RESUMO
OBJECTIVES: Bruton's tyrosine kinase (Btk) is required for B lymphocyte and myeloid cell contributions to pathology in murine models of arthritis. Here, we examined the potential contributions of synovial Btk expression and activation to inflammation in rheumatoid arthritis (RA). MATERIALS AND METHODS: Btk was detected by immunohistochemistry and digital image analysis in synovial tissue from biologically naive RA (n=16) and psoriatic arthritis (PsA) (n=12) patients. Cell populations expressing Btk were identified by immunofluorescent double labelling confocal microscopy, quantitative (q-) PCR and immunoblotting. The effects of a Btk-specific inhibitor, RN486, on gene expression in human macrophages and RA synovial tissue explants (n=8) were assessed by qPCR, ELISA and single-plex assays. RESULTS: Btk was expressed at equivalent levels in RA and PsA synovial tissue, restricted to B lymphocytes, monocytes, macrophages and mast cells. RN486 significantly inhibited macrophage IL-6 production induced by Fc receptor and CD40 ligation. RN486 also reduced mRNA expression of overlapping gene sets induced by IgG, CD40 ligand (CD40L) and RA synovial fluid, and significantly suppressed macrophage production of CD40L-induced IL-8, TNF, MMP-1 and MMP-10, LPS-induced MMP-1, MMP-7 and MMP-10 production, and spontaneous production of IL-6, PDGF, CXCL-9 and MMP-1 by RA synovial explants. CONCLUSIONS: Btk is expressed equivalently in RA and PsA synovial tissue, primarily in macrophages. Btk activity is needed to drive macrophage activation in response to multiple agonists relevant to inflammatory arthritis, and promotes RA synovial tissue cytokine and MMP production. Pharmacological targeting of Btk may be of therapeutic benefit in the treatment of RA and other inflammatory diseases.
Assuntos
Artrite Reumatoide/metabolismo , Isoquinolinas/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Membrana Sinovial/metabolismo , Adulto , Tirosina Quinase da Agamaglobulinemia , Idoso , Artrite Reumatoide/genética , Linfócitos B/metabolismo , Feminino , Expressão Gênica , Humanos , Imuno-Histoquímica , Interleucina-6/metabolismo , Macrófagos/efeitos dos fármacos , Masculino , Mastócitos/metabolismo , Microscopia Confocal , Pessoa de Meia-Idade , Proteínas Tirosina Quinases/efeitos dos fármacos , Proteínas Tirosina Quinases/metabolismoRESUMO
Organ transplant patients are often treated with immunosuppressants, such as the calcineurin phosphatase inhibitor, cyclosporin A, to block T cell-mediated graft rejection. The calcium release-activated calcium (CRAC/ORAI) channels, which act upstream of calcineurin, are essential for calcium entry and CD4(+) T-cell activation. Although cyclosporine A has also been shown to inhibit FoxP3(+) Tregs both in vitro and in vivo, the role of ORAI channel inhibition in natural Tregs (nTregs) or inducible Tregs (iTregs) has not been investigated. We found that, despite inhibition of calcium influx through the ORAI channels, ORAI channel inhibitors were unable to repress FoxP3 expression in mouse and human nTregs, whereas FoxP3 expression was inhibited in iTregs. In contrast, cyclosporin A inhibited FoxP3 expression in both nTregs and iTregs. We also generated mice with a T cell-specific, conditional knockout of ORAI1 and found that the mice have normal nTreg development and suppressive activity. Moreover, iTregs derived from ORAI1 conditional knockout mice develop normally and are still susceptible to ORAI channel inhibition. Our data indicate that unlike CD4(+) T cells and iTregs, nTregs are resistant to ORAI-mediated inhibition. Targeting ORAI channels potentially offers a novel way to inhibit pathologic T cells, while sparing nTreg-mediated tolerance.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/metabolismo , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Animais , Ciclosporina/farmacologia , Relação Dose-Resposta a Droga , Fatores de Transcrição Forkhead/antagonistas & inibidores , Fatores de Transcrição Forkhead/genética , Humanos , Camundongos , Relação Estrutura-Atividade , Linfócitos T Reguladores/metabolismoRESUMO
OBJECTIVE: Bruton's tyrosine kinase (BTK) plays a critical role in B cell development and function. We recently described a selective BTK inhibitor, RN486, that blocks B cell receptor (BCR) and Fcγ receptor signaling and is efficacious in animal models of arthritis. The aim of this study was to examine the potential efficacy of BTK in systemic lupus erythematosus (SLE), using an NZB × NZW mouse model of spontaneous SLE. METHODS: Mice received RN486 or its vehicle (administered in chow) at a final concentration of 30 mg/kg for 8 weeks, starting at 32 weeks of age. RESULTS: The administration of RN486 completely stopped disease progression, as determined by histologic and functional analyses of glomerular nephritis. The efficacy was associated with striking inhibition of B cell activation, as demonstrated by a significant reduction in CD69 expression in response to BCR crosslinking. RN486 markedly reduced the secretion of IgG anti-double-stranded DNA (anti-dsDNA) secretion, as determined by enzyme-linked immunosorbent and enzyme-linked immunospot assays. Flow cytometric analysis demonstrated depletion of CD138(high) B220(low) plasma cells in the spleen. RN486 inhibited secretion of IgG anti-dsDNA but not IgM anti-dsDNA, suggesting that pharmacologic blockade of BTK resembles the reported transgenic expression of low levels of endogenous BTK in B cells. In addition, RN486 may also impact the effector function of autoantibodies, as evidenced by a significant reduction in immune complex-mediated activation of human monocytes in vitro and down-regulation of the expression of macrophage-related and interferon-inducible genes in both the kidneys and spleens of treated mice. CONCLUSION: Collectively, our data suggest that BTK inhibitors may simultaneously target autoantibody-producing and effector cells in SLE, thus constituting a promising therapeutic alternative for this disease.
