulating MerTK expression in humans. We found that MerTK expression is heterogenous among macrophage subsets, being mostly restricted to anti-inflammatory M2c (CD14(+)CD16(+)CD163(+)CD204(+)CD206(+)CD209(-)) cells, differentiated by M-CSF or glucocorticoids. Small numbers of MerTK(+) "M2c-like" cells are also detectable among circulating CD14(bright)CD16(+) monocytes. MerTK expression levels adapt to changing immunologic environment, being suppressed in M1 and M2a macrophages and in dendritic cells. Remarkably, although glucocorticoid-induced differentiation is IL-10 independent, M-CSF-driven M2c polarization and related MerTK upregulation require IL-10. However, neither IL-10 alone nor TGF-ß are sufficient to fully differentiate M2c (CD16(+)CD163(+)MerTK(+)) macrophages. M-CSF and IL-10, both released by T lymphocytes, may thus be required together to promote regulatory T cell-mediated induction of anti-inflammatory monocytes-macrophages. MerTK enables M2c macrophages to clear early ACs more efficiently than other macrophage subsets, and it mediates AC clearance by CD14(bright)CD16(+) monocytes. Moreover, M2c cells release Gas6, which in turn amplifies IL-10 secretion via MerTK. IL-10-dependent induction of the Gas6/MerTK pathway may, therefore, constitute a positive loop for M2c macrophage homeostasis and a critical checkpoint for maintenance of anti-inflammatory conditions. Our findings give new insight into human macrophage polarization and favor a central role for MerTK in regulation of macrophage functions. Eliciting M2c polarization can have therapeutic utility for diseases such as lupus, in which a defective AC clearance contributes to initiate and perpetuate the pathological process.
Assuntos
Apoptose/imunologia , Movimento Celular/imunologia , Polaridade Celular/imunologia , Macrófagos/enzimologia , Macrófagos/imunologia , Proteínas Proto-Oncogênicas/biossíntese , Receptores Proteína Tirosina Quinases/biossíntese , Apoptose/genética , Comunicação Autócrina/genética , Comunicação Autócrina/imunologia , Diferenciação Celular/imunologia , Movimento Celular/genética , Polaridade Celular/genética , Células Cultivadas , Células Dendríticas/citologia , Células Dendríticas/enzimologia , Células Dendríticas/imunologia , Humanos , Imunofenotipagem , Macrófagos/citologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/fisiologia , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/fisiologia , Ativação Transcricional/imunologia , c-Mer Tirosina QuinaseRESUMO
B cells are subjected to selection at multiple checkpoints during their development. The selection of Ab H chains is difficult to study because of the large diversity of the CDR3. To study the selection of individual Ab H chain V region genes (V(H)), we performed CDR3 spectratyping of â¼ 75-300 rearrangements per individual V(H) in C57BL6/J mice. We measured the fraction of rearrangements that were in-frame in B cell DNA. We demonstrate that individual V(H)s have different fractions of in-frame rearrangements (IF fractions) ranging from 10 to 90% and that these IF fractions are reproducible in different mice. For most V(H)s, the IF fraction in pro-B cells approximated 33% and then shifted to the nearly final (mature) B cell value by the cycling pre-B cell stage. The frequency of high in-frame (IF) V(H) usage increased in cycling pre-B cells compared with that in pro-B cells, whereas this did not occur for low IF V(H)s. The IF fraction did not shift as much in BCR-expressing B cells and was minimally affected by L chain usage for most V(H). High IF clan II/III V(H)s share more positively charged CDR2 sequences, whereas high IF clan I J558 CDR2 sequences are diverse. These data indicate that individual V(H)s are subjected to differential selection, that V(H) IF fraction is mainly established through pre-BCR-mediated selection, that it may operate differently in clan I versus II/III V(H)s, and that it has a lasting influence on the Ab repertoire.
