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1.
Clin Exp Immunol ; 216(3): 240-251, 2024 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-38363980

RESUMO

The spleen plays a role in innate and adaptive immunity, and autoimmune diseases like rheumatoid arthritis (RA). We investigated the effect of splenectomy in early and moderate stages of autoimmune arthritis in a mouse model. To induce recombinant human G1-induced arthritis (GIA), BALB/c mice were immunized intraperitoneally three times in 4-week intervals with the rhG1 antigen. Mice were splenectomized on day 7 (SPE1) or day 35 (SPE2) after the initiation of immunization; tested for clinical severity, joint radiological and histological changes, serum levels of inflammatory cytokines and autoantibodies, and rhG1-specific immune responses; and compared to those in control mice with spleen left intact. Circulating Tregs and T-helper subset ratios in the spleen and inguinal lymph nodes (LNs) were also examined using flow cytometry. The onset of severe inflammatory response was significantly delayed in SPE1 and SPE2 groups compared to control mice at early stages of GIA, which was associated with increased circulating Tregs. After the third immunization, as disease progressed, the severity scores were robustly increased in all mice. Nevertheless, in splenectomized mice, we observed reduced joint deterioration and cartilage damage, more Th2 cells in LNs, and reduced levels of pro-inflammatory cytokines and autoantibodies in their sera. Mesenteric LN cells of splenectomized mice exhibited weaker response in vitro against the rhG1 antigen compared to control mice spleen. In conclusion, splenectomy in the early stages of GIA delayed the inflammatory response, suggesting a protective effect against the development and progression of severe destructive arthritis.


Assuntos
Artrite Reumatoide , Autoanticorpos , Citocinas , Camundongos Endogâmicos BALB C , Esplenectomia , Linfócitos T Reguladores , Animais , Camundongos , Linfócitos T Reguladores/imunologia , Autoanticorpos/imunologia , Autoanticorpos/sangue , Humanos , Artrite Reumatoide/imunologia , Artrite Reumatoide/cirurgia , Baço/imunologia , Feminino , Artrite Experimental/imunologia , Linfonodos/imunologia , Modelos Animais de Doenças , Articulações/patologia , Articulações/imunologia , Articulações/cirurgia , Células Th2/imunologia , Inflamação/imunologia , Proteínas Recombinantes/imunologia
2.
Int J Mol Sci ; 24(12)2023 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-37372957

RESUMO

The natural autoantibody (natAAb) network is thought to play a role in immune regulation. These IgM antibodies react with evolutionary conserved antigens; however, they do not lead to pathological tissue destruction as opposed to pathological autoantibodies (pathAAb). The exact relation between the natAAbs and pathAAbs is still not completely understood; therefore, in the present study, we set out to measure nat- and pathAAb levels against three conserved antigens in a spontaneous autoimmune disease model: the NZB mouse strain which develops autoimmune hemolytic anemia (AIHA) from six months of age. There was an age dependent increase in the natAAb levels in the serum against Hsp60, Hsp70, and the mitochondrial citrate synthase until 6-9 months of age, followed by a gradual decrease. The pathological autoantibodies appeared after six months of age, which corresponded with the appearance of the autoimmune disease. The changes in nat/pathAAb levels were coupled with decreasing B1- and increasing plasma cell and memory B cell percentages. Based on this, we propose that there is a switch from natAAbs towards pathAAbs in aged NZB mice.


Assuntos
Anemia Hemolítica Autoimune , Doenças Autoimunes , Camundongos , Animais , Autoanticorpos
3.
Clin Exp Immunol ; 209(2): 201-214, 2022 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-35576510

RESUMO

The spleen is the largest secondary lymphoid organ which is involved in the development of B cells and also in systemic (auto)immune responses. Using the recombinant human G1 domain-induced arthritis (GIA) model in splenectomized and control BALB/c mice, we investigated the role of the spleen in the induction and pathogenesis of autoimmune arthritis. Splenectomized mice developed GIA with a similar clinical picture to the control group. However, we observed significant alterations in the humoral and cellular immune responses in splenectomized mice. In the sera of the splenectomized mice, we found lower pro-inflammatory cytokine and anti-rhG1 IgM levels, but higher IL-4, anti-rhG1 IgG1 and anti-CCP and RF antibodies. The arthritis induction in the splenectomized group was associated with a significant expansion of activated helper T cells and an increase in the proportion of the circulating B1 and marginal zone B cell subsets. Importantly, immunization of the splenectomized mice with rhG1 induced the formation of germinal centers in the inguinal- and mesenteric lymph nodes (i/mLNs) which showed an active immune response to rhG1. Finally, both B and T cells from the mLNs of the splenectomized mice showed decreased intracellular Ca2+ signaling than those of the control group. Collectively, these findings indicate that the presence of the spleen is not critical for the induction of GIA, and in its absence the autoimmune arthritis is most likely promoted through the compensatory activity of the i/mLNs. However, our data implies the immunological role of the spleen in arthritis which could be further assessed in human RA.


Assuntos
Artrite , Doenças Autoimunes , Animais , Modelos Animais de Doenças , Humanos , Imunidade , Imunoglobulina G , Inflamação , Camundongos , Camundongos Endogâmicos BALB C , Esplenectomia
4.
Apoptosis ; 25(9-10): 715-729, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32737651

RESUMO

Earlier we have reported that thymic regulatory T cells (Treg) are resistant to in vivo glucocorticoid hormone (GC)-induced apoptosis, while the most GC-sensitive DP thymocytes died through the activation of mitochondrial apoptotic pathway. Here we analyzed the apoptosis-inducing effect of high dose (10-6 M) in vitro dexamethasone (DX) treatment in mouse thymic- and splenic Tregs and CD4+ T cells. Activation of both extrinsic and intrinsic apoptotic pathways started after 2 h of DX treatment in CD4 SP thymocytes and was 3 × higher than in CD4+ splenocytes, while in Treg cells, weak activation of the extrinsic apoptotic pathway started only after 3 h. We also investigated the expression of 21 apoptosis-related molecules using a protein array and found higher level of both pro-and anti-apoptotic molecules in Tregs compared to CD4+ T cells. 4 h in vitro DX treatment induced upregulation of most apoptosis-related molecules both in Tregs and CD4+ T cells, except for the decrease of Bcl-2 expression in CD4+ T cells. We found high basal cytosolic Ca2+ levels in untreated Treg cells, which further increased after DX treatment, while the specific TCR-induced Ca2+ signal was lower in Tregs than in CD4+ T cells. Our results suggest that in the background of the relative apoptosis resistance of Treg cells to GCs might be their high basal cytosolic Ca2+ level and upregulated Bcl-2 expression. In contrast, downregulation of Bcl-2 expression in CD4+ T cells can explain their higher, DX-induced apoptosis sensitivity.


Assuntos
Glucocorticoides/farmacologia , Hormônios/farmacologia , Mitocôndrias/efeitos dos fármacos , Linfócitos T Reguladores/efeitos dos fármacos , Animais , Apoptose/genética , Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Citocromos c/genética , Dexametasona/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glucocorticoides/metabolismo , Humanos , Camundongos , Mitocôndrias/imunologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/imunologia , Transdução de Sinais/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Timócitos/efeitos dos fármacos
5.
Int J Mol Sci ; 21(17)2020 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-32859051

RESUMO

B cells play a crucial role in the pathogenesis of rheumatoid arthritis. In Nkx2-3-deficient mice (Nkx2-3-/-) the spleen's histological structure is fundamentally changed; therefore, B cell homeostasis is seriously disturbed. Based on this, we were curious, whether autoimmune arthritis could be induced in Nkx2-3-/- mice and how B cell activation and function were affected. We induced arthritis with immunization of recombinant human proteoglycan aggrecan G1 domain in Nkx2-3-/- and control BALB/c mice. We followed the clinical picture, characterized the radiological changes, the immune response, and intracellular Ca2+ signaling of B cells. Incidence of the autoimmune arthritis was lower, and the disease severity was milder in Nkx2-3-/- mice than in control BALB/c mice. The radiological changes were in line with the clinical picture. In Nkx2-3-/- mice, we measured decreased antigen-induced proliferation and cytokine production in spleen cell cultures; in the sera, we found less anti-CCP-IgG2a, IL-17 and IFNγ, but more IL-1ß, IL-4 and IL-6. B cells isolated from the lymph nodes of Nkx2-3-/- mice showed decreased intracellular Ca2+ signaling compared to those isolated from BALB/c mice. Our findings show that the transcription factor Nkx2-3 might regulate the development of autoimmune arthritis most likely through modifying B cell activation.