Assuntos
Linfócitos B/patologia , Glomerulonefrite/tratamento farmacológico , Glomérulos Renais/patologia , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Proteínas Tirosina Quinases/antagonistas & inibidores , Tirosina Quinase da Agamaglobulinemia , Animais , Complexo Antígeno-Anticorpo/metabolismo , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Regulação para Baixo , Glomerulonefrite/metabolismo , Glomerulonefrite/patologia , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/metabolismo , Lectinas Tipo C/metabolismo , Lúpus Eritematoso Sistêmico/metabolismo , Lúpus Eritematoso Sistêmico/patologia , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos NZB , Receptores de IgG/metabolismoRESUMO
BACKGROUND: Thymic stromal lymphopoietin (TSLP) pathway blockade is a potential strategy for asthma treatment because the main activities of TSLP are activation of myeloid dendritic cells (mDCs) and modulation of cytokine production by mast cells. TSLP-activated mDCs prime the differentiation of naive T cells into inflammatory TH2 cells. OBJECTIVE: We sought to investigate mechanisms underlying the development of allergic lung inflammation in cynomolgus monkeys using gene expression profiling and to assess the effect of thymic stromal lymphopoietin receptor (TSLPR) blockade in this model. METHODS: An mAb against human TSLPR was generated and confirmed to be cross-reactive to cynomolgus monkey. Animals were dosed weekly with either vehicle or anti-TSLPR mAb for 6 weeks, and their responses to allergen challenge at baseline, week 2, and week 6 were assessed. RESULTS: After 6 weeks of treatment, anti-TSLPR mAb-treated animals showed reduced bronchoalveolar lavage (BAL) fluid eosinophil counts, reduced airway resistance in response to allergen challenge, and reduced IL-13 cytokine levels in BAL fluid compared with values seen in vehicle-treated animals. Expression profiling of BAL fluid cells collected before and after challenge showed a group of genes upregulated by allergen challenge that strongly overlapped with 11 genes upregulated in dendritic cells (DCs) when in vitro stimulated by TSLP (TSLP-DC gene signature). The number of genes differentially expressed in response to challenge was reduced in antibody-treated animals after 6 weeks relative to vehicle-treated animals. Expression of the TSLP-DC gene signature was also significantly reduced in antibody-treated animals. CONCLUSION: These results demonstrate promising efficacy for TSLPR blockade in an allergic lung inflammation model in which TSLP activation of mDCs might play a key role.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Asma/terapia , Modelos Animais de Doenças , Hipersensibilidade/terapia , Inflamação/terapia , Receptores de Citocinas/antagonistas & inibidores , Animais , Anticorpos Monoclonais/imunologia , Asma/imunologia , Cricetinae , Citocinas/imunologia , Citocinas/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Feminino , Humanos , Hipersensibilidade/imunologia , Inflamação/imunologia , Macaca fascicularis/imunologia , Receptores de Citocinas/imunologia , Células Th2/imunologia , Células Th2/metabolismo , Linfopoietina do Estroma do TimoRESUMO
Genetic mutation and pharmacological inhibition of Bruton's tyrosine kinase (Btk) both have been shown to prevent the development of collagen-induced arthritis (CIA) in mice, providing a rationale for the development of Btk inhibitors for treating rheumatoid arthritis (RA). In the present study, we characterized a novel Btk inhibitor, 6-cyclopropyl-8-fluoro-2-(2-hydroxymethyl-3-{1-methyl-5-[5-(4-methyl-piperazin-1-yl)-pyridin-2-ylamino]-6-oxo-1,6-dihydro-pyridin-3-yl}-phenyl)-2H-isoquinolin-1-one (RN486), in vitro and in rodent models of immune hypersensitivity and arthritis. We demonstrated that RN486 not only potently and selectively inhibited the Btk enzyme, but also displayed functional activities in human cell-based assays in multiple cell types, blocking Fcε receptor cross-linking-induced degranulation in mast cells (IC(50) = 2.9 nM), Fcγ receptor engagement-mediated tumor necrosis factor α production in monocytes (IC(50) = 7.0 nM), and B cell antigen receptor-induced expression of an activation marker, CD69, in B cells in whole blood (IC(50) = 21.0 nM). RN486 displayed similar functional activities in rodent models, effectively preventing type I and type III hypersensitivity responses. More importantly, RN486 produced robust anti-inflammatory and bone-protective effects in mouse CIA and rat adjuvant-induced arthritis (AIA) models. In the AIA model, RN486 inhibited both joint and systemic inflammation either alone or in combination with methotrexate, reducing both paw swelling and inflammatory markers in the blood. Together, our findings not only demonstrate that Btk plays an essential and conserved role in regulating immunoreceptor-mediated immune responses in both humans and rodents, but also provide evidence and mechanistic insights to support the development of selective Btk inhibitors as small-molecule disease-modifying drugs for RA and potentially other autoimmune diseases.