Assuntos
Regiões Determinantes de Complementaridade/metabolismo , DNA/metabolismo , Células Precursoras de Linfócitos B/metabolismo , Hipermutação Somática de Imunoglobulina/fisiologia , Animais , Regiões Determinantes de Complementaridade/genética , Regiões Determinantes de Complementaridade/imunologia , DNA/genética , DNA/imunologia , Regulação da Expressão Gênica/fisiologia , Camundongos , Células Precursoras de Linfócitos B/citologia , Células Precursoras de Linfócitos B/imunologia , Proteínas Proto-Oncogênicas c-bcr/biossíntese , Proteínas Proto-Oncogênicas c-bcr/genética , Proteínas Proto-Oncogênicas c-bcr/imunologiaRESUMO
Tuberous sclerosis is an autosomal dominant disorder characterized by involvement of skin, nervous system, kidneys, and lungs. It results from mutations in 1 of 2 genes: TSC1 (encoding hamartin) or TSC2 (encoding tuberin), leading to dysregulation and activation of the mammalian target of rapamycin (mTOR) pathway. Constitutive activation of mTOR signaling has recently been reported in systemic lupus erythematosus (SLE), and inhibition of this pathway may benefit patients with SLE nephritis. We report a case of a young woman with tuberous sclerosis who developed fulminant SLE, with lower extremity edema, massive proteinuria, and class IV lupus glomerulonephritis. She died despite treatment with high-dose steroids, plasmapheresis, and cyclophosphamide. Although there are no prior reports of coexistence of these 2 rare diseases, this case is of considerable interest because of the possibility that activation of mTOR by the TSC mutations may have led to activation of the immune system and the development of unusually severe SLE.
Assuntos
Lúpus Eritematoso Sistêmico/diagnóstico , Lúpus Eritematoso Sistêmico/epidemiologia , Esclerose Tuberosa/diagnóstico , Esclerose Tuberosa/epidemiologia , Adulto , Comorbidade , Ciclofosfamida/uso terapêutico , Evolução Fatal , Feminino , Humanos , Lúpus Eritematoso Sistêmico/terapia , Plasmaferese , Esteroides/uso terapêutico , Serina-Treonina Quinases TOR/fisiologia , Esclerose Tuberosa/terapiaRESUMO
PURPOSE: Primary Sjögren's syndrome (SjS) is an autoimmune disorder characterized by lymphocytic infiltration of the salivary and lacrimal glands, B-cell clonal expansions and an increased risk of lymphoma. In order to understand the role of B cells in this disorder, the antibody repertoire and B-cell maturation were studied in a mouse model of SjS called B6.Aec1/2. METHODS: B6.Aec1/2 serum was analyzed for antibodies by ELISA and immunoprecipitation, B-cell development by flow cytometry, and antibody gene rearrangements by CDR3 spectratyping and quantitative PCR. In order to test the functional consequences of the observed defects, B6.Aec1/2 mice were crossed with anti-dsDNA antibody heavy chain knock-in mice (B6.56R). RESULTS: B6.Aec1/2 mice exhibit B-cell clonal expansions, have altered serum immunoglobulin levels and spontaneously produce multireactive autoantibodies. B6.Aec1/2 mice also have decreased numbers of bone marrow pre-B cells and decreased frequencies of kappa light chain gene deletion. These findings suggest that B6.Aec1/2 mice have a defective early B-cell tolerance checkpoint. B6.56R.Aec1/2 mice unexpectedly had lower anti-dsDNA antibody levels than B6.56R mice and less salivary gland infiltration than B6.Aec1/2 mice. CONCLUSIONS: These data suggest that the early tolerance checkpoint defect in B6.Aec1/2 mice is not sufficient to promulgate disease in mice with pre-formed autoantibodies, such as B6.56R. Rather, B6.Aec1/2 mice may require a diverse B-cell repertoire for efficient T-B-cell collaboration and disease propagation. These findings imply that therapies aimed at reducing B-cell diversity or T-B interactions may be helpful in treating SjS.
Assuntos
Linfócitos B/imunologia , Modelos Animais de Doenças , Tolerância Imunológica , Síndrome de Sjogren/imunologia , Animais , Autoanticorpos/sangue , Linfócitos B/citologia , Diferenciação Celular , Cromatina/imunologia , DNA/imunologia , Feminino , Imunoglobulinas/sangue , Imunoglobulinas/imunologia , Camundongos , Camundongos TransgênicosRESUMO
Sjogren's syndrome (SS) is characterized by infiltration of exocrine glands with T and B lymphocytes, leading to glandular dysfunction and frequently accompanied by hypergammaglobulinemia and autoantibodies. The role of T cells, which predominate in the lesions, has attracted much interest. CD4 T cells seem to be responding to autoantigens on apoptotic cells, such as the Ro and La antigens, or to the cytoskeletal antigen α-fodrin. Physical injury to ocular surfaces may also lead to T cell mediated responses to self antigens and perpetuate disease. Within the salivary glands, T cell responsiveness is further promoted by the special capacity of salivary epithelial tissue to provide costimulation and enhanced antigen presentation. Cytokines are key mediators of the T cell contribution to pathology, with roles attributed both to Th1 and Th2 cells. Recently, striking data implicate Th17 cells in the stimulation of B cells, and a role for the related cytokine IL-21 produced by follicular T helper cells is now appreciated. Dysfunction of T regulatory cells has been shown to have a role in the exuberant production of cytokines by Th17 cells. Beyond their role in provoking B cell hyperactivity and immunoglobulin secretion, T cells are directly involved in destruction of glands through Fas and perforin-mediated cytotoxicity. Animal models of SS have confirmed the role of T cell derived cytokines in disease and support a role for effector-memory cells in pathogenesis. Further elucidation of the role of T cells will open avenues for better treatment of SS, whose current management is still mainly supportive.