Assuntos
Agrecanas/química , Artrite Experimental/genética , Artrite Reumatoide/genética , Linfócitos B/metabolismo , Proteínas de Homeodomínio/genética , Fatores de Transcrição/genética , Agrecanas/efeitos adversos , Agrecanas/imunologia , Animais , Artrite Experimental/induzido quimicamente , Artrite Experimental/metabolismo , Artrite Reumatoide/induzido quimicamente , Artrite Reumatoide/metabolismo , Sinalização do Cálcio , Células Cultivadas , Citocinas/metabolismo , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Domínios Proteicos , Índice de Gravidade de Doença , Baço/citologia , Baço/metabolismo
6.
J Neuroinflammation ; 15(1): 335, 2018 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-30509328

RESUMO

OBJECTIVE: The regulatory role of capsaicin-sensitive peptidergic sensory nerves has been shown in acute inflammation, but little is known about their involvement in T/B-cell driven autoimmune arthritis. This study integratively characterized the function of these nerve endings in the proteoglycan-induced chronic arthritis (PGIA), a translational model of rheumatoid arthritis. METHODS: Peptidergic afferents were defunctionalized by resiniferatoxin (RTX) pretreatment in BALB/c mice, PGIA was induced by repeated antigen challenges. Hind paw volume, arthritis severity, grasping ability and the mechanonociceptive threshold were monitored during the 17-week experiment. Myeloperoxidase activity, vascular leakage and bone turnover were evaluated by in vivo optical imaging. Bone morphology was assessed using micro-CT, the intertarsal small joints were processed for histopathological analysis. RESULTS: Following desensitization of the capsaicin-sensitive afferents, ankle edema, arthritis severity and mechanical hyperalgesia were markedly diminished. Myeloperoxidase activity was lower in the early, but increased in the late phase, whilst plasma leakage and bone turnover were not altered. Desensitized mice displayed similar bone spurs and erosions, but increased trabecular thickness of the tibia and bony ankylosis of the spine. Intertarsal cartilage thickness was not altered in the model, but desensitization increased this parameter in both the non-arthritic and arthritic groups. CONCLUSION: This is the first integrative in vivo functional and morphological characterization of the PGIA mouse model, wherein peptidergic afferents have an important regulatory function. Their overall effect is proinflammatory by increasing acute inflammation, immune cell activity and pain. Meanwhile, their activation decreases spinal ankylosis, arthritis-induced altered trabecularity, and cartilage thickness in small joints.


Assuntos
Artrite Reumatoide/induzido quimicamente , Artrite Reumatoide/patologia , Capsaicina/farmacologia , Proteoglicanas/toxicidade , Fármacos do Sistema Sensorial/farmacologia , Limiar Sensorial/efeitos dos fármacos , Animais , Tornozelo/diagnóstico por imagem , Cartilagem/patologia , Modelos Animais de Doenças , Diterpenos/farmacologia , Feminino , Membro Posterior/efeitos dos fármacos , Membro Posterior/fisiopatologia , Camundongos , Camundongos Endogâmicos BALB C , Neurotoxinas/farmacologia , Peptídeos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Índice de Gravidade de Doença , Coluna Vertebral/diagnóstico por imagem
7.
Apoptosis ; 22(2): 239-253, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27888447

RESUMO

Glucocorticoids (GC) are important in the regulation of selection and apoptosis of CD4+CD8+ double-positive (DP) thymocytes. The pronounced GC-sensitivity of DP thymocytes, observed earlier, might be due to the combination of classical (genomic) and alternative (non-genomic) glucocorticoid receptor (GR) signaling events modifying activation or apoptotic pathways. In particular, the previously demonstrated mitochondrial translocation of activated GR in DP thymocytes offered a fascinating explanation for their pronounced GC-induced apoptosis sensitivity. However, the fine molecular details how the mitochondrial translocation of GR might regulate apoptosis remained unclear. Therefore, in the present study, we intended to examine which apoptotic pathways could be involved in GC-induced thymocyte apoptosis. Furthermore we investigated the potential relationship between the GR and Bcl-2 proteins. Using an in vitro test system, thymocytes from 4-week-old BALB/c mice, were treated with the GC-analogue dexamethasone (DX). Bax accumulated in mitochondria upon DX treatment. Mitochondrial GR showed association with members of the Bcl-2 family: Bak, Bim, Bcl-xL. Elevated Cytochrome C, and active caspase-3, -8, and -9 levels were detected in thymocytes after DX treatment. These results support the hypothesis that in early phases of GC-induced thymocyte apoptosis, the mitochondrial pathway plays a crucial role, confirmed by the release of Cytochrome C and the activation of caspase-9. The activation of caspase-8 was presumably due to cross-talk between apoptotic signaling pathways. We propose that the GC-induced mitochondrial accumulation of Bax and the interaction between the GR and Bim, Bcl-xL and Bak could play a role in the regulation of thymocyte apoptosis.


