RESUMO
Molecular steps that activate store-operated calcium entry (SOCE) via Orai channel supramolecular complex remain incompletely defined. We have earlier shown that α-SNAP regulates the on-site functional assembly and calcium selectivity of Orai1 channels. Here we investigate the molecular basis of its association with Orai, Stim and find that the affinity of α-SNAP for Orai and Stim is substantially higher than previously reported affinities between Stim and Orai sub-domains. α-SNAP binds the coiled-coil 3 (CC3) sub-domain of Stim1. Mutations of Tryptophan 430 in Stim1-CC3 disrupted α-SNAP association and SOCE, demonstrating a novel α-SNAP dependent function for this crucial subdomain. Further, α-SNAP binds the hinge region near the C-terminus of Orai1 and an additional broad region near the N-terminus and Valine 262 and Leucine 74 were necessary for these respective interactions, but not Orai, Stim co-clustering. Thus, high affinity interactions with α-SNAP are necessary for imparting functionality to Stim, Orai clusters and induction of SOCE.
Assuntos
Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/metabolismo , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Clonagem Molecular , Fibroblastos/citologia , Fibroblastos/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Camundongos , Mutação , Proteínas de Neoplasias/química , Proteína ORAI1/química , Ligação Proteica , Molécula 1 de Interação Estromal/químicaRESUMO
T cell effector functions require sustained calcium influx. However, the signaling and phenotypic consequences of non-specific sodium permeation via calcium channels remain unknown. α-SNAP is a crucial component of Orai1 channels, and its depletion disrupts the functional assembly of Orai1 multimers. Here we show that α-SNAP hypomorph, hydrocephalus with hopping gait, Napahyh/hyh mice harbor significant defects in CD4 T cell gene expression and Foxp3 regulatory T cell (Treg) differentiation. Mechanistically, TCR stimulation induced rapid sodium influx in Napahyh/hyh CD4 T cells, which reduced intracellular ATP, [ATP]i. Depletion of [ATP]i inhibited mTORC2 dependent NFκB activation in Napahyh/hyh cells but ablation of Orai1 restored it. Remarkably, TCR stimulation in the presence of monensin phenocopied the defects in Napahyh/hyh signaling and Treg differentiation, but not IL-2 expression. Thus, non-specific sodium influx via bonafide calcium channels disrupts unexpected signaling nodes and may provide mechanistic insights into some divergent phenotypes associated with Orai1 function.
Assuntos
Trifosfato de Adenosina/metabolismo , Antígenos CD4/biossíntese , Expressão Gênica , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Transdução de Sinais , Sódio/metabolismo , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/metabolismo , Animais , Linfócitos T CD4-Positivos/fisiologia , Cátions/metabolismo , Diferenciação Celular , Camundongos Endogâmicos C57BL , Camundongos Knockout , Linfócitos T Reguladores/fisiologiaRESUMO
Orai1 forms a highly calcium-selective pore of the calcium release activated channel, and α-SNAP is necessary for its function. Here we show that α-SNAP regulates on-site assembly of Orai1 dimers into calcium-selective multimers. We find that Orai1 is a dimer in resting primary mouse embryonic fibroblasts but displays variable stoichiometry in the plasma membrane of store-depleted cells. Remarkably, α-SNAP depletion induces formation of higher-order Orai1 oligomers, which permeate significant levels of sodium via Orai1 channels. Sodium permeation in α-SNAP-deficient cells cannot be corrected by tethering multiple Stim1 domains to Orai1 C-terminal tail, demonstrating that α-SNAP regulates functional assembly and calcium selectivity of Orai1 multimers independently of Stim1 levels. Fluorescence nanoscopy reveals sustained coassociation of α-SNAP with Stim1 and Orai1, and α-SNAP-depleted cells show faster and less constrained mobility of Orai1 within ER-PM junctions, suggesting Orai1 and Stim1 coentrapment without stable contacts. Furthermore, α-SNAP depletion significantly reduces fluorescence resonance energy transfer between Stim1 and Orai1 N-terminus but not C-terminus. Taken together, these data reveal a unique role of α-SNAP in the on-site functional assembly of Orai1 subunits and suggest that this process may, in part, involve enabling crucial low-affinity interactions between Orai1 N-terminus and Stim1.
Assuntos
Proteína ORAI1/metabolismo , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/metabolismo , Animais , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Membrana Celular/metabolismo , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Humanos , Transporte de Íons , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Transporte Proteico , Canais de Sódio/genética , Canais de Sódio/metabolismo , Molécula 1 de Interação Estromal/metabolismoRESUMO
Store-operated calcium entry (SOCE) by calcium release activated calcium (CRAC) channels constitutes a primary route of calcium entry in most cells. Orai1 forms the pore subunit of CRAC channels and Stim1 is the endoplasmic reticulum (ER) resident Ca(2+) sensor. Upon store-depletion, Stim1 translocates to domains of ER adjacent to the plasma membrane where it interacts with and clusters Orai1 hexamers to form the CRAC channel complex. Molecular steps enabling activation of SOCE via CRAC channel clusters remain incompletely defined. Here we identify an essential role of α-SNAP in mediating functional coupling of Stim1 and Orai1 molecules to activate SOCE. This role for α-SNAP is direct and independent of its known activity in NSF dependent SNARE complex disassembly. Importantly, Stim1-Orai1 clustering still occurs in the absence of α-SNAP but its inability to support SOCE reveals that a previously unsuspected molecular re-arrangement within CRAC channel clusters is necessary for SOCE. DOI:http://dx.doi.org/10.7554/eLife.00802.001.
Assuntos
Cálcio/metabolismo , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/metabolismo , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/fisiologia , Animais , Drosophila , Humanos , Transporte de Íons , Fatores de Transcrição NFATC/metabolismoRESUMO
Lectin has been isolated from mycelia of Aspergillus terricola by single step purification on porcine stomach mucin-Sepharose 4B affinity column. Lectin could be effectively purified with 75% recovery and 4.47-fold increase in specific activity. Lectin migrated as a single band on SDS-PAGE with an apparent molecular mass of 32.5 kDa. Sugar inhibition assay revealed that the lectin did not strongly interact with most carbohydrates and their derivatives tested while strong binding affinity to D-glucose, D-sucrose, N-acetyl-D-galactosamine, asialofetuin, porcine stomach mucin, and bovine submaxillary mucin was indicated. Neuraminidase and protease treatment to erythrocytes enhanced lectin titre. Lectin activity was stable within the pH range of 7.0-10.5. A. terricola lectin displayed remarkable thermostability and remained unaffected upon incubation at 70 degrees C for 2.5 h. Lectin did not require metal ions for its activity. Incubation with denaturants (urea, thiourea, and guanidine-HCl) substantially reduced lectin activity. Carbohydrate analysis revealed that it is a glycoprotein with 9.76% total sugars.
Assuntos
Aspergillus/química , Lectinas/isolamento & purificação , Micélio/química , Temperatura , Animais , Aspergillus/efeitos dos fármacos , Bovinos , Cromatografia de Afinidade , Ácido Edético/farmacologia , Eletroforese em Gel de Poliacrilamida , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Hemaglutinação/efeitos dos fármacos , Testes de Hemaglutinação , Humanos , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Lectinas/metabolismo , Micélio/efeitos dos fármacos , Desnaturação Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Sus scrofaRESUMO
Activation of store operated Ca(2+) entry involves stromal interaction molecule 1 (STIM1), localized to the endoplasmic reticulum (ER), and calcium channel subunit (Orai1/CRACM1), localized to the plasma membrane. Confocal microscopy shows that thapsigargin-mediated depletion of ER Ca(2+) stores in RBL mast cells causes a redistribution of STIM1, labeled with monomeric red fluorescent protein (mRFP), to micrometer-scale ER-plasma membrane junctions that contain Orai1/CRACM1, labeled with monomeric Aequorea coerulescens green fluorescent protein (AcGFP). Using fluorescence resonance energy transfer (FRET), we determine that this visualized coredistribution is accompanied by nanoscale interaction of STIM1-mRFP and AcGFP-Orai1/CRACM1. We find that antigen stimulation of immunoglobulin E receptors causes much less Orai1/CRACM1 and STIM1 association, but strong interaction is observed under conditions that prevent refilling of ER stores. Stimulated association monitored by FRET is inhibited by sphingosine derivatives in parallel with inhibition of Ca(2+) influx. Similar structural and functional effects are caused by mutation of acidic residues in the cytoplasmic segment of Orai1/CRACM1, suggesting a role for electrostatic interactions via these residues in the coupling of Orai1/CRACM1 to STIM1. Our results reveal dynamic molecular interactions between STIM1 and Orai1/CRACM1 that depend quantitatively on electrostatic interactions and on the extent of store depletion.
Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Membrana Celular/metabolismo , Glicoproteínas de Membrana/metabolismo , Animais , Canais de Cálcio/genética , Linhagem Celular , Inibidores Enzimáticos/metabolismo , Transferência Ressonante de Energia de Fluorescência , Glicoproteínas de Membrana/genética , Proteína ORAI1 , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Esfingosina/química , Esfingosina/metabolismo , Eletricidade Estática , Molécula 1 de Interação Estromal , Tapsigargina/metabolismoRESUMO
Calcium acts as a second messenger in many cell types, including lymphocytes. Resting lymphocytes maintain a low concentration of Ca2+. However, engagement of antigen receptors induces calcium influx from the extracellular space by several routes. A chief mechanism of Ca2+ entry in lymphocytes is through store-operated calcium (SOC) channels. The identification of two important molecular components of SOC channels, CRACM1 (the pore-forming subunit) and STIM1 (the sensor of stored calcium), has allowed genetic and molecular manipulation of the SOC entry pathway. In this review, we highlight advances in the understanding of Ca2+ signaling in lymphocytes with special emphasis on SOC entry. We also discuss outstanding questions and probable future directions of the field.
Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Linfócitos/imunologia , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Animais , Apresentação de Antígeno , Degranulação Celular , Humanos , Ativação Linfocitária , Linfócitos/metabolismo , Mastócitos/fisiologia , Camundongos , Proteína ORAI1 , Receptores de Detecção de Cálcio/metabolismo , Molécula 1 de Interação Estromal , Timo/imunologia , Timo/metabolismoRESUMO
CRACM1 (also called Orai1) constitutes the pore subunit of store-operated calcium release-activated calcium channels. A point mutation in the gene encoding CRACM1 is associated with severe combined immunodeficiency disease in humans. Here we generated CRACM1-deficient mice in which beta-galactosidase activity 'reported' CRACM1 expression. CRACM1-deficient mice were smaller in size. Mast cells derived from CRACM1-deficient mice showed grossly defective degranulation and cytokine secretion, and the allergic reactions elicited in vivo were inhibited in CRACM1-deficient mice. We detected robust CRACM1 expression in skeletal muscles and some regions of the brain, heart and kidney but not in the lymphoid regions of thymus and spleen. In contrast, we found CRACM2 expression to be much higher in mouse T cells. In agreement with those findings, the store-operated calcium influx and development and proliferation of CRACM1-deficient T cells was unaffected. Thus, CRACM1 is crucial in mouse mast cell effector function, but mouse T cell calcium release-activated calcium channels are functional in the absence of CRACM1.
Assuntos
Canais de Cálcio/fisiologia , Mastócitos/imunologia , Animais , Cálcio/metabolismo , Canais de Cálcio/biossíntese , Degranulação Celular , Proliferação de Células , Células Cultivadas , Citocinas/metabolismo , Mastócitos/metabolismo , Camundongos , Camundongos Knockout , Proteína ORAI1 , Proteína ORAI2 , Especificidade de Órgãos , Subunidades Proteicas/biossíntese , Subunidades Proteicas/fisiologia , Linfócitos T/citologia , Linfócitos T/metabolismoRESUMO
Store-operated calcium (SOC) entry is the major route of calcium influx in non-excitable cells, especially immune cells. The best characterized store-operated current, I(CRAC), is carried by calcium release activated calcium (CRAC) channels. The existence of the phenomenon of store-operated calcium influx was proposed almost two decades ago. However, in spite of rigorous research by many laboratories, the identity of the key molecules participating in the process has remained a mystery. In all these years, multiple different approaches have been adopted by countless researchers to identify the molecular players in this fundamental process. Along the way, many crucial discoveries have been made, some of which have been summarized here. The last couple of years have seen significant breakthroughs in the field-identification of STIM1 as the store Ca(2+) sensor and CRACM1 (Orai1) as the pore-forming subunit of the CRAC channel. The field is now actively engaged in deciphering the gating mechanism of CRAC channels. We summarize here the latest progress in this direction.
Assuntos
Canais de Cálcio/metabolismo , Animais , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/fisiologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiologia , Mastócitos/imunologia , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Proteína ORAI1 , Canais de Cátion TRPC/genética , Canais de Cátion TRPC/fisiologiaRESUMO
Receptor-mediated Ca(2+) release from the endoplasmic reticulum (ER) is often followed by Ca(2+) entry through Ca(2+)-release-activated Ca(2+) (CRAC) channels in the plasma membrane . