RESUMO
The Src homology region 2 domain-containing phosphatase-1 (SHP-1) is an intracellular tyrosine phosphatase that plays a negative regulatory role in immune cell signaling. Absent or diminished SHP-1 catalytic activity results in reduced bone mass with enhanced bone resorption. Here, we sought to investigate if Shp1 overexpression leads to increased bone mass and improved mechanical properties. Male and female wildtype (WT) and SHP1-transgenic (Tg) mice at 28, 56, and 84 days of age were compared. We applied microcomputed tomography to assess femoral cortical bone geometry and trabecular architecture and 3-point mechanical bending to assess mid-diaphyseal structural and estimated material properties. Serum OPG, RANKL, P1NP, and CTX-1 concentrations were measured by enzyme-linked immunoassay. The majority of transgene effects were restricted to the 28-day-old mice. Trabecular bone volume per total volume, trabecular number, and connectivity density were greater in 28-day-old female SHP1-Tg mice when compared to WTs. SHP1-Tg female mice showed increased total and medullary areas, with no difference in cortical area and thickness. Cortical tissue mineral density was strongly genotype-dependent. Failure load, yield load, ultimate stress, and yield stress were all lower in 28-day-old SHP1-Tg females. In 28-day-old SHP1-Tg females, circulating levels of OPG and P1NP were higher and RANKL levels were lower than WT controls. Our study demonstrates a role for SHP-1 in early postnatal bone development; SHP-1 overexpression negatively impacted whole bone strength and material properties in females.
Assuntos
Desenvolvimento Ósseo , Proteínas Tirosina Fosfatases , Camundongos , Masculino , Feminino , Animais , Microtomografia por Raio-X , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases/metabolismo , Camundongos TransgênicosRESUMO
The extracellular matrix in asthmatic lungs contains abundant low-molecular-weight hyaluronan, and this is known to promote antigen presentation and allergic responses. Conversely, high-molecular-weight hyaluronan (HMW-HA), typical of uninflamed tissues, is known to suppress inflammation. We investigated whether HMW-HA can be adapted to promote tolerance to airway allergens. HMW-HA was thiolated to prevent its catabolism and was tethered to allergens via thiol linkages. This platform, which we call "XHA," delivers antigenic payloads in the context of antiinflammatory costimulation. Allergen/XHA was administered intranasally to mice that had been sensitized previously to these allergens. XHA prevents allergic airway inflammation in mice sensitized previously to either ovalbumin or cockroach proteins. Allergen/XHA treatment reduced inflammatory cell counts, airway hyperresponsiveness, allergen-specific IgE, and T helper type 2 cell cytokine production in comparison with allergen alone. These effects were allergen specific and IL-10 dependent. They were durable for weeks after the last challenge, providing a substantial advantage over the current desensitization protocols. Mechanistically, XHA promoted CD44-dependent inhibition of nuclear factor-κB signaling, diminished dendritic cell maturation, and reduced the induction of allergen-specific CD4 T-helper responses. XHA and other potential strategies that target CD44 are promising alternatives for the treatment of asthma and allergic sinusitis.
Assuntos
Alérgenos/imunologia , Ácido Hialurônico/química , Ácido Hialurônico/farmacologia , Tolerância Imunológica/efeitos dos fármacos , Animais , Anti-Inflamatórios/farmacologia , Células da Medula Óssea/citologia , Diferenciação Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Reagentes de Ligações Cruzadas/metabolismo , Células Dendríticas/efeitos dos fármacos , Receptores de Hialuronatos/metabolismo , Imunização , Interleucina-10 , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Peso Molecular , NF-kappa B/metabolismo , Pneumonia/imunologia , Pneumonia/patologia , Pneumonia/fisiopatologia , Transporte Proteico/efeitos dos fármacos , Compostos de Sulfidrila/metabolismoRESUMO
Rheumatoid arthritis (RA) is one of the most common autoimmune disorders characterized by the chronic and progressive inflammation of various organs, most notably the synovia of joints leading to joint destruction, a shorter life expectancy, and reduced quality of life. Although we have substantial information about the pathophysiology of the disease with various groups of immune cells and soluble mediators identified to participate in the pathogenesis, several aspects of the altered immune functions and regulation in RA remain controversial. Animal models are especially useful in such scenarios. Recently research focused on IL-17 and IL-17 producing cells in various inflammatory diseases such as in RA and in different rodent models of RA. These studies provided occasionally contradictory results with IL-17 being more prominent in some of the models than in others; the findings of such experimental setups were sometimes inconclusive compared to the human data. The aim of this review is to summarize briefly the recent advancements on the role of IL-17, particularly in the different rodent models of RA.