Assuntos
Apoptose/genética , Proteína 11 Semelhante a Bcl-2/genética , Receptores de Glucocorticoides/genética , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína X Associada a bcl-2/genética , Animais , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Caspases/genética , Diferenciação Celular/genética , Citocromos c/genética , Citocromos c/metabolismo , Glucocorticoides/metabolismo , Camundongos , Mitocôndrias/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Receptores de Glucocorticoides/metabolismo , Transdução de Sinais/genética , Timócitos/metabolismo
8.
Mediators Inflamm ; 2016: 6145810, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26903711

RESUMO

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 , Humanos
9.
Curr Protoc ; 4(5): e1053, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38752927

RESUMO

The recombinant human proteoglycan aggrecan-G1 domain (rhG1)-induced arthritis (GIA) mouse model is a complex model of rheumatoid arthritis (RA). In GIA, autoimmune arthritis is induced by repeated intraperitoneal immunization of genetically susceptible BALB/c mice with the rhG1 antigen emulsified in the adjuvant dimethyldioctadecylammonium (DDA). This article describes the steps for producing and purifying the rhG1 antigen, the immunization protocol, methods for following the clinical picture of arthritis, and the evaluation of relevant laboratory parameters. In this model, the autoimmune arthritis develops stepwise, similar to RA: First is the preclinical stage (after the first immunization, days 0-20) with no sign of inflammation but detectable T and B cell activation; next, the stage of early arthritis (after the second immunization, days 21-41), where the first definitive signs of arthritis appear together with autoantibody production; and then the severe late-stage arthritis (after the third immunization, after day 42), which presents with massive inflammation of the limbs, leading to cartilage and bone destruction and finally ankylosis. The protocols described here provide sufficient information for investigators to use the GIA model to study different aspects of autoimmune arthritis. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Induction of recombinant human proteoglycan aggrecan-G1 domain (rhG1)-induced arthritis (GIA) Support Protocol 1: Production of rhG1-Xa-mFc2a fusion protein with CHOK1 mammalian expression system Support Protocol 2: Purification of the rhG1-Xa-mFc2a fusion protein by affinity chromatography Support Protocol 3: Preparation of DDA adjuvant Support Protocol 4: Clinical assessment of arthritis Support Protocol 5: Measurement of serum antibody levels and cytokines Support Protocol 6: Measurement of rhG1-induced proliferation and cytokine production in spleen cell culture Support Protocol 7: Histological assessment of arthritic limbs Support Protocol 8: Evaluation of arthritis with micro-computed tomography.


Assuntos
Agrecanas , Modelos Animais de Doenças , Camundongos Endogâmicos BALB C , Proteínas Recombinantes , Animais , Agrecanas/metabolismo , Camundongos , Humanos , Artrite Reumatoide/imunologia , Artrite Experimental/imunologia , Artrite Experimental/patologia
10.
Arthritis Rheum ; 63(5): 1312-21, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21305522

RESUMO

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.


Assuntos
Agrecanas/farmacologia , Artrite Experimental/imunologia , Artrite Reumatoide/imunologia , Cartilagem Articular/imunologia , Proteoglicanas/farmacologia , Agrecanas/imunologia , Análise de Variância , Animais , Artrite Experimental/sangue , Artrite Experimental/induzido quimicamente , Artrite Reumatoide/sangue , Artrite Reumatoide/induzido quimicamente , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Imunização , Interleucinas/sangue , Interleucinas/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Proteoglicanas/imunologia , Fator Reumatoide/sangue , Linfócitos T
11.
Pharmaceuticals (Basel) ; 15(1)2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-35056114

RESUMO

Transient Receptor Potential Ankyrin 1 (TRPA1) has been reported to influence neuroinflammation and lymphocyte function. We analysed the immune phenotype and activation characteristics of TRPA1-deficient mice (knockout-KO) generated by targeted deletion of the pore-loop domain of the ion channel. We compared TRPA1 mRNA and protein expression in monocyte and lymphocyte subpopulations isolated from primary and secondary lymphatic organs of wild type (WT) and KO mice. qRT-PCR and flow cytometric studies indicated a higher level of TRPA1 in monocytes than in lymphocytes, but both were orders of magnitude lower than in sensory neurons. We found lower CD4+/CD8+ thymocyte ratios, diminished CD4/CD8 rates, and B cell numbers in the KO mice. Early activation marker CD69 was lower in CD4+ T cells of KO, while the level of CD8+/CD25+ cells was higher. In vitro TcR-mediated activation did not result in significant differences in CD69 level between WT and KO splenocytes, but lower cytokine (IL-1ß, IL-6, TNF-α, IL-17A, IL-22, and RANTES) secretion was observed in KO splenocytes. Basal intracellular Ca2+ level and TcR-induced Ca2+ signal in T lymphocytes did not differ significantly, but interestingly, imiquimod-induced Ca2+ level in KO thymocytes was higher. Our results support the role of TRPA1 in the regulation of activation, cytokine production, and T and B lymphocytes composition in mice.