Assuntos
Autoanticorpos/imunologia , Linfócitos B/imunologia , Glândulas Salivares/imunologia , Síndrome de Sjogren/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Apoptose/imunologia , Autoanticorpos/biossíntese , Autoantígenos/imunologia , Linfócitos B/metabolismo , Linfócitos B/patologia , Comunicação Celular/imunologia , Humanos , Interleucinas/biossíntese , Interleucinas/imunologia , Ativação Linfocitária , Camundongos , Perforina/genética , Perforina/imunologia , Glândulas Salivares/metabolismo , Glândulas Salivares/patologia , Síndrome de Sjogren/metabolismo , Síndrome de Sjogren/patologia , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/patologia , Receptor fas/genética , Receptor fas/imunologiaRESUMO
Activation and migration of marginal zone B (MZB) cells into follicular (FO) regions of the spleen has been proposed as one of the mechanisms that regulate the development of autoreactive B cells. The mer receptor tyrosine kinase (Mertk) mediates apoptotic cell clearance and regulates activation and cytokine secretion. In the well-studied class II chronic GVH model of bm12 cells into B6 hosts, we observed that Mertk deficient B6 mice did not generate autoantibodies in response to this allogeneic stimulus. We posited that Mertk is important in MHC-II-mediated B cell signaling. In the present study, we show that B cells from Mertk(-/-) mice but not WT B6 mice exhibited decreased calcium mobilization and tyrosine phosphorylation when stimulated by MHC-II cross-linking. The finding that Mertk was important for class II signaling in B cells was further supported by the preponderance of a-allotype autoantibodies in cGVH in RAG-KO mice reconstituted with a mixture of bone marrow from Mertk(-/-) mice (b-allotype) and C20 mice (a-allotype). MZB cells from Mertk(-/-) mice were unable to down regulate surface CD1d expression and subsequent inclusion in the MZ, associated with significantly lower germinal center responses compared to MZB cells from WT. Moreover, Mertk(-/-) mice treated with an anti-CD1d down regulating antibody responded significantly to bm12 cells, while no response was observed in Mertk(-/-) mice treated with control antibodies. Taken together, these findings extend the role of Mertk to include CD1d down regulation on MZB cells, a potential mechanism limiting B cell activation in cGVH.
Assuntos
Antígenos CD1d/imunologia , Linfócitos B/imunologia , Centro Germinativo/imunologia , Doença Enxerto-Hospedeiro/imunologia , Proteínas Proto-Oncogênicas/imunologia , Receptores Proteína Tirosina Quinases/imunologia , Animais , Antígenos CD1d/genética , Autoanticorpos/biossíntese , Autoanticorpos/imunologia , Linfócitos B/patologia , Cálcio/imunologia , Cálcio/metabolismo , Doença Crônica , Expressão Gênica/imunologia , Centro Germinativo/patologia , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/patologia , Humanos , Alótipos de Imunoglobulina/genética , Alótipos de Imunoglobulina/imunologia , Ativação Linfocitária , Camundongos , Camundongos Knockout , Fosforilação , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/deficiência , Receptores Proteína Tirosina Quinases/genética , Transdução de Sinais , Baço/imunologia , Baço/patologia , c-Mer Tirosina QuinaseRESUMO
BACKGROUND: The TAM receptors (tyro3, axl and mer) and their ligands (vitamin K-dependent proteins-Gas6 and Protein S) are crucial modulators of inflammation, which may be relevant in chronic kidney disease (CKD). Gas6 and axl have multiple roles in mediating vascular atherosclerosis and injury, thrombosis and inflammation, yet nothing is known about the Gas6-axl pathway in humans with CKD. Given the prevalence of chronic inflammation and vascular disease in this population, we measured TAM ligands in patients with various levels of renal function. METHODS: Gas6 and protein S were quantified in the plasma by ELISA in three patient groups: end-stage renal disease on chronic hemodialysis (HD), CKD and normal controls. RESULTS: Significantly increased levels of Gas6 and protein S were found in CKD patients compared with normal controls (P < 0.01 and <0.001, respectively). In HD patients, Gas6 levels were elevated compared with