Assuntos
Artrite Reumatoide/metabolismo , Doenças Autoimunes/metabolismo , Interleucina-17/metabolismo , Células Th17/metabolismo , Animais , Artrite Reumatoide/imunologia , Doenças Autoimunes/imunologia , Modelos Animais de Doenças , HumanosRESUMO
Hyaluronan (HA) deposition is often correlated with mucosal inflammatory responses, where HA mediates both protective and pathological responses. By modifying the HA matrix, Tnfip6 (TNF-α-induced protein-6; also known as TSG-6 (TNF-stimulated gene-6)) is thought to potentiate anti-inflammatory and anti-plasmin effects that are inhibitory to leukocyte extravasation. In this study, we examined the role of endogenous TSG-6 in the pathophysiological responses associated with acute allergic pulmonary inflammation. Compared with wild-type littermate controls, TSG-6(-/-) mice exhibited attenuated inflammation marked by a significant decrease in pulmonary HA concentrations measured in the bronchoalveolar lavage and lung tissue. Interestingly, despite the equivalent induction of both humoral and cellular Th2 immunity and the comparable levels of cytokines and chemokines typically associated with eosinophilic pulmonary inflammation, airway eosinophilia was significantly decreased in TSG-6(-/-) mice. Most importantly, contrary to their counterpart wild-type littermates, TSG-6(-/-) mice were resistant to the induction of airway hyperresponsiveness and manifested improved lung mechanics in response to methacholine challenge. Our study demonstrates that endogenous TSG-6 is dispensable for the induction of Th2 immunity but is essential for the robust increase in pulmonary HA deposition, propagation of acute eosinophilic pulmonary inflammation, and development of airway hyperresponsiveness. Thus, TSG-6 is implicated in the experimental murine model of allergic pulmonary inflammation and is likely to contribute to the pathogenesis of asthma.
Assuntos
Moléculas de Adesão Celular/metabolismo , Eosinofilia/metabolismo , Regulação da Expressão Gênica , Ácido Hialurônico/metabolismo , Pulmão/metabolismo , Animais , Asma/metabolismo , Hiper-Reatividade Brônquica/imunologia , Lavagem Broncoalveolar , Ensaio de Imunoadsorção Enzimática/métodos , Matriz Extracelular/metabolismo , Feminino , Ácido Hialurônico/química , Inflamação , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Células Th2/metabolismoRESUMO
The covalent transfer of heavy chains (HCs) from inter-α-inhibitor (IαI) to hyaluronan (HA) via the protein product of tumor necrosis factor-stimulated gene-6 (TSG-6) forms the HC-HA complex, a pathological form of HA that promotes the adhesion of leukocytes to HA matrices. The transfer of HCs to high molecular weight (HMW) HA is a reversible event whereby TSG-6 can shuffle HCs from one HA molecule to another. Therefore, HMW HA can serve as both an HC acceptor and an HC donor. In the present study, we show that transfer of HCs to low molecular weight HA oligosaccharides is an irreversible event where subsequent shuffling does not occur, i.e. HA oligosaccharides from 8 to 21 monosaccharide units in length can serve as HC acceptors, but are unable to function as HC donors. We show that the HC-HA complex is present in the synovial fluid of mice subjected to systemic and monoarticular mouse models of rheumatoid arthritis. Furthermore, we demonstrate that HA oligosaccharides can be used, with TSG-6, to irreversibly shuffle HCs from pathological, HMW HC-HA to HA oligosaccharides, thereby restoring HC-HA matrices from the inflamed joint to their normal state, unmodified with HCs. This process was also effective for HC-HA in the synovial fluid of human rheumatoid arthritis patients (in vitro).
Assuntos
Moléculas de Adesão Celular/metabolismo , Ácido Hialurônico/química , Oligossacarídeos/química , alfa-Globulinas/química , Animais , Carboidratos/química , Eletroforese/métodos , Matriz Extracelular/metabolismo , Humanos , Cadeias Pesadas de Imunoglobulinas/química , Inflamação , Cinética , Leucócitos/metabolismo , Camundongos , Modelos Moleculares , Conformação Molecular , Proteínas Recombinantes/químicaRESUMO
An increasing number of studies show that besides the inherited genetic architecture (that is, genomic DNA), various environmental factors significantly contribute to the etiology of rheumatoid arthritis. Epigenetic factors react to external stimuli and form bridges between the environment and the genetic information-harboring DNA. Epigenetic mechanisms are implicated in the final interpretation of the encoded genetic information by regulating gene expression, and alterations in their profile influence the activity of the immune system. Overall, epigenetic mechanisms further increase the well-known complexity of rheumatoid arthritis by providing additional subtle contributions to rheumatoid arthritis susceptibility. Although there are controversies regarding the involvement of epigenetic and genetic factors in rheumatoid arthritis etiology, it is becoming obvious that the two systems (genetic and epigenetic) interact with each other and are ultimately responsible for rheumatoid arthritis development. Here, epigenetic factors and mechanisms involved in rheumatoid arthritis are reviewed and new, potential therapeutic targets are discussed.
Assuntos
Artrite Reumatoide/etiologia , Artrite Reumatoide/genética , Epigênese Genética/genética , Animais , Artrite Reumatoide/diagnóstico , Cromatina/genética , Metilação de DNA/genética , Humanos , Mutação/genéticaRESUMO
OBJECTIVE: To identify epigenetic factors that are implicated in the pathogenesis of rheumatoid arthritis (RA), and to explore the therapeutic potential of the targeted inhibition of these factors. METHODS: Polymerase chain reaction (PCR) arrays were used to investigate the expression profile of genes that encode key epigenetic regulator enzymes. Mononuclear cells from RA patients and mice were monitored for gene expression changes, in association with arthritis development in murine models of RA. Selected genes were further characterized by quantitative reverse transcription-PCR, Western blot, and flow cytometry methods. The targeted inhibition of the up-regulated enzymes was studied in arthritic mice. RESULTS: A set of genes with arthritis-specific expression was identified by the PCR arrays. Aurora kinases A and B, both of which were highly expressed in arthritic mice and treatment-naive RA patients, were selected for detailed analysis. Elevated aurora kinase expression was accompanied by increased phosphorylation of histone H3, which promotes proliferation of T lymphocytes. Treatment with VX-680, a pan-aurora kinase inhibitor, promoted B cell apoptosis, provided significant protection against disease onset, and attenuated inflammatory reactions in arthritic mice. CONCLUSION: Arthritis development is accompanied by changes in expression of a number of epigenome-modifying enzymes. Drug-induced down-regulation of the aurora kinases, among other targets, seems to be sufficient to treat experimental arthritis. Development of new therapeutics that target aurora kinases can potentially improve RA management.
Assuntos
Artrite Experimental/enzimologia , Artrite Reumatoide/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Apoptose/efeitos dos fármacos , Artrite Experimental/genética , Artrite Experimental/prevenção & controle , Artrite Reumatoide/genética , Aurora Quinases , Linfócitos B/efeitos dos fármacos , Western Blotting , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Epigênese Genética , Feminino , Citometria de Fluxo , Expressão Gênica/efeitos dos fármacos , Perfilação da Expressão Gênica , Histonas/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Fosforilação/genética , Fosforilação/fisiologia , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/efeitos dos fármacos , Regulação para CimaRESUMO
BACKGROUND: Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of innate immune cells with a granulocyte-like or monocyte-like phenotype and a unique ability to suppress T-cell responses. MDSCs have been shown to accumulate in cancer patients, but recent studies suggest that these cells are also present in humans and animals suffering from autoimmune diseases. We previously identified MDSCs in the synovial fluid (SF) of mice with experimental autoimmune arthritis. The goal of the present study was to identify MDSCs in the SF of patients with rheumatoid arthritis (RA). METHODS: RA SF cells were studied by flow cytometry using antibodies to MDSC cell surface markers as well as by analysis of cell morphology. The suppressor activity of RA SF cells toward autologous peripheral blood T cells was determined ex vivo. We employed both antigen-nonspecific (anti-CD3/CD28 antibodies) and antigen-specific (allogeneic cells) induction systems to test the effects of RA SF cells on the proliferation of autologous T cells. RESULTS: SF from RA patients contained MDSC-like cells, the majority of which showed granulocyte (neutrophil)-like phenotype and morphology. RA SF cells significantly suppressed the proliferation of anti-CD3/CD28-stimulated autologous T cells upon co-culture. When compared side by side, RA SF cells had a more profound inhibitory effect on the alloantigen-induced than the anti-CD3/CD28-induced proliferation of autologous T cells. CONCLUSION: MDSCs are present among RA SF cells that are commonly regarded as inflammatory neutrophils. Our results suggest that the presence of neutrophil-like MDSCs in the SF is likely beneficial, as these cells have the ability to limit the expansion of joint-infiltrating T cells in RA.
Assuntos
Artrite Reumatoide/imunologia , Imunidade Inata/imunologia , Leucócitos Mononucleares/imunologia , Líquido Sinovial/citologia , Líquido Sinovial/imunologia , Linfócitos T/imunologia , Adulto , Artrite Reumatoide/patologia , Técnicas de Cocultura , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Projetos PilotoRESUMO
Rheumatoid arthritis (RA) is recognized as an autoimmune joint disease driven by T cell responses to self (or modified self or microbial mimic) antigens that trigger and aggravate the inflammatory condition. Newer treatments of RA employ monoclonal antibodies or recombinant receptors against cytokines or immune cell receptors as well as small-molecule Janus kinase (JAK) inhibitors to systemically ablate the cytokine or cellular responses that fuel inflammation. Unlike these treatments, a therapeutic vaccine, such as CEL-4000, helps balance adaptive immune homeostasis by promoting antigen-specific regulatory rather than inflammatory responses, and hence modulates the immunopathological course of RA. In this review, we discuss the current and proposed therapeutic products for RA, with an emphasis on antigen-specific therapeutic vaccine approaches to the treatment of the disease. As an example, we describe published results of the beneficial effects of CEL-4000 vaccine on animal models of RA. We also make a recommendation for the design of appropriate clinical studies for these newest therapeutic approaches, using the CEL-4000 vaccine as an example. Unlike vaccines that create or boost a new immune response, the clinical success of an immunomodulatory therapeutic vaccine for RA lies in its ability to redirect autoreactive pro-inflammatory memory T cells towards rebalancing the "runaway" immune/inflammatory responses that characterize the disease. Human trials of such a therapy will require alternative approaches in clinical trial design and implementation for determining safety, toxicity, and efficacy. These approaches include adaptive design (such as the Bayesian optimal design (BOIN), currently employed in oncological clinical studies), and the use of disease-related biomarkers as indicators of treatment success.
Assuntos
Artrite Reumatoide , Vacinas , Animais , Humanos , Teorema de Bayes , Citocinas/uso terapêutico , Vacinas/uso terapêutico , Resultado do TratamentoRESUMO
T cells orchestrate joint inflammation in rheumatoid arthritis (RA), but B cells/B cell-derived factors are also involved in disease pathogenesis. The goal of this study was to understand the role of antigen-specific T and B cells in the pathological events of arthritis, which is impossible to study in humans due to the small number of antigen-specific cells. To determine the significance of antigen-specific lymphocytes and antibodies in the development of an autoimmune mouse model of RA, we generated TCR transgenic (TCR-Tg) mice specific for the dominant arthritogenic epitope of cartilage proteoglycan (PG) and performed a series of combined transfers of T cells, B cells and autoantibodies into BALB/c.Scid mice. The adoptive transfer of highly purified T cells from naive TCR-Tg, arthritic TCR-Tg or arthritic wild-type mice induced arthritis in SCID recipients, but the onset and severity of the disease were dependent on the sequential events of the T cell-supported reconstitution of PG-specific B cells and autoantibodies. The presence of activated PG-specific T cells was critical for disease induction, establishing a unique milieu for the selective homeostasis of autoantibody-producing B cells. In this permissive environment, anti-PG autoantibodies bound to cartilage and induced activation of the complement cascade, leading to irreversible cartilage destruction in affected joints. These findings may lead to a better understanding of the complex molecular and cellular mechanisms of RA.
Assuntos
Artrite Reumatoide/imunologia , Autoanticorpos/imunologia , Linfócitos B/imunologia , Homeostase/imunologia , Linfócitos T/imunologia , Transferência Adotiva , Agrecanas/imunologia , Animais , Formação de Anticorpos , Artrite Experimental/imunologia , Autoantígenos/imunologia , Humanos , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Camundongos TransgênicosRESUMO
OBJECTIVE: To determine whether myeloid cells (such as granulocytes) present in the synovial fluid (SF) of arthritic joints have an impact on adaptive immunity. Specifically, we investigated the effects of SF cells harvested from the joints of mice with proteoglycan-induced arthritis (PGIA), on dendritic cell (DC) maturation and antigen-specific T cell proliferation. METHODS: We monitored DC maturation (MHCII and CD86 expression) by flow cytometry upon coculture of DCs with SF cells or spleen myeloid cells from mice with PGIA. The effects of these myeloid cells on T cell proliferation were studied using T cells purified from PG-specific T cell receptor (TCR)-transgenic (Tg) mice. Phenotype analysis of myeloid cells was performed by immunostaining, reverse transcription-polymerase chain reaction, Western blotting, and biochemical assays. RESULTS: Inflammatory SF cells significantly suppressed the maturation of DCs upon coculture. PG-TCR-Tg mouse T cells cultured with antigen-loaded DCs showed dramatic decreases in proliferation in the presence of SF cells. Spleen myeloid cells from arthritic mice did not have suppressive effects. SF cells were unable to suppress CD3/CD28-stimulated proliferation of the same T cells, suggesting a DC-dependent mechanism. SF cells exhibited all of the characteristics of myeloid-derived suppressor cells (MDSCs) and exerted suppression primarily through the production of nitric oxide and reactive oxygen species by granulocyte-like cells. CONCLUSION: SF in the joints of mice with PGIA contains a population of granulocytic MDSCs that potently suppress DC maturation and T cell proliferation. These MDSCs have the potential to limit the expansion of autoreactive T cells, thus breaking the vicious cycle of autoimmunity and inflammation.
Assuntos
Artrite Experimental/imunologia , Artrite Reumatoide/imunologia , Proliferação de Células , Células Dendríticas/imunologia , Células Mieloides/imunologia , Líquido Sinovial/imunologia , Linfócitos T/imunologia , Animais , Artrite Experimental/metabolismo , Artrite Reumatoide/metabolismo , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Ativação Linfocitária/imunologia , Camundongos , Camundongos Transgênicos , Células Mieloides/citologia , Células Mieloides/metabolismo , Líquido Sinovial/citologia , Líquido Sinovial/metabolismo , Linfócitos T/metabolismoRESUMO
OBJECTIVE: Synovial inflammation, a feature of both osteoarthritis (OA) and meniscal injury, is hypothesized to be triggered in part via stimulation of Toll-like receptors (TLRs). We undertook this study to test whether a TLR-2- or TLR-4-stimulating factor in synovial fluid (SF) from patients with early knee OA with meniscal injury could lead to inflammatory activation of synoviocytes. METHODS: SF was obtained from patients with early OA cartilage damage undergoing arthroscopic meniscal procedures. SF was used to stimulate primary cultures of fibroblast-like synoviocytes (FLS) and cell lines transfected with TLR-2 or TLR-4. SF was used either alone or in combination with a TLR-2 stimulus (palmitoyl-3-cysteine-serine-lysine-4 [Pam3CSK4]) or a TLR-4 stimulus (lipopolysaccharide [LPS]). In blocking experiments, SF was preincubated with anti-CD14 antibody. RESULTS: SF from these patients did not stimulate interleukin-8 (IL-8) release from TLR transfectants. Compared with SF on its own, SF (at concentrations of 0.09-25%) in combination with TLR-2 or TLR-4 ligands resulted in significant augmentation of IL-8 release from both transfectants and primary FLS. Soluble CD14 (sCD14), a coreceptor for TLRs, was measured in SF from patients with early OA at levels comparable to those in patients with advanced OA and patients with rheumatoid arthritis. Blockade with anti-CD14 antibody abolished the ability of SF to augment IL-8 production in response to LPS, and diminished Pam3CSK4 responses. CONCLUSION: SF augments FLS responses to TLR-2 and TLR-4 ligands. This effect was largely due to sCD14. Our results demonstrate that sCD14 in the setting of OA and meniscal injury sensitizes FLS to respond to inflammatory stimuli such as TLR ligands.
Assuntos
Receptores de Lipopolissacarídeos/metabolismo , Osteoartrite do Joelho/metabolismo , Líquido Sinovial/metabolismo , Membrana Sinovial/metabolismo , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Idoso , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Feminino , Humanos , Lipopeptídeos/farmacologia , Lipopolissacarídeos/farmacologia , Masculino , Pessoa de Meia-Idade , Osteoartrite do Joelho/patologia , Líquido Sinovial/efeitos dos fármacos , Membrana Sinovial/efeitos dos fármacos , Membrana Sinovial/patologia , Receptor 2 Toll-Like/agonistas , Receptor 4 Toll-Like/agonistasRESUMO
The T-lineage restricted protein THEMIS has been shown to play a critical role in T cell development. THEMIS, via its distinctive CABIT domains, inhibits the catalytic activity of the tyrosine phosphatase SHP1 (PTPN6). SHP1 and THEMIS bind to the ubiquitous cytosolic adapter GRB2, and the purported formation of a tri-molecular THEMIS-GRB2-SHP1 complex facilitates inactivation of SHP1 by THEMIS. The importance of this function of GRB2 among its numerous documented activities is unclear as GRB2 binds to multiple proteins and participates in several signaling responses in thymocytes. Here, we show that similar to Themis-/- thymocytes, the primary molecular defect in GRB2-deficient thymocytes is increased catalytically active SHP1 and the developmental block in GRB2-deficient thymocytes is alleviated by deletion or inhibition of SHP1 and is exacerbated by SHP1 overexpression. Thus, the principal role of GRB2 during T cell development is to promote THEMIS-mediated inactivation of SHP1 thereby enhancing the sensitivity of TCR signaling in CD4+CD8+ thymocytes to low affinity positively selecting self-ligands.
Assuntos
Proteína Adaptadora GRB2 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Receptores de Antígenos de Linfócitos T , Timócitos , Diferenciação Celular , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Timócitos/metabolismo , Proteína Adaptadora GRB2/metabolismoRESUMO
The T cell lineage-restricted protein THEMIS plays a critical role in T cell development at the positive selection stage. In the SHP1 activation model, THEMIS is proposed to enhance the activity of the tyrosine phosphatase SHP1 (encoded by Ptpn6), thereby dampening T cell antigen receptor (TCR) signaling and preventing the inappropriate negative selection of CD4+CD8+ thymocytes by positively selecting ligands. In contrast, in the SHP1 inhibition model, THEMIS is proposed to suppress SHP1 activity, rendering CD4+CD8+ thymocytes more sensitive to TCR signaling initiated by low-affinity ligands to promote positive selection. We sought to resolve the controversy regarding the molecular function of THEMIS. We found that the defect in positive selection in Themis-/- thymocytes was ameliorated by pharmacologic inhibition of SHP1 or by deletion of Ptpn6 and was exacerbated by SHP1 overexpression. Moreover, overexpression of SHP1 phenocopied the Themis-/- developmental defect, whereas deletion of Ptpn6, Ptpn11 (encoding SHP2), or both did not result in a phenotype resembling that of Themis deficiency. Last, we found that thymocyte negative selection was not enhanced but was instead impaired in the absence of THEMIS. Together, these results provide evidence favoring the SHP1 inhibition model, supporting a mechanism whereby THEMIS functions to enhance the sensitivity of CD4+CD8+ thymocytes to TCR signaling, enabling positive selection by low-affinity, self-ligand-TCR interactions.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Timócitos , Linfócitos T CD8-Positivos , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Animais , Peptídeos e Proteínas de Sinalização Intercelular/genéticaRESUMO
TSG-6 (TNF-α-stimulated gene/protein 6), a hyaluronan (HA)-binding protein, has been implicated in the negative regulation of inflammatory tissue destruction. However, little is known about the tissue/cell-specific expression of TSG-6 in inflammatory processes, due to the lack of appropriate reagents for the detection of this protein in vivo. Here, we report on the development of a highly sensitive detection system and its use in cartilage proteoglycan (aggrecan)-induced arthritis, an autoimmune murine model of rheumatoid arthritis. We found significant correlation between serum concentrations of TSG-6 and arthritis severity throughout the disease process, making TSG-6 a better biomarker of inflammation than any of the other arthritis-related cytokines measured in this study. TSG-6 was present in arthritic joint tissue extracts together with the heavy chains of inter-α-inhibitor (IαI). Whereas TSG-6 was broadly detectable in arthritic synovial tissue, the highest level of TSG-6 was co-localized with tryptases in the heparin-containing secretory granules of mast cells. In vitro, TSG-6 formed complexes with the tryptases murine mast cell protease-6 and -7 via either heparin or HA. In vivo TSG-6-tryptase association could also be detected in arthritic joint extracts by co-immunoprecipitation. TSG-6 has been reported to suppress inflammatory tissue destruction by enhancing the serine protease-inhibitory activity of IαI against plasmin. TSG-6 achieves this by transferring heavy chains from IαI to HA, thus liberating the active bikunin subunit of IαI. Because bikunin is also present in mast cell granules, we propose that TSG-6 can promote inhibition of tryptase activity via a mechanism similar to inhibition of plasmin.