12.
Sci Rep ; 12(1): 5808, 2022 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-35388084

RESUMO

Rat pheochromocytoma (PC12) cells were treated with the proteasome inhibitor MG-132 and morphological changes were recorded. Initially, neuronal differentiation was induced but after 24 h signs of morphological deterioration became apparent. We performed nuclear staining, flow cytometry and WST-1 assay then analyzed signal transduction pathways involving Akt, p38 MAPK (Mitogen-Activated Protein Kinase), JNK (c-Jun N-terminal Kinase), c-Jun and caspase-3. Stress signaling via p38, JNK and c-Jun was active even after 24 h of MG-132 treatment, while the survival-mediating Akt phosphorylation declined and the executor of apoptosis (caspase-3) was activated by that time and apoptosis was also observable. We examined subcellular localization of stress signaling components, applied kinase inhibitors and dominant negative H-Ras mutant-expressing PC12 cells in order to decipher connections of stress-mediating pathways. Our results are suggestive of that treatment with the proteasome inhibitor MG-132 has a biphasic nature in PC12 cells. Initially, it induces neuronal differentiation but prolonged treatments lead to apoptosis.


Assuntos
Leupeptinas , Inibidores de Proteassoma , Neoplasias das Glândulas Suprarrenais , Animais , Apoptose/fisiologia , Caspase 3 , Ativação Enzimática , Proteínas Quinases JNK Ativadas por Mitógeno , Células PC12 , Feocromocitoma , Inibidores de Proteassoma/farmacologia , Proteínas Proto-Oncogênicas c-akt , Ratos , Proteínas Quinases p38 Ativadas por Mitógeno
13.
Arthritis Rheum ; 62(10): 2984-94, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20564001

RESUMO

OBJECTIVE: To investigate whether genetic preponderance of a T cell receptor (TCR) recognizing an arthritogenic peptide of human cartilage proteoglycan (PG) is sufficient for development of arthritis. METHODS: We performed a longitudinal study using BALB/c mice expressing a TCR that recognizes the arthritogenic ATEGRVRVNSAYQDK peptide of human cartilage PG. PG-specific TCR-transgenic (PG-TCR-Tg) mice were inspected weekly for peripheral arthritis until 12 months of age. Peripheral joints were examined histologically, and T cell responses, T cell activation markers, serum cytokines, and autoantibodies were measured. Apoptosis and signaling studies were performed in vitro on T cells from aged PG-TCR-Tg mice. RESULTS: Spontaneous arthritis developed as early as 5-6 months of age, and the incidence increased to 40-50% by 12 months of age. Progressive inflammation began with cartilage and bone erosions in the interphalangeal joints, and later expanded to the proximal joints of the front and hind paws. Spontaneous arthritis was associated with a high proportion of activated CD4+ T cells, enhanced interferon-γ and interleukin-17 (IL-17) production, and elevated levels of serum autoantibodies. PG-TCR-Tg mice lacking IL-4 developed arthritis earlier and at a higher incidence than IL-4-sufficient mice. Antigen-specific activation-induced cell death was diminished in vitro in CD4+ T cells of PG-TCR-Tg mice with spontaneous arthritis, especially in those lacking IL-4. CONCLUSION: The presence of CD4+ T cells expressing a TCR specific for an arthritogenic PG epitope is sufficient to trigger spontaneous autoimmune inflammation in the joints of BALB/c mice. IL-4 appears to be a negative regulator of this disease, through attenuation of activation-induced cell death.