controls (P < 0.001) and positively associated with low albumin (r = 0.33; P = 0.01), dialysis vintage (r = 0.36; P = 0.008) and IV iron administration (r = 0.33; P = 0.01). The levels of Gas6 rose with CKD stage and were inversely associated with estimated GFR (P < 0.0001). CONCLUSIONS: Dysregulation of circulating Gas6 is associated with renal disease and inversely proportional to renal function. Low albumin and higher IV iron administration were associated with higher Gas6 levels, suggesting a possible connection between inflammation and oxidative stress mediated by iron. Protein S levels were also elevated in CKD patients, but the relevance of this finding needs to be further investigated.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/sangue , Falência Renal Crônica/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Precursores de Proteínas/sangue , Proteína S/análise , Protrombina , Diálise RenalRESUMO
Germinal centers (GCs) are specialized microenvironments that generate high-affinity Ab-forming cells (AFCs) and memory B cells. Many B cells undergo apoptosis during B cell clonal selection in GCs. Although the factors that regulate the AFC and GC responses are not precisely understood, it is widely believed that dysregulated AFCs and GCs contribute to autoimmunity. The Mer receptor tyrosine kinase (Mer) facilitates macrophage clearance of apoptotic cells. The Tyro-3, Axl, and Mer receptors, including Mer, suppress TLRs and cytokine-mediated inflammatory responses. We report in this study that tingible body macrophages (TBMφs) in GCs express Mer. Compared to C57BL/6 (B6) controls, Mer-deficient (Mer(-/-)) mice had significantly higher AFC, GC, and Th1-skewed IgG2 Ab (especially IgG2c) responses against the T cell-dependent Ag (4-hydroxy-3-nitrophenyl) acetyl-chicken γ globulin. Mer(-/-) mice had a significantly higher percentage of GC B cells on days 9, 14, and 21 postimmunization compared with B6 controls. Significantly increased numbers of apoptotic cells accumulated in Mer(-/-) GCs than in B6 GCs, whereas the number of TBMφs remained similar in both strains. Our data are the first, to our knowledge, to demonstrate a critical role for Mer in GC apoptotic cell clearance by TBMφs and have interesting implications for Mer in the regulation of B cell tolerance operative in the AFC and GC pathways.
Assuntos
Células Produtoras de Anticorpos/imunologia , Apoptose/imunologia , Macrófagos/imunologia , Proteínas Proto-Oncogênicas/imunologia , Receptores Proteína Tirosina Quinases/imunologia , Animais , Formação de Anticorpos/imunologia , Células Produtoras de Anticorpos/metabolismo , Separação Celular , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Centro Germinativo/citologia , Centro Germinativo/imunologia , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Baço/citologia , Baço/imunologia , c-Mer Tirosina QuinaseRESUMO
Studies in autoantibody transgenic mice have demonstrated receptor editing rearrangements at Ab H and L chain loci. However, the physiologic role of H chain editing (V(H) replacement and rearrangement on the second allele) has been called into question. It is unclear if additional rounds of H chain rearrangement are driven by BCR specificity. In this study, we analyze the manner in which B cells undergo additional H chain rearrangements in an anti-DNA H chain knock-in mouse, B6.56R. We find that rearrangements in 56R(+) B cells tend to involve the D gene locus on both alleles and the most J(H)-proximal V(H) gene segments on the endogenous allele. As a result, some B cells exhibit V(D)J rearrangements on both H chain alleles, yet allelic exclusion is tightly maintained in mature 56R B cells. As B cells mature, a higher proportion expresses the nontransgenic H chain allele. Rearrangements on both H chain alleles exhibit junctional diversity consistent with TdT-mediated N-addition, and TdT RNA is expressed exclusively at the pro-B cell stage in B6.56R. Collectively, these findings favor a single, early window of H chain rearrangement in B6.56R that precedes the expression of a functional BCR. B cells that happen to successfully rearrange another H chain may be favored in the periphery.