Assuntos
Artrite Experimental/tratamento farmacológico , Artrite Experimental/imunologia , Hipersensibilidade/imunologia , Hipersensibilidade/prevenção & controle , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Tirosina Quinases/antagonistas & inibidores , Tirosina Quinase da Agamaglobulinemia , Animais , Artrite Experimental/enzimologia , Células Cultivadas , Feminino , Humanos , Hipersensibilidade/enzimologia , Masculino , Mastócitos/efeitos dos fármacos , Mastócitos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos WistarRESUMO
The transcription factor interferon regulatory factor 5 (IRF5) plays essential roles in pathogen-induced immunity downstream of Toll-, nucleotide-binding oligomerization domain-, and retinoic acid-inducible gene I-like receptors and is an autoimmune susceptibility gene. Normally, inactive in the cytoplasm, upon stimulation, IRF5 undergoes posttranslational modification(s), homodimerization, and nuclear translocation, where dimers mediate proinflammatory gene transcription. Here, we report the rational design of cell-penetrating peptides (CPPs) that disrupt IRF5 homodimerization. Biochemical and imaging analysis shows that IRF5-CPPs are cell permeable, noncytotoxic, and directly bind to endogenous IRF5. IRF5-CPPs were selective and afforded cell type- and species-specific inhibition. In plasmacytoid dendritic cells, inhibition of IRF5-mediated interferon-α production corresponded to a dose-dependent reduction in nuclear phosphorylated IRF5 [p(Ser462)IRF5], with no effect on pIRF5 levels. These data support that IRF5-CPPs function downstream of phosphorylation. Together, data support the utility of IRF5-CPPs as novel tools to probe IRF5 activation and function in disease.
Assuntos
Peptídeos Penetradores de Células , Peptídeos Penetradores de Células/genética , Peptídeos Penetradores de Células/metabolismo , Peptídeos Penetradores de Células/farmacologia , Células Dendríticas/metabolismo , Regulação da Expressão Gênica , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , FosforilaçãoRESUMO
Platelet microparticles (pMPs) are small membrane-coated vesicles that are released from the plasma membrane upon platelet activation. In the joint fluid of patients with rheumatoid arthritis, pMP can interact with and activate fibroblast-like synoviocytes (FLS), which are important effector cells that mediate both immune activation and joint destruction. The signaling process by which engagement of glycoprotein VI (GPVI), a surface glycoprotein receptor for collagen which is expressed on platelets, triggers pMP generation is poorly understood, but has been suggested to involve Spleen Tyrosine Kinase (SYK), best known as an upstream activator of Bruton's Tyrosine Kinase (BTK) in B cells. In this study, we showed that activation of human platelets triggered by convulxin or collagen, specific ligands for GPVI receptor, or alternatively by antibody-mediated cross-linking of another platelet receptor, C type lectin-like receptor 2 (CLEC2), resulted in phosphorylation of BTK and downstream effector, phospholipase Cγ2 (PLCγ2). A potent and selective BTK inhibitor, RN486, inhibited GPVI- or CLEC2-mediated PLCγ2 phosphorylation and pMP production in a dose-dependent manner. BTK is also an essential effector of B cell receptor (BCR)-induced B cell signaling. Consistent with the biology, the IC50s of BTK inhibitors with varying potencies in a BCR-dependent B cell activation marker assay correlated with those in the GPVI-mediated PLCγ2 phosphorylation. In a co-culture system consisting of human primary synovial FLS and activated human platelets, convulxin stimulation resulted in elevated production of pro-inflammatory cytokines, IL-6 and IL-8, an effect which was dose-dependently blocked by RN486. The effects are specific as RN486 abrogated platelet aggregation induced by GPVI ligands but not by other platelet surface receptor agonists. Taken together, our data further support the potential therapeutic utility of BTK inhibitors in RA therapy, by inhibiting GPVI-mediated platelet activation and thus subsequent amplification of inflammation driven by pMP-induced FLS cytokines production.