Assuntos
Artrite Reumatoide/imunologia , Linfócitos T CD4-Positivos/imunologia , Morte Celular/imunologia , Interleucina-4/imunologia , Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Animais , Artrite Experimental/imunologia , Epitopos de Linfócito T/imunologia , Interleucina-4/deficiência , Estudos Longitudinais , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Proteoglicanas/imunologia
14.
Int Immunol ; 21(5): 511-22, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19254958

RESUMO

Proteoglycan (PG) aggrecan-induced arthritis (PGIA) is a murine model of rheumatoid arthritis (RA). Although BALB/c and DBA/2 mice share the same MHC (H-2d) haplotype, the BALB/c strain is susceptible to PGIA, while DBA/2 mice are resistant. Therefore, these two inbred mouse strains provide an opportunity to study arthritis susceptibility factors excluding the effects of MHC-associated genetic components. The goal of this study was to monitor changes in the cellular composition and activation state following intra-peritoneal (i.p.) immunization to induce PGIA; additionally, we sought to identify new susceptibility factors by comparing PG-induced immune responses in BALB/c and DBA/2 mice. Upon i.p. PG injection, resident naive B1 cells are replaced by both T cells and conventional B cells in the peritoneum of BALB/c mice. These peritoneal T cells produce IFNgamma and IL-17, cytokines shown to be important in RA and corresponding arthritis models. Moreover, peritoneal cells can adoptively transfer PGIA to SCID mice, demonstrating their arthritogenic properties. Our results indicate that repeatedly injected antigen leads to the recruitment and activation of immune cells in the peritoneum; these cells then trigger the effector phase of the disease. The migration and activation of T(h)1/T(h)17 cells in the peritoneal cavity in response to PG immunization, which did not occur in the arthritis-resistant DBA/2 strain, may be critical factors of arthritis susceptibility in BALB/c mice.


Assuntos
Artrite Experimental/imunologia , Interleucina-17/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Agrecanas/imunologia , Animais , Polaridade Celular , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Camundongos SCID , Lavagem Peritoneal , Peritônio/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Auxiliares-Indutores/metabolismo
15.
Int Immunol ; 21(11): 1269-76, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19737783

RESUMO

Glucocorticoid receptor (GR) signaling plays an important role in the selection and apoptosis of thymocytes. Besides nuclear translocation, mitochondrial translocation of the ligand-bound GR in lymphoid cells was also shown, which might determine glucocorticoid (GC)-induced apoptosis sensitivity. In the present work, we followed the ligand-induced GR trafficking in CD4+CD8+ double-positive (DP) thymocytes. Using confocal microscopy, we found that upon short-term in vitro GC analog [dexamethasone (DX)] treatment, the GR translocates into the mitochondria but not into the nucleus in DP cells. We also analyzed the GR redistribution in cytosolic, nuclear and mitochondrial fractions of unseparated thymocytes by western blot and confirmed that in DX-treated cells a significant fraction of the GR translocates into the mitochondria. DX reduced the mitochondrial membrane potential of DP cells within 30 min, measured by flow cytometry, which refers to a direct modulatory activity of mitochondrial GR translocation. The abundant mitochondrial GR found in DP cells well correlates with their high GC-induced apoptosis sensitivity.


Assuntos
Apoptose/efeitos dos fármacos , Mitocôndrias/metabolismo , Receptores de Glucocorticoides/metabolismo , Linfócitos T/imunologia , Timo/imunologia , Animais , Apoptose/imunologia , Linhagem Celular Tumoral , Dexametasona/farmacologia , Glucocorticoides/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Mitocôndrias/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos
16.
J Ovarian Res ; 13(1): 25, 2020 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-32138790

RESUMO

BACKGROUND: Ovarian hyperstimulation syndrome (OHSS) is a rare, yet severe, iatrogenic complication of ovulation induction therapy during assisted reproductive procedures. Our group previously detected atypical cells in the ascitic fluid of OHSS patients, although no malignancy developed during follow up. Here, the aim was to perform a comparative analysis of the cytokines present in the abdominal fluid of patients affected by OHSS versus patients with advanced ovarian cancer, a benign adnexal mass, or ovarian endometriosis. METHODS: This prospective, non-randomized study was conducted at the Clinical Center of the University of Pecs Department of Obstetrics and Gynecology/Reproductive Center between October 2016 and March 2018. Abdominal fluid samples were obtained from 76 patients and subjected to Luminex analysis. The samples were collected from patients with OHSS (OHSS; n = 16), advanced ovarian cancer (OC; n = 22), a benign adnexal mass (BAM; n = 21), or ovarian endometriosis (EM; n = 17). Data were subjected to the non-parametric Kruskal-Wallis test and Spearman's rank correlation coefficient to identify statistical differences between the four study groups. RESULTS: Leukocytosis and hemoconcentration were detected in the peripheral blood of OHSS patients. Abdominal fluid analysis further revealed significantly higher levels of interleukin (IL)-6, IL-8, IL-10, and transforming growth factor (TGF)-ß in both the OHSS and OC groups compared to the BAM and EM groups. The highest concentration of vascular endothelial growth factor (VEGF) was detected in the OC group, while a significantly lower level was detected in the OHSS group. Moreover, VEGF levels in OC and OHSS groups were significantly elevated compared to the levels in the BAM and EM groups. CONCLUSIONS: Vasoactive and hematogenic cytokines were present at higher levels in both the OHSS and OC abdominal fluid samples compared to the fluid samples obtained from the peritoneal cavity of the BAM patients. It is possible that these cytokines play an important role in the formation of ascites.