Assuntos
Artrite/metabolismo , Moléculas de Adesão Celular/metabolismo , Heparina/metabolismo , Triptases/metabolismo , alfa-Globulinas/imunologia , alfa-Globulinas/metabolismo , Animais , Artrite/imunologia , Biomarcadores/metabolismo , Células CHO , Moléculas de Adesão Celular/imunologia , Cricetinae , Cricetulus , Fibrinolisina/imunologia , Fibrinolisina/metabolismo , Heparina/imunologia , Humanos , Articulações/imunologia , Articulações/metabolismo , Camundongos , Triptases/imunologiaRESUMO
Existing literature demonstrates that fibroblast growth factor-2 (FGF-2) exerts opposing, contradictory biological effects on cartilage homeostasis in different species. In human articular cartilage, FGF-2 plays a catabolic and anti-anabolic role in cartilage homeostasis, driving homeostasis toward degeneration and osteoarthritis (OA). In murine joints, however, FGF-2 has been identified as an anabolic mediator as ablation of the FGF-2 gene demonstrated increased susceptibility to OA. There have been no previous studies specifically addressing species-specific differences in FGF-2-mediated biological effects. In this study, we provide a mechanistic understanding by which FGF-2 exerts contradictory biological effects in human versus murine tissues. Using human articular cartilage (ex vivo) and a medial meniscal destabilization (DMM) animal model (in vivo), species-specific expression patterns of FGFR receptors (FGFRs) are elucidated between human and murine articular cartilage. In the murine OA model followed by intra-articular injection of FGF-2, we further correlate FGFR profiles to changes in behavioral pain perception, proteoglycan content in articular cartilage, and production of inflammatory (CD11b) and angiogenic (VEGF) mediators in synovium lining cells. Our results suggest that the fundamental differences in cellular responses between human and murine tissues may be secondary to distinctive expression patterns of FGFRs that eventually determine biological outcomes in the presence of FGF-2. The complex interplay of FGFRs and the downstream signaling cascades induced by FGF-2 in human cartilage should add caution to the use of this particular growth factor for biological therapy in the future.
Assuntos
Cartilagem Articular/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Osteoartrite/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Adulto , Idoso , Animais , Artralgia/metabolismo , Antígeno CD11b/biossíntese , Humanos , Inflamação , Camundongos , Pessoa de Meia-Idade , Neovascularização Fisiológica/efeitos dos fármacos , Osteoartrite/patologia , Proteoglicanas/biossíntese , Especificidade da Espécie , Membrana Sinovial/metabolismo , Fator A de Crescimento do Endotélio Vascular/biossínteseRESUMO
Recent imaging studies on intact lymph nodes (LNs) of naïve T cell receptor (TCR)-transgenic mice have reported that T cells reduce their motility upon contact with relevant antigen-presenting cells (APCs). Using in vivo two-photon imaging of T cells in joint-draining (JD) LNs, we examined whether similar changes in T cell motility are observed in wild type mice. Co-transfer of T cells from naïve mice and antigen-experienced T cells from mice with proteoglycan (PG)-induced arthritis into naïve or arthritic recipients resulted in prolonged interactions of antigen-experienced T cells with APCs upon intra-articular antigen (PG) injection, indicating that T cells from arthritic wild type mice recapitulate the motile behavior reported in naïve TCR-transgenic mice. However, naïve T cells also engaged in stable interactions with APCs in the JDLNs of arthritic recipients, suggesting an enhanced ability of APCs in the JDLNs of arthritic hosts to present antigen to either naïve or antigen-experienced T cells.
Assuntos
Células Apresentadoras de Antígenos/patologia , Artrite Experimental/patologia , Artrite Reumatoide/imunologia , Articulações/patologia , Linfonodos/patologia , Linfócitos T/fisiologia , Animais , Apresentação de Antígeno , Células Apresentadoras de Antígenos/imunologia , Artrite Experimental/induzido quimicamente , Artrite Experimental/imunologia , Artrite Reumatoide/patologia , Movimento Celular , Modelos Animais de Doenças , Feminino , Humanos , Injeções Intra-Articulares , Articulações/imunologia , Linfonodos/imunologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência por Excitação Multifotônica , Proteoglicanas/isolamento & purificação , Proteoglicanas/farmacologia , Linfócitos T/imunologia , Linfócitos T/transplanteRESUMO
We found a spontaneous autosomal mutation in a mouse leading to neutrophil infiltration with ulceration in the upper dermis of homozygous offspring. These animals had increased neutrophil numbers, associated with normal lymphocyte count, in peripheral blood and bone marrow, suggesting a myeloproliferative disorder; however, granulocyte precursor proliferation in bone marrow was actually reduced (because circulating neutrophils were less susceptible to apoptosis). Neutrophil infiltration of the skin and other organs and high serum levels of immunoglobulins and autoantibodies, cytokines, and acute-phase proteins were additional abnormalities, all of which could be reduced by high-dose corticosteroid treatment or neutrophil depletion by antibodies. Use of genome-wide screening localized the mutation within an 0.4-Mbp region on mouse chromosome 6. We identified insertion of a B2 element in exon 6 of the Ptpn6 gene (protein tyrosine phosphatase, non-receptor type 6; also known as Shp-1). This insertion involves amino acid substitutions that significantly reduced the enzyme activity in mice homozygous for the mutation. Disease onset was delayed, and the clinical phenotype was milder than the phenotypes of other Ptpn6-mutants described in motheaten (me, mev) mice; we designated this new genotype as Ptpn6(meB2/meB2) and the phenotype as meB2. This new phenotype encompasses an autoinflammatory disease showing similarities to many aspects of the so-called neutrophilic dermatoses, a heterogeneous group of skin diseases with unknown etiology in humans.
Assuntos
Doenças Hereditárias Autoinflamatórias/genética , Neutrófilos/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Proteína Tirosina Fosfatase não Receptora Tipo 6/fisiologia , Dermatopatias/metabolismo , Corticosteroides/farmacologia , Animais , Autoanticorpos/química , Mapeamento Cromossômico , Homozigoto , Humanos , Imunoglobulinas/química , Inflamação , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , MutaçãoRESUMO
We have found a B2 repeat insertion in the gene encoding protein tyrosine phosphatase nonreceptor type 6 (PTPN6) in a mouse that developed a skin disorder with clinical and histopathological features resembling those seen in human neutrophilic dermatoses. Neutrophilic dermatoses are a group of complex heterogeneous autoinflammatory diseases that all demonstrate excessive neutrophil infiltration of the skin. Therefore, we tested the cDNA and genomic DNA sequences of PTPN6 from patients with Sweet's syndrome (SW) and pyoderma gangrenosum and found numerous novel splice variants in different combinations. Isoforms resulting from deletions of exons 2, 5, 11, and 15 and retention of intron 1 or 5 were the most common in a patients with a familial case of SW, who had a neonatal onset of an inflammatory disorder with skin lesions and a biopsy specimen consistent with SW. These isoforms were associated with a heterozygous E441G mutation and a heterozygous 1.7-kbp deletion in the promoter region of the PTPN6 gene. Although full-length PTPN6 was detected in all other patients with either pyoderma gangrenosum or SW, it was always associated with splice variants: a partial deletion of exon 4 with the complete deletion of exon 5, alterations that were not detected in healthy controls. The defect in transcriptional regulation of the hematopoietic PTPN6 appears to be involved in the pathogenesis of certain subsets of the heterogeneous group of neutrophilic dermatoses.
Assuntos
Mutação , Neutrófilos/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Dermatopatias/genética , Adulto , Idoso , Processamento Alternativo , Sequência de Bases , Citocinas/metabolismo , Éxons , Feminino , Deleção de Genes , Heterozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Monoéster Fosfórico Hidrolases/química , Proteína Tirosina Fosfatase não Receptora Tipo 6/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
OBJECTIVE: To develop a simplified and relatively inexpensive version of cartilage proteoglycan-induced arthritis (PGIA), an autoimmunity model of rheumatoid arthritis (RA), and to evaluate the extent to which this new model replicates the disease parameters of PGIA and RA. METHODS: Recombinant human G1 domain of human cartilage PG containing "arthritogenic" T cell epitopes was generated in a mammalian expression system and used for immunization of BALB/c mice. The development and progression of arthritis in recombinant human PG G1-immunized mice (designated recombinant human PG G1-induced arthritis [GIA]) was monitored, and disease parameters were compared with those in the parent PGIA model. RESULTS: GIA strongly resembled PGIA, although the clinical symptoms and immune responses in mice with GIA were more uniform than in those with PGIA. Mice with GIA showed evidence of stronger Th1 and Th17 polarization than those with PGIA, and anti-mouse PG autoantibodies were produced in different isotype ratios in the 2 models. Rheumatoid factor (RF) and anti-cyclic citrullinated peptide (anti-CCP) antibodies were detected in both models; however, serum levels of IgG-RF and anti-CCP antibodies were different in GIA and PGIA, and both parameters correlated better with disease severity in GIA than in PGIA. CONCLUSION: GIA is a novel model of seropositive RA that exhibits all of the characteristics of PGIA. Although the clinical phenotypes are similar, GIA and PGIA are characterized by different autoantibody profiles, and the 2 models may represent 2 subtypes of seropositive RA, in which more than 1 type of autoantibody can be used to monitor disease severity and response to treatment.