Assuntos
Plaquetas/metabolismo , Micropartículas Derivadas de Células/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Proteínas Tirosina Quinases/metabolismo , Sinovite/metabolismo , Tirosina Quinase da Agamaglobulinemia , Artrite Reumatoide/metabolismo , Linfócitos B/imunologia , Catálise , Técnicas de Cocultura , Humanos , Interleucina-6/biossíntese , Interleucina-8/biossíntese , Lectinas Tipo C/metabolismo , Ativação Linfocitária/imunologia , Fosfolipase C gama/metabolismo , Fosforilação , Ativação Plaquetária , Agregação Plaquetária/efeitos dos fármacos , Glicoproteínas da Membrana de Plaquetas/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Receptores de Antígenos de Linfócitos B/metabolismoRESUMO
Spleen tyrosine kinase (SYK) and Bruton's tyrosine kinase (BTK) are key mediators in coupling cell surface receptors, such as the B-cell receptor (BCR), to downstream signaling events affecting diverse biological functions. There is therefore tremendous interest in the development of pharmacological inhibitors targeting the SYK-BTK axis for the treatment of inflammatory disorders and hematological malignancies. A good pharmacodynamic (PD) assay, ideally a blood-based assay that measures proximal events, is warranted for evaluation of such inhibitors. In platelets, collagen-induced activation of membrane glycoprotein GPVI is dependent on the SYK-BTK axis. Here, we report the development of a novel immunoassay that uses the dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) to measure GPVI-mediated phosphorylation of phospholipase C γ2 (PLCγ2), a direct substrate of SYK and BTK, in platelets. The assay was validated using SYK or BTK inhibitors and generated IC50 correlated with those from the BCR-induced B-cell activation assay. Furthermore, this assay showed good stability and uniformity over a period of 24 h in different donors. Interestingly, compound IC50 values using blood from patients with rheumatoid arthritis were slightly higher compared with those produced using samples from healthy donors. This novel platelet PLCγ2 phosphorylation-based immunoassay should serve as a promising PD assay for preclinical and clinical development of inhibitors targeting the SYK-BTK axis.
Assuntos
Ensaios Enzimáticos/métodos , Imunoensaio/métodos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Fosfolipase C gama/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Tirosina Quinase da Agamaglobulinemia , Antígenos CD/análise , Antígenos de Diferenciação de Linfócitos T/análise , Artrite Reumatoide/sangue , Artrite Reumatoide/metabolismo , Plaquetas/citologia , Plaquetas/metabolismo , Neoplasias Hematológicas/metabolismo , Humanos , Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lectinas Tipo C/análise , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , Quinase SykRESUMO
INTRODUCTION: Spleen tyrosine kinase (SYK) is a key integrator of intracellular signals triggered by activated immunoreceptors, including Bcell receptors (BCR) and Fc receptors, which are important for the development and function of lymphoid cells. Given the clinical efficacy of Bcell depletion in the treatment of rheumatoid arthritis and multiple sclerosis, pharmacological modulation of Bcells using orally active small molecules that selectively target SYK presents an attractive alternative therapeutic strategy. METHODS: A SYK inhibitor was developed and assayed in various in vitro systems and in the mouse model of collagen-induced arthritis (mCIA). RESULTS: A novel ATP-competitive inhibitor of SYK, 6-[(1R,2S)-2-Amino-cyclohexylamino]-4-(5,6-dimethyl-pyridin-2-ylamino)-pyridazine-3-carboxylic acid amide, designated RO9021, with an adequate kinase selectivity profile and oral bioavailability, was developed. In addition to suppression of BCR signaling in human peripheral blood mononuclear cells (PBMC) and whole blood, FcγR signaling in human monocytes, and FcϵR signaling in human mast cells, RO9021 blocked osteoclastogenesis from mouse bone marrow macrophages in vitro. Interestingly, Toll-like Receptor (TLR) 9 signaling in human Bcells was inhibited by RO9021, resulting in decreased levels of plasmablasts, immunoglobulin (Ig) M and IgG upon B-cell differentiation. RO9021 also potently inhibited type I interferon production by human plasmacytoid dendritic cells (pDC) upon TLR9 activation. This effect is specific to TLR9 as RO9021 did not inhibit TLR4- or JAK-STAT-mediated signaling. Finally, oral administration of RO9021 inhibited arthritis progression in the mCIA model, with observable pharmacokinetics (PK)-pharmacodynamic (PD) correlation. CONCLUSIONS: Inhibition of SYK kinase activity impinges on various innate and adaptive immune responses. RO9021 could serve as a starting point for the development of selective SYK inhibitors for the treatment of inflammation-related and autoimmune-related disorders.
Assuntos
Imunidade Adaptativa/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Administração Oral , Aminopiridinas/química , Aminopiridinas/farmacologia , Animais , Artrite Experimental/metabolismo , Artrite Experimental/prevenção & controle , Doenças Autoimunes/tratamento farmacológico , Doenças Autoimunes/metabolismo , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Citometria de Fluxo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células Jurkat , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Modelos Moleculares , Estrutura Molecular , Osteoclastos/citologia , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Ligação Proteica , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/química , Estrutura Terciária de Proteína , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismo , Piridazinas/química , Piridazinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/administração & dosagem , Bibliotecas de Moléculas Pequenas/química , Quinase SykRESUMO
Store operated calcium entry (SOCE) downstream of T cell receptor (TCR) activation in T lymphocytes has been shown to be mediated mainly through the Calcium Release Activated Calcium (CRAC) channel. Here, we compared the effects of a novel, potent and selective CRAC current inhibitor, 2,6-Difluoro-N-{5-[4-methyl-1-(5-methyl-thiazol-2-yl)-1,2,5,6-tetrahydro-pyridin-3-yl]-pyrazin-2-yl}-benzamide (RO2959), on T cell effector functions with that of a previously reported CRAC channel inhibitor, YM-58483, and a calcineurin inhibitor Cyclosporin A (CsA). Using both electrophysiological and calcium-based fluorescence measurements, we showed that RO2959 is a potent SOCE inhibitor that blocked an IP3-dependent current in CRAC-expressing RBL-2H3 cells and CHO cells stably expressing human Orai1 and Stim1, as well as SOCE in human primary CD4(+) T cells triggered by either TCR stimulation or thapsigargin treatment. Furthermore, we demonstrated that RO2959 completely inhibited cytokine production as well as T cell proliferation mediated by TCR stimulation or MLR (mixed lymphocyte reaction). Lastly, we showed by gene expression array analysis that RO2959 potently blocked TCR triggered gene expression and T cell functional pathways similar to CsA and another calcineurin inhibitor FK506. Thus, both from a functional and transcriptional level, our data provide evidence that RO2959 is a novel and selective CRAC current inhibitor that potently inhibits human T cell functions.
Assuntos
Linfócitos T CD4-Positivos/efeitos dos fármacos , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/metabolismo , Anilidas/farmacologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Células CHO , Calcineurina/metabolismo , Inibidores de Calcineurina , Cálcio/metabolismo , Canais de Cálcio/genética , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Cricetinae , Ciclosporina/farmacologia , Citocinas/genética , Citocinas/metabolismo , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Humanos , Ativação Linfocitária/efeitos dos fármacos , Teste de Cultura Mista de Linfócitos/métodos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína ORAI1 , Ratos , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Molécula 1 de Interação Estromal , Tacrolimo/farmacologia , Tiadiazóis/farmacologiaRESUMO
The expression of the cannabinoid peripheral cannabinoid receptor (CB(2)) receptor on peripheral immune cells suggests that compounds specific for CB(2) might be effective anti-inflammatory agents. In this report, we present the initial biological profiling of a novel triaryl bis-sulfone, Sch.336 (N-[1(S)-[4-[[4-methoxy-2-[(4-methoxyphenyl)sulfonyl]phenyl]-sulfonyl]phenyl]ethyl]methanesulfonamide), which is selective for the human cannabinoid CB(2) receptor (hCB(2)). Sch.336 is an inverse agonist at hCB(2), as shown by its ability to decrease guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding to membranes containing hCB(2), by the ability of GTPgammaS to left-shift Sch.336 binding to hCB(2) in these membranes, and by the compound's ability to increase forskolin-stimulated cAMP levels in CHO cells expressing hCB(2). In these systems, Sch.336 displays a greater potency than that reported for the CB(2)-selective dihydropyrazole, SR144528 (N-[(1S)-endo-1,3,3-trimethylbicyclo [2.2.1]heptan2-yl]-5-(4-chloro-3-methylphenyl)-1-[(4-methylphenyl)methyl]-1H-pyrazole-3-carboxamide). In vitro, Sch.336 impairs the migration of CB(2)-expressing recombinant cell lines to the cannabinoid agonist 2-arachidonylglycerol. In vivo, the compound impairs migration of cells to cannabinoid agonist HU210 [(6aR)-trans-3-(1,1-dimethylheptyl)-6a,7,10,10a-tetrahydro-1-hydroxy-6,6-dimethyl-6H-dibenzo [b,d] pyran-9-methanol]. Oral administration of the Sch.336 significantly inhibited leukocyte trafficking in several rodent in vivo models, induced either by specific chemokines or by antigen challenge. Finally, oral administration of Sch.336 blocked ovalbumin-induced lung eosinophilia in mice, a disease model for allergic asthma. We conclude that selective cannabinoid CB(2) inverse agonists may serve as novel immunomodulatory agents in the treatment of a broad range of acute and chronic inflammatory disorders in which leukocyte recruitment is a hallmark of disease pathology.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Canfanos/farmacologia , Canabinoides/farmacologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Leucócitos Mononucleares/efeitos dos fármacos , Pirazóis/farmacologia , Receptor CB2 de Canabinoide/agonistas , Animais , Anti-Inflamatórios não Esteroides/uso terapêutico , Células CHO , Canfanos/uso terapêutico , Canabinoides/uso terapêutico , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Cricetinae , Cricetulus , AMP Cíclico/metabolismo , Feminino , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Hipersensibilidade Tardia/tratamento farmacológico , Leucócitos Mononucleares/metabolismo , Camundongos , Camundongos Endogâmicos , Ligação Proteica , Eosinofilia Pulmonar/tratamento farmacológico , Pirazóis/uso terapêutico , Receptor CB2 de Canabinoide/biossínteseRESUMO
Studies to characterize the endogenous expression and pharmacology of peripheral human cannabinoid receptor (hCB2) have been hampered by the dearth of authentic anti-hCB2 antibodies and the lack of radioligands with CB2 selectivity. We recently described a novel CB2 inverse agonist, N-[1(S)-[4-[[4-methoxy-2-[(4methoxyphenyl)sulfonyl] phenyl]sulfonyl] phenyl]ethyl]methane-sulfonamide (Sch225336), that binds hCB2 with high affinity and excellent selectivity versus hCB1. The precursor primary amine of Sch225336 was prepared and reacted directly with [(35)S]mesyl chloride (synthesized from commercially obtained [(35)S]methane sulfonic acid) to generate [(35)S]Sch225336. [(35)S]Sch225336 has high specific activity (>1,400 Ci/mmol) and affinity for hCB2 (65 pm). Using [(35)S]Sch225336, we assayed hemopoietic cells and cell lines to quantitate the expression and pharmacology of hCB2. Lastly, we used [(35)S]Sch225336 for detailed autoradiographic analysis of CB2 in lymphoid tissues. Based on these data, we conclude that [(35)S]Sch225336 represents a unique radioligand for the study of CB2 endogenously expressed in blood cells and tissues.
Assuntos
Receptor CB2 de Canabinoide/agonistas , Receptor CB2 de Canabinoide/análise , Sulfonamidas/metabolismo , Animais , Autorradiografia , Western Blotting , Células CHO , Cricetinae , Células HL-60 , Humanos , Linfócitos/química , Ensaio Radioligante , Baço/químicaRESUMO
A novel class of cannabinoid CB2 receptor ligands is described. These triaryl bis-sulfones are nanomolar inhibitors of the CB2 receptor and show high selectivity over the cannabinoid CB1 receptor. One example of this new class decreases ligand-induced GTPgammaS binding to recombinant CB2 cell membranes, identifying the compound as a CB2-selective inverse agonist.
Assuntos
Receptor CB2 de Canabinoide/antagonistas & inibidores , Sulfonas/síntese química , Sulfonas/farmacologia , Amidas/química , Amidas/farmacologia , Canabinoides/farmacologia , Membrana Celular/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Ligantes , Relação Estrutura-Atividade , Sulfonamidas/química , Sulfonamidas/farmacologia , Ureia/química , Ureia/farmacologiaRESUMO
The human P2Y6 receptor (hP2Y6) is a member of the G protein-coupled pyrimidinergic P2 receptor family that responds specifically to the extracellular nucleotide uridine diphosphate (UDP). Recently, the hP2Y6 receptor has been reported to mediate monocyte IL-8 production in response to UDP or lipopolysaccharide (LPS), but the role of hP2Y6 in regulating other pro-inflammatory cytokines or mediators is largely unknown. We demonstrate here that UDP specifically induces soluble TNF-alpha and IL-8 production in a promonocytic U937 cell line stably transfected with hP2Y6. However, we did not detect IL-1alpha, IL-1beta, IL-6, IL-10, IL-18, and PGE2 in the conditioned media from the same cell line. These results distinguish UDP/P2Y6 signaling from LPS signaling. Interestingly, UDP induces the production of IL-8, but not TNF-alpha, in human astrocytoma 1321N1 cell lines stably transfected with hP2Y6. Therefore, the immune effect of UDP/P2Y6 signaling on the production of proinflammatory cytokines is selective and dependent on cell types. We further identify that UDP can also induce the production of proinflammatory chemokines MCP-1 and IP-10 in hP2Y6 transfected promonocytic U937 cell lines, but not astrocytoma 1321N1 cell lines stably transfected with hP2Y6. From the Taqman analysis, UDP stimulation significantly upregulates the mRNA levels of IL-8, IP-10, and IL-1beta, but not TNF-alpha. Taken together, these new findings expand the pro-inflammatory biology of UDP mediated by the P2Y6 receptor.
Assuntos
Quimiocinas/metabolismo , Citocinas/metabolismo , Monócitos/efeitos dos fármacos , Receptores Purinérgicos P2/fisiologia , Difosfato de Uridina/farmacologia , Sequência de Bases , Linhagem Celular Tumoral , Quimiocinas/genética , Citocinas/genética , Primers do DNA , Humanos , Monócitos/metabolismo , RNA Mensageiro/genéticaRESUMO
The KCNN4 potassium-ion channel has been reported to play an important role in regulating antigen-induced T cell effector functions in vitro. This study presents the first evidence that a selective KCNN4 blocker, TRAM-34, confers protection against experimental autoimmune encephalomyelitis (EAE) in the mouse model. Treatment with the KCNN4 blocker did not prevent infiltration of T cells in the spinal cord, but resulted in the reduction of both the protein and the message levels of TNF-alpha and IFN-gamma as well as the message levels of several other pro-inflammatory molecules in the spinal cord. Plasma concentrations of TRAM-34 within a 24-h period were between the in vitro IC(50) and IC(90) values for the KCNN4 channel. The effect of TRAM-34 was reversible, as indicated by the development of clinical EAE symptoms within 48 h after withdrawal of treatment. In summary, our data support the idea that KCNN4 channels play a critical role in the immune response during the development of MOG-induced EAE in C57BL/6 mice.
Assuntos
Encefalomielite Autoimune Experimental/tratamento farmacológico , Canais de Potássio Cálcio-Ativados/antagonistas & inibidores , Animais , Movimento Celular/imunologia , Movimento Celular/fisiologia , Encefalomielite Autoimune Experimental/prevenção & controle , Inflamação/imunologia , Canais de Potássio Ativados por Cálcio de Condutância Intermediária , Camundongos , RNA Mensageiro/metabolismo , Medula Espinal/imunologia , Medula Espinal/fisiologiaRESUMO
OBJECTIVE: Collagen-induced arthritis (CIA) in the rhesus monkey is a nonhuman primate model of rheumatoid arthritis (RA). The close phylogenetic relationship between humans and the rhesus monkey makes this model useful for the preclinical safety and efficacy testing of new therapies that are inactive in animals more distinctly related to humans. In this study, we tested the therapeutic potential of a novel, small molecular weight antagonist of CCR5, SCH-X, in this model. METHODS: CIA was induced in 10 rhesus monkeys. The animals were allocated to receive SCH-X or saline as the control (n = 5 in each group). Treatment was initiated on the day of CIA induction and continued for 45 days. Monkeys were monitored before and 63 days after CIA induction for macroscopic signs of clinical arthritis, such as soft-tissue swelling and body weight. Furthermore, markers of inflammation and joint degradation were monitored to follow the disease course. RESULTS: Only 2 of 5 animals in the SCH-X-treated group displayed prominent soft-tissue swelling, compared with all 5 saline-treated monkeys. In addition to the suppression of joint inflammation, treatment with SCH-X resulted in a reduction in joint destruction, as demonstrated by lower rates of urinary excretion of collagen crosslinks, with confirmation by histology. Whereas in all saline-treated monkeys, marked erosion of joint cartilage was observed, this was absent in 4 of the 5 SCH-X-treated monkeys. CONCLUSION: The systemic effects of treatment with SCH-X were a suppressed acute-phase reaction (reduction in C-reactive protein level) in the 3 treated monkeys with CIA that remained asymptomatic, and an altered antibody response toward type II collagen. The results suggest that the CCR5 antagonist SCH-X might have a strong clinical potential for treatment during periods of active inflammation, as seen in RA.
Assuntos
Artrite Experimental/prevenção & controle , Antagonistas dos Receptores CCR5 , Animais , Artrite Experimental/induzido quimicamente , Artrite Experimental/patologia , Cartilagem Articular/patologia , Colágeno Tipo II , Seguimentos , Macaca mulatta , MasculinoRESUMO
Cloned T regulatory type 1 (Tr1) cells produce IL-10, TGF-beta, IFN-gamma, and very low or non-detectable levels of IL-2 and IL-4, following TCR-mediated activation. In addition, upon TCR stimulation, Tr1 cell clones up-regulate activation markers but show low proliferative responses, partially due to the suppressive effect of autocrine IL-10 and TGF-beta. Here we show that Tr1 cells have growth requirements different from those of Th1 and Th2 cells. Exogenous IL-15, and to a lesser extent IL-2, induce and support the proliferation of Tr1 cells in the absence of TCR activation. This strong cytokine response correlates with high constitutive levels of the IL-2/15Rbeta and common gamma chains expressed by Tr1 cell clones. Furthermore, suboptimal doses of IL-15, in combination with IL-2, induce a significant growth (median value: 25-fold increase in cell number) of Tr1 cell clones during a culture period of 11 days, which leads to an in vitro expansion of Tr1 cell clones comparable to that of Th1 and Th2 cell clones. Tr1 cell clones cultured in IL-15 continue to secrete immunosuppressive cytokines and to proliferate poorly upon reactivation via TCR. These findings indicate that Tr1 cells are constitutively capable of responding to cytokines and mainly to IL-15. This growth factor enables a significant in vitro expansion of Tr1 cells facilitating further biological and biochemical characterization of this unique T cell subset.
Assuntos
Citocinas/farmacologia , Ativação Linfocitária , Receptores de Antígenos de Linfócitos T/fisiologia , Linfócitos T/fisiologia , Células Clonais , Citocinas/biossíntese , Humanos , Interleucina-15/farmacologia , Interleucina-2/farmacologia , Interleucina-4/farmacologia , Interleucina-7/farmacologia , Receptores de Interleucina-2/análiseRESUMO
Gallic acid (GA) and several gallate derivatives were identified as inhibitors of fucosyltransferase VII (FucT VII). The inhibition by GA and (-)-epigallocatechin gallate (EGCG) is time-dependent and irreversible. GA and EGCG showed inhibition with IC(50) of 60 and 700 nM, respectively, after pre-incubation with FucT VII in the presence of MnCl(2). Absence of MnCl(2) results in significantly weaker inhibition. Complexation of Mn(2+) with GA, EGCG, and gallate esters was observed. Such complexation, however, is not rate-limiting for the inhibition of FucT VII. Therefore, time-dependent inhibition of fucosyltransferases by GA and EGCG is likely due to the slow inactivation by the inhibitors or Mn-inhibitor complex. Although Mg(2+) or Ca(2+) can replace Mn(2+) for FucT VII activation, none forms a complex with GA or EGCG and hence results in weaker inhibition of FucT VII. GA and EGCG also inhibit FucT IV and alpha2,3-(N)-sialyltransferase in the low micromolar range. The structure-function divergence could be observed, as EGCG, but not GA or gallate esters, inhibits Zn(2+) containing metalloproteases such as TNFalpha convertase, matrix metalloproteases 2 and 7.
Assuntos
Catequina/análogos & derivados , Fucosiltransferases/antagonistas & inibidores , Ácido Gálico/farmacologia , Catequina/metabolismo , Catequina/farmacologia , Ácido Elágico/metabolismo , Ácido Elágico/farmacologia , Enzimas/metabolismo , Fucosiltransferases/metabolismo , Ácido Gálico/análogos & derivados , Ácido Gálico/metabolismo , Humanos , Manganês/metabolismoRESUMO
Interleukin-17 (IL-17) is a CD4 T-cell-derived proinflammatory cytokine. We investigated the effects of locally produced IL-17 by tumors as a means to evaluate its biologic function. Although recombinant IL-17 protein or retroviral transduction of IL-17 gene into tumors did not affect in vitro proliferation, IL-17 transfectants grew more rapidly in vivo when compared with controls. Immunostaining for Factor VIII revealed that tumors transduced with IL-17 had significantly higher vascular density when compared with controls. IL-17 indeed elicited neovascularization in rat cornea. In addition, angiogenic activity present in the conditioned media of CD4 T cells was markedly suppressed by neutralizing monoclonal antibody to IL-17. IL-17 had no direct effect on the growth of vascular endothelial cells, whereas IL-17 significantly stimulated migration. IL-17 also markedly promoted the cord formation of vascular endothelial cells. In addition, IL-17 up-regulated elaboration of a variety of proangiogenic factors by fibroblasts as well as tumor cells. These findings reveal a novel role for IL-17 as a CD4 T-cell-derived mediator of angiogenesis that stimulates vascular endothelial cell migration and cord formation and regulates production of a variety of proangiogenic factors. Furthermore, they suggest that inhibition of biologic action of IL-17 may have therapeutic benefits when applied to angiogenesis-related disorders.
Assuntos
Interleucina-17/farmacologia , Neoplasias Experimentais/patologia , Neovascularização Patológica/induzido quimicamente , Animais , Divisão Celular/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , Córnea/irrigação sanguínea , Córnea/efeitos dos fármacos , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Feminino , Substâncias de Crescimento/biossíntese , Humanos , Interleucina-17/genética , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Experimentais/etiologia , Ratos , Transdução Genética , Veias Umbilicais/citologia , Regulação para Cima/efeitos dos fármacosRESUMO
We previously reported that interleukin-10 (IL-10) and transforming growth factor (TGF)-beta treatment of primary mixed lymphocyte reaction (MLR) cultures resulted in secondary alloantigen-specific hyporesponsiveness and protection from graft-versus-host disease (GVHD) lethality. Here, we report that CD4+ T cells recovered from the IL-10- and TGF-beta-treated primary MLR cultures have immunoregulatory function. Tolerized cells significantly inhibited proliferation of naive alloreactive CD4+ T cells in a primary MLR. Inhibition of the naive alloresponse was observed with as few as 1 tolerized cell to 10 naive responder cells. Tolerized cells were able to significantly reduce GVHD lethality when injected with naive alloreactive CD4+ T cells into major histocombatibility class (MHC) II disparate recipients. Rigorous CD25 depletion of the primary MLR had no effect on generation of a regulatory capacity, suggesting that the regulatory cells likely originated from CD4+CD25- T cells. Immune suppression was mediated independently of IL-10 and TGF-beta production, as neutralizing antibodies for IL-10, IL-10R, and TGF-beta were unable to revert suppression, and IL-10- deficient CD4+ T cells were able to mediate in vitro and in vivo suppression. The generation of immunoregulatory cells from a CD4+CD25- population during tolerization with IL-10 and TGF-beta provides an additional mechanism to prevent GVHD lethality by T cells that may escape full tolerance induction.