Assuntos
Rearranjo Gênico de Cadeia Pesada de Linfócito B/genética , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/genética , Imunoglobulina M/química , Imunoglobulina M/genética , Animais , Anticorpos Antinucleares/biossíntese , Anticorpos Antinucleares/química , Anticorpos Antinucleares/genética , Subpopulações de Linfócitos B/citologia , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Técnicas de Introdução de Genes/métodos , Imunoglobulina D/biossíntese , Imunoglobulina D/química , Imunoglobulina D/genética , Cadeias Pesadas de Imunoglobulinas/biossíntese , Imunoglobulina M/biossíntese , Região Variável de Imunoglobulina/biossíntese , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos B/biossíntese , Receptores de Antígenos de Linfócitos B/genética , Células-Tronco/citologia , Células-Tronco/imunologia , Células-Tronco/metabolismo , Fatores de TempoRESUMO
Mice lacking the membrane tyrosine kinase c-mer have been shown to have altered macro-phage cytokine production and defective phagocytosis of apoptotic cells despite normal phagocytosis of other particles. We show here that c-mer-deficient mice have impaired clearance of infused apoptotic cells and that they develop progressive lupus-like autoimmunity, with antibodies to chromatin, DNA, and IgG. The autoimmunity appears to be driven by endogenous antigens, with little polyclonal B cell activation. These mice should be an excellent model for studying the role of apoptotic debris as an immunogenic stimulus for systemic autoimmunity.
Assuntos
Apoptose/imunologia , Autoimunidade/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Proteínas Tirosina Quinases/deficiência , Proteínas Proto-Oncogênicas/deficiência , Receptores Proteína Tirosina Quinases , Animais , Autoanticorpos/sangue , Linfócitos B/imunologia , Cardiolipinas/imunologia , Cromatina/imunologia , DNA/imunologia , Modelos Animais de Doenças , Feminino , Corantes Fluorescentes , Mesângio Glomerular/patologia , Imunoglobulina G/imunologia , Lúpus Eritematoso Sistêmico/complicações , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Fagocitose/imunologia , Proteínas Tirosina Quinases/genética , Proteinúria/complicações , Proteinúria/patologia , Proteínas Proto-Oncogênicas/genética , Fator Reumatoide/sangue , Rodaminas , c-Mer Tirosina QuinaseRESUMO
The murine chronic GVH (cGVH) model of SLE is induced by allo-recognition of foreign major histocompatibility complex (MHC) class II determinants. Previous studies have shown that syngeneic CD4(+) T cells are needed during B cell development in order to induce cGVH response in CD4KO mice. Our present studies show that B cells require "nurturing" by CD4 T cells through much of their ontogeny in order to respond to allo-signaling and become autoreactive. The nurturing process does not require antigen-specific cognate interactions between CD4 T cells and B cells. It is mediated by IL-4, but not IL-10, IL-6 and IFN-gamma. The CD4 T cell nurturing may be supplanted by large doses of IL-4 and/or by agonistic anti-CD40 mAb. Understanding the mechanism of this "nurturing" process may yield clues to the role of CD4 T cells in lupus and in host defense in general.
Assuntos
Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular/imunologia , Doença Enxerto-Hospedeiro/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Animais , Anticorpos Antinucleares/sangue , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Autoanticorpos/sangue , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Antígenos CD4/genética , Linfócitos T CD4-Positivos/transplante , Antígenos CD40/agonistas , Antígenos CD40/imunologia , Diferenciação Celular/efeitos dos fármacos , Citocinas/genética , Modelos Animais de Doenças , Feminino , Doença Enxerto-Hospedeiro/patologia , Doença Enxerto-Hospedeiro/urina , Antígenos de Histocompatibilidade Classe II/genética , Proteínas de Homeodomínio/genética , Interleucina-4/genética , Interleucina-4/farmacologia , Lúpus Eritematoso Sistêmico/patologia , Lúpus Eritematoso Sistêmico/urina , Ativação Linfocitária/imunologia , Depleção Linfocítica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes , Proteinúria/diagnóstico , Receptores de Antígenos de Linfócitos T/genética , Pele/patologiaRESUMO
The Mer receptor tyrosine kinase is strongly expressed in the glomerulus. We wondered if this molecule might modify immune-mediated glomerular disease through its functions as a receptor for apoptotic cells and immunoregulatory molecule. Mer-knockout (KO) mice showed decreased survival rate and greatly increased proteinuria and serum urea levels compared to wild type (WT) mice by day 3 after injection of NTS. Their glomeruli were hyperplastic and later became necrotic. In the glomerulus of WT mice, a significant increase of Mer expression was observed. Apoptotic bodies were evident in NTS-treated Mer-KO kidneys, but not in normal controls. NTS-treated Mer-KO mice had massive neutrophil infiltration and inflammatory cytokine expression. Mer thus has a critical role in attenuating renal inflammation, both as a receptor for apoptotic cells and as a molecule that downregulates inflammation.