Assuntos
Ascite/metabolismo , Líquido Ascítico/metabolismo , Citocinas/metabolismo , Síndrome de Hiperestimulação Ovariana/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores , Citocinas/sangue , Feminino , Fertilização in vitro/efeitos adversos , Humanos , Pessoa de Meia-Idade , Síndrome de Hiperestimulação Ovariana/sangue , Síndrome de Hiperestimulação Ovariana/etiologia , Indução da Ovulação
17.
Immun Ageing ; 6: 8, 2009 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-19519881

RESUMO

BACKGROUND: Rheumatoid arthritis (RA) most often begins in females in the fourth-fifth decade of their life, suggesting that the aging of the immune system (immunosenescence) has a major role in this disease. Therefore, in the present study, we sought to investigate the effect of age on arthritis susceptibility in BALB/c mice using the proteoglycan (PG)-induced arthritis (PGIA) model of RA. RESULTS: We have found that young, 1-month-old female BALB/c mice are resistant to the induction of PGIA, but with aging they become susceptible. PG-induced T cell responses decline with age, whereas there is a shift toward Th1 cytokines. An age-dependent decrease in T cell number is associated with an increased ratio of the memory phenotype, and lower CD28 expression. Antigen-presenting cells shifted from macrophages and myeloid dendritic cells in young mice toward B cells in older mice. The regulatory/activated T cell ratio decreases in older mice after PG injections indicating impaired regulation of the immune response. CONCLUSION: We conclude that immunosenescence could alter arthritis susceptibility in a very complex manner including both adaptive and innate immunities, and it cannot be determined by a single trait. Cumulative alterations in immunoregulatory functions closely resemble human disease, which makes this systemic autoimmune arthritis model of RA even more valuable.

18.
Cells ; 8(5)2019 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-31137740

RESUMO

T cells play an essential role in the pathogenesis of both human rheumatoid arthritis (RA) and its murine models. A key molecule in T cell activation is ZAP-70, therefore we aimed to investigate the effects of partial ZAP-70 deficiency on the pathogenesis of recombinant human G1(rhG1)-induced arthritis (GIA), a well-established mouse model of RA. Arthritis was induced in BALB/c and ZAP-70+/- heterozygous mice. Disease progression was monitored using a scoring system and in vivo imaging, antigen-specific proliferation, cytokine and autoantibody production was measured and T cell apoptotic pathways were analyzed. ZAP-70+/- mice developed a less severe arthritis, as shown by both clinical picture and in vitro parameters (decreased T cell proliferation, cytokine and autoantibody production). The amount of cleaved Caspase-3 increased in arthritic ZAP-70+/- T cells, with no significant changes in cleaved Caspase-8 and -9 levels; although expression of Bim, Bcl-2 and Cytochrome C showed alterations. Tyrosine phosphorylation was less pronounced in arthritic ZAP-70+/- T cells and the amount of Cbl-b-a negative regulator of T cell activation-decreased as well. We hypothesize that the less severe disease seen in the partial absence of ZAP-70 might be caused by the decreased T cell activation accompanied by increased apoptosis.


Assuntos
Apoptose/imunologia , Artrite Reumatoide/metabolismo , Autoimunidade , Ativação Linfocitária/imunologia , Linfócitos T/imunologia , Proteína-Tirosina Quinase ZAP-70/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Agrecanas/farmacologia , Animais , Artrite Reumatoide/induzido quimicamente , Autoanticorpos/metabolismo , Proteína 11 Semelhante a Bcl-2/metabolismo , Caspase 3/metabolismo , Células Cultivadas , Citocromos c/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Feminino , Técnicas de Inativação de Genes , Humanos , Fragmentos Fc das Imunoglobulinas/farmacologia , Imunoglobulina G/química , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Baço/citologia , Baço/patologia , Proteína-Tirosina Quinase ZAP-70/genética
19.
Immunobiology ; 213(1): 39-50, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18207026

RESUMO

Glucocorticoid hormone (GC) production by thymic epithelial cells influences TcR signalling in DP thymocytes and modifies their survival. In the present work, we focused on exploring details of GC effects on DP thymocyte apoptosis with or without parallel TcR activation in AND transgenic mice, carrying TcR specific for pigeon cytochrome C, in vivo. Here we show that the glucocorticoid receptor (GR) protein level was the lowest in DP thymocytes, and it was slightly down-regulated by GC analogue, anti-CD3, PCC and combined treatments as well. Exogenous GC analogue treatment or TcR stimulation alone lead to marked DP cell depletion, coupled with a significant increase of early apoptotic cell ratio (AnnexinV staining), marked abrogation of the mitochondrial function in DP cells (CMXRos staining), and significant decrease in the Bcl-2(high) DP thymocyte numbers, respectively. On the other hand, the simultaneous exposure to these two proapototic signals effectively reversed all the above-described changes. The parallel analysis of CD4 SP cell numbers, AnnexinV, CMXRos, Bcl-2 and GR stainings revealed, that the GR and TcR signals were not antagonistic on the mature thymocytes. These data provide experimental evidence in TcR transgenic mice, in vivo, that when TcR activation and GR signals are present simultaneously, they rescue double positive thymocytes from programmed cell death. The two separate signalling pathways merge in DP thymocytes at such important apoptosis regulating points as the Bcl-2 and GR, showing that their balanced interplay is essential in DP cell survival.


Assuntos
Apoptose , Dexametasona/farmacologia , Mitocôndrias/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Glucocorticoides/metabolismo , Linfócitos T/fisiologia , Animais , Anexina A5/metabolismo , Complexo CD3/imunologia , Citocromos c/imunologia , Camundongos , Camundongos Transgênicos , Mitocôndrias/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia
20.
Immunobiology ; 223(4-5): 422-431, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29223294

RESUMO

OBJECTIVE: Despite the fact that glucocorticoids (GC) are important therapeutic tools, their effects on regulatory T cells (Treg) are not well defined. The aim of our work was to investigate how GCs influence in vivo the thymic (tTreg) and peripheral Treg (pTreg) differentiation, survival and cytokine production. METHODS: Tregs were detected with flow cytometry in lymphatic organs of 4-6 weeks old BALB/c mice after repeated (2-4days), high-dose in vivo GC treatment using CD4/CD25 cell surface and Foxp3/IL-10/TGFß/glucocorticoid receptor (GR) intracellular staining. Cytokine, Foxp3, and GR mRNA levels of sorted CD4+CD25high T cells were analyzed using RT-PCR. Foxp3 and GR localization in Treg cells was investigated with confocal microscopy. RESULTS: GC treatment of mice resulted in increased relative tTreg frequency in the thymus, which was due to decreased total thymocyte numbers with unchanged absolute tTreg cell count. In contrast the relative pTreg cell ratio in secondary lymphatic organs decreased or showed no changes after GC treatment, while the absolute number of pTregs decreased. Elevated intracellular IL-10+ and TGFß+ tTreg and pTreg ratios were measured in GC-treated animals, accompanied with elevated Foxp3 mRNA expression. In addition, GC treatment caused increased TGFß and IL-35 mRNA expression in CD4+CD25high+ splenic and elevated IL-10 mRNA level in thymic tTregs. GR expression of thymic tTreg cells was lower than in pTregs. GC treatment caused an opposite change in GR levels, elevating GR in tTregs but decreasing it in pTregs. We observed a nuclear localization of GR in both tTregs and pTregs, which showed high colocalization (∼60%) with Foxp3 transcription factor. These data suggest an interaction of these two transcription factors with further increase due to GC treatment in splenic pTregs. CONCLUSION: Our data show selective survival of tTregs and elevated production of immunosuppressive cytokines by Treg cells after GC treatment, which may contribute to the immunosuppressive effects of GCs.


Assuntos
Fatores de Transcrição Forkhead/metabolismo , Glucocorticoides/uso terapêutico , Linfócitos T Reguladores/imunologia , Timo/imunologia , Animais , Circulação Sanguínea , Sobrevivência Celular , Células Cultivadas , Fatores de Transcrição Forkhead/genética , Terapia de Imunossupressão , Interleucina-10/metabolismo , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Receptores de Glucocorticoides/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Regulação para Cima
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