RESUMO
High homocysteine (HCy) levels are associated with lymphocyte-mediated inflammatory responses that are sometimes in turn related to hypoxia. Because adenosine is a potent lymphocyte suppressor produced in hypoxic conditions and shares metabolic pathways with HCy, we addressed the influence of high HCy levels on the hypoxia-induced, adenosine-mediated, alteration of lymphocyte viability. We treated mitogen-stimulated human lymphocytes isolated from healthy individuals and the human lymphoma T-cell line CEM with cobalt chloride (CoCl2 )to reproduce hypoxia. We found that CoCl2 -altered cell viability was dose-dependently reversed using HCy. In turn, the HCy effect was inhibited using DL-propargylglycine, a specific inhibitor of the hydrogen sulphide (H2 S)-synthesizing enzyme cystathionine-γ-lyase involved in HCy catabolism. We then addressed the intracellular metabolic pathway of adenosine and HCy, and the role of the adenosine A2A receptor (A2 A R). We observed that: (i) hypoxic conditions lowered the intracellular concentration of HCy by increasing adenosine production, which resulted in high A2 A R expression and 3', 5'-cyclic adenosine monophosphate production; (ii) increasing intracellular HCy concentration reversed the hypoxia-induced adenosinergic signalling despite high adenosine concentration by promoting both S-adenosylhomocysteine and H2 S production; (iii) DL-propargylglycine that inhibits H2 S production abolished the HCy effect. Together, these data suggest that high HCy levels prevent, via H2 S production and the resulting down-regulation of A2 A R expression, the hypoxia-induced adenosinergic alteration of lymphocyte viability. We point out the relevance of these mechanisms in the pathophysiology of cardiovascular diseases.
Assuntos
Cobalto/toxicidade , Homocisteína/farmacologia , Sulfeto de Hidrogênio/farmacologia , Linfócitos/citologia , Adenosina/metabolismo , Adulto , Alcinos/farmacologia , Hipóxia Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Glicina/análogos & derivados , Glicina/farmacologia , Homocisteína/metabolismo , Humanos , Hiper-Homocisteinemia/metabolismo , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Pessoa de Meia-Idade , Modelos Biológicos , Transdução de Sinais/efeitos dos fármacosRESUMO
The nucleoside adenosine acts on the nervous and cardiovascular systems via the A2A receptor (A2AR). In response to oxygen level in tissues, adenosine plasma concentration is regulated in particular via its synthesis by CD73 and via its degradation by adenosine deaminase (ADA). The cell-surface endopeptidase CD26 controls the concentration of vasoactive and antioxidant peptides and hence regulates the oxygen supply to tissues and oxidative stress response. Although overexpression of adenosine, CD73, ADA, A2AR, and CD26 in response to hypoxia is well documented, the effects of hyperoxic and hyperbaric conditions on these elements deserve further consideration. Rats and a murine Chem-3 cell line that expresses A2AR were exposed to 0.21 bar O2, 0.79 bar N2 (terrestrial conditions; normoxia); 1 bar O2 (hyperoxia); 2 bar O2 (hyperbaric hyperoxia); 0.21 bar O2, 1.79 bar N2 (hyperbaria). Adenosine plasma concentration, CD73, ADA, A2AR expression, and CD26 activity were addressed in vivo, and cAMP production was addressed in cellulo. For in vivo conditions, 1) hyperoxia decreased adenosine plasma level and T cell surface CD26 activity, whereas it increased CD73 expression and ADA level; 2) hyperbaric hyperoxia tended to amplify the trend; and 3) hyperbaria alone lacked significant influence on these parameters. In the brain and in cellulo, 1) hyperoxia decreased A2AR expression; 2) hyperbaric hyperoxia amplified the trend; and 3) hyperbaria alone exhibited the strongest effect. We found a similar pattern regarding both A2AR mRNA synthesis in the brain and cAMP production in Chem-3 cells. Thus a high oxygen level tended to downregulate the adenosinergic pathway and CD26 activity. Hyperbaria alone affected only A2AR expression and cAMP production. We discuss how such mechanisms triggered by hyperoxygenation can limit, through vasoconstriction, the oxygen supply to tissues and the production of reactive oxygen species.
Assuntos
Adenosina/genética , Dipeptidil Peptidase 4/genética , Hiperóxia/genética , Transdução de Sinais/genética , 5'-Nucleotidase/genética , Adenosina Desaminase/genética , Animais , Linhagem Celular , AMP Cíclico/genética , Regulação para Baixo/genética , Masculino , Camundongos , Estresse Oxidativo/genética , Oxigênio/metabolismo , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Receptor A2A de Adenosina/genéticaRESUMO
Hypoxia affects inflammation by modulating T-cell activation via the adenosinergic system. We supposed that, in turn, inflammation influences cell hypoxic behavior and that stimulation of T-cells in inflammatory conditions involves the concerted action of the nuclear factor κB (NF-κB) and the related hypoxia-inducible factor 1α (HIF-1α) on the adenosinergic system. We addressed this hypothesis by monitoring both transcription factors and four adenosinergic signaling parameters - namely adenosine, adenosine deaminase (ADA), adenosine A2A receptor (A2AR) and cAMP - in T-cells stimulated using phorbol myristate acetate and phytohemagglutinin and submitted to hypoxic conditions which were mimicked using CoCl2 treatment. We found that cell viability was more altered in stimulated than in resting cells under hypoxia. Detailed analysis showed that: i) NF-κB activation remained at basal level in resting hypoxic cells but greatly increased following stimulation, stimulated hypoxic cells exhibiting the higher level; ii) HIF-1α production induced by hypoxia was boosted via NF-κB activation in stimulated cells whereas hypoxia increased HIF-1α production in resting cells without further activating NF-κB; iii) A2AR expression and cAMP production increased in stimulated hypoxic cells whereas adenosine level remained unchanged due to ADA regulation; and iv) the presence of H2S, an endogenous signaling molecule in inflammation, reversed the effect of stimulation on cell viability by down-regulating the activity of transcription factors and adenosinergic immunosuppression. We also found that: i) the specific A2AR agonist CGS-21680 increased the suppressive effect of hypoxia on stimulated T-cells, the antagonist ZM-241385 exhibiting the opposite effect; and ii) Rolipram, a selective inhibitor of cAMP-specific phosphodiesterase 4, and 8-Br-cAMP, a cAMP analog which preferentially activates cAMP-dependent protein kinase A (PKA), increased T-cell immunosuppression whereas H-89, a potent and selective inhibitor of cAMP-dependent PKA, restored cell viability. Together, these data indicate that inflammation enhances T-cell sensitivity to hypoxia via NF-κB activation. This process upregulates A2AR expression and enhances cAMP production and PKA activation, resulting in adenosinergic T-cell immunosuppression that can be modulated via H2S.
Assuntos
Hipóxia Celular , NF-kappa B/metabolismo , Receptor A2A de Adenosina/metabolismo , Sulfitos/toxicidade , Linfócitos T/metabolismo , Agonistas do Receptor A2 de Adenosina/farmacologia , Antagonistas do Receptor A2 de Adenosina/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cobalto/toxicidade , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Terapia de Imunossupressão , Isoquinolinas/farmacologia , Receptor A2A de Adenosina/química , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , Linfócitos T/citologia , Triazinas/farmacologia , Triazóis/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
Some ligand-receptor couples involve spare receptors, which are apparent when a maximal response is achieved with only a small fraction of the receptor population occupied. This situation favours cross-reactions with low-affinity ligands, which may be detrimental for cell signaling. In the case of the adenosine A2A receptors (A2AR), which have an immunosuppressive effect on lymphocytes through cAMP production, the presence of spare A2AR remains to be established. We examined the situation using patients over-expressing lymphocyte A2AR and an agonist-like mAb to A2AR. We found that maximal mAb binding and functional response varied among the patients whereas the dissociation constant and half-maximal effective concentration had similar mean values (0.19 and 0.18 µM, respectively). Lymphocyte A2AR expression was correlated to plasma adenosine level and A2AR occupation but not to A2AR response. These results are consistent with a lack of a reserve of functional A2AR on human lymphocytes as a general rule and suggest that the amount and functional state of the expressed A2AR determine the maximal level of the lymphocyte response to adenosine.
RESUMO
We examined the short-course expression of various parameters involved in the adenosinergic signalling of a human T cell line during in vitro decrease of the medium culture oxygen tension mimicking in vivo hypoxia. Fall of 92 mmHg in oxygen tension of culture medium induced in CEM, a CD4+ human T cell line, a continuous production of hypoxia-inducing factor-1α with a plateau value at 9 h, a rapid increase in adenosine production peaking at 3 h and a decrease in adenosine deaminase peaking at 6 h. The adenosine A(2A) receptor (A(2A)R) protein level of CEM cells was enhanced with a peak at 6 h. Intracellular 3',5'-cyclic adenosine monophosphate accumulated in CEM cells with a maximal level at 9 h. These results show that a human-cultured T cells line can upregulate its own adenosine production and A(2A)R expression during exposure to acute hypoxia. Hypoxia-increased stimulation of the adenosinergic signalling of T cells may have immunosuppressive properties and, consequently, A(2A)R agonists may have therapeutic relevance.
Assuntos
Adenosina/metabolismo , Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/metabolismo , Hipóxia Celular , Linhagem Celular , Meios de Cultura , AMP Cíclico/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Receptor A2A de Adenosina/metabolismo , Linfócitos T/efeitos dos fármacos , Fatores de Tempo , Regulação para Cima/efeitos dos fármacosRESUMO
Adenosine is a modulator of nociceptive pathways, both at the spinal and supraspinal levels. Adenosine A(1) and A(2A) receptors (A(1)R, A(2A)R) are expressed in the basal ganglia where they are the target of caffeine, the most widely use psychoactive drug which acts as an antagonist to both types of receptors. Given the controversial role of A(2A)R versus A(1)R in modulating pain in brain areas, mice received intracerebroventricular injection of Adonis, an agonist-like monoclonal antibody with high specificity for the A(2A)R and were subjected to behavioral tests investigating nociceptive thresholds. We report that Adonis led to a significant dose-dependent increase in hot-plate and tail-flick latencies in mice and that such increase was prevented by caffeine and ZM 241385, a specific A(2A)R antagonist. The Adonis antinociceptive effects were also inhibited by naloxone, a non selective antagonist for opioid receptors, suggesting that Adonis acts, at least in part, through the stimulation of the endogenous opioid system. These results confirm the A(2A)R as a target for pain control and Adonis as a potential drug with therapeutic interest.
Assuntos
Analgésicos/administração & dosagem , Anticorpos Monoclonais/administração & dosagem , Limiar da Dor/efeitos dos fármacos , Dor/metabolismo , Receptor A2A de Adenosina/metabolismo , Animais , Western Blotting , Injeções Intraventriculares , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Immunocompetent cells express various G-protein-coupled receptors that transduce extracellular signals across the plasma membrane. Among them, CXCR4 and CCR5 chemokines receptors and adenosine A(2A) receptors (A(2A)R) are involved in inflammatory processes. Considering that A(2A)R activation may have incidence on CXCR4 and CCR5 protein expression through heterologous desensitization process, we tested Adonis, an agonist-like monoclonal antibody to A(2A)R on CD4+ CEM T-cells. We found that Adonis inhibited the CEM cell growth, upregulated A(2A)R and downregulated CXCR4 and CCR5 without modifying the CD4 expression. By reducing the expression of CXCR4 and CCR5 chemokines receptors utilized as entry co-receptors by HIV-1 during viral infection of CD4 expressing cells, Adonis stimulation of A(2A)R appears as a valuable means to treat infected cells.
Assuntos
Agonistas do Receptor A2 de Adenosina/farmacologia , Anticorpos Monoclonais/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Receptor A2A de Adenosina/biossíntese , Receptores CXCR4/biossíntese , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo , Humanos , Receptor A2A de Adenosina/genética , Receptor A2A de Adenosina/imunologia , Receptores CCR5/biossíntese , Receptores CCR5/genética , Receptores CXCR4/genética , Transdução de SinaisRESUMO
The second extracellular loop of the A(2A) receptor (A(2A)R) of adenosine was used to immunize mice for production of Adonis, an IgM monoclonal antibody. Adonis bound to the immunogen peptide and the native receptor in ELISA with K(D) values in 6.51-12.35 nM range. It recognized a linear epitope of 7 amino acids (LFEDVVP) at the C-terminal part of the external loop. Adonis revealed a 45-kDa band in lysate of human peripheral blood mononuclear cells in Western blotting in denaturing conditions. This served to monitor the up-regulation of the A(2A)R expression by caffeine. Adonis stimulated the cAMP production and inhibited the cell proliferation of an A(2A)R transfected stable cell line. These results confirm the immunogenicity and the functional relevance of the second extracellular loop of the A(2A)R. They suggest that Adonis may be of clinical use in various pathological situations to measure the regulation of the A(2A)R expression and to act as A(2A)R agonist drug.
Assuntos
Agonistas do Receptor A2 de Adenosina , Anticorpos Monoclonais/biossíntese , Receptor A2A de Adenosina/imunologia , Adulto , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Western Blotting , Linhagem Celular , Proliferação de Células , AMP Cíclico/biossíntese , Mapeamento de Epitopos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Peptídeos/química , TitulometriaRESUMO
In the processes underlying thyroid autoimmunity, thyrocytes probably act as antigen-presenting cells exposing T-cell epitopes to intrathyroid lymphocytes. To study the interactions between lymphocytes and thyrocytes, which are arranged in a tight, polarized monolayer, we developed a new in vitro model based on human thyrocytes grown on the underside of a filter placed in a bicameral chamber. Thyrocytes from Graves' disease glands were plated onto the upper face of a 8-mum-pore polyethylene terephthalate culture insert filter placed in the inverted position and grown for 24 h before the insert was returned to the normal position for a week in the cell culture plate wells. Thyrocytes grown in the presence of thyroid stimulating hormone, forming a homogeneous monolayer on the underside of the filter, reached confluence after 8 days in vitro. The cells developed a transepithelial electrical resistance >1,000 Omega.cm(2), and the ZO-1 tight junction protein showed a junctional pattern of distribution. Thyrocytes showed a polarized pattern of thyroperoxidase and thyroid stimulating hormone receptor expression in the apical and basolateral positions, respectively. They were also found to aberrantly express DR class II human leukocyte antigen and an Fc immunoglobulin receptor (FcgammaRIIB2) in the basolateral and apical positions, respectively. Autologous intrathyroidal T lymphocytes cocultured for 24 h across the filter with the thyrocyte monolayer proliferated and remained in the upper chamber without any leakage occurring through the epithelial barrier, which makes this model particularly suitable for studying the cell-cell interactions involved in antigen processing.
Assuntos
Comunicação Celular/imunologia , Cultura em Câmaras de Difusão/métodos , Linfócitos/citologia , Glândula Tireoide/citologia , Glândula Tireoide/imunologia , Polaridade Celular/fisiologia , Células Cultivadas , Cultura em Câmaras de Difusão/instrumentação , Células Epiteliais/citologia , Células Epiteliais/imunologia , Filtração , Humanos , Técnicas In Vitro , Junções Íntimas/fisiologiaRESUMO
Biosynthesis of thyroid hormones is an oxidative process that generates reactive oxygen species (ROS) and involves thyroperoxidase (TPO) that is one of the main autoantigens involved in autoimmune thyroid diseases. The ectodomain of TPO consists of a large N-terminal myeloperoxidase-like module followed by a complement control protein (CCP)-like module and an epidermal growth factor-like module. The presence of these two additional gene modules suggests that they may play some crucial, hitherto unsuspected role associated with thyroid function. Because the CCP module is a constituent of the molecules involved in the activation of C4 complement component, we investigated the possibility that C4 may bind to TPO and activate the complement pathway in autoimmune conditions. We showed that TPO via its CCP module directly activated complement without any mediation by Ig. We suggested that this additional complement pathway requires the production of ROS and specially hydroxyl radicals that aggregate TPO and oxidize methionines of C4. Moreover, we found, in patients with Hashimoto's thyroiditis, that thyrocytes overexpress C4 and all the downstream components of the complement pathway. These results indicate that TPO has some as yet unknown function, which may contribute along with other mechanisms to the massive cell destruction observed in Hashimoto's thyroiditis. Investigating this complement pathway, therefore, would provide an excellent means of reaching a better understanding of the etiology of other degenerative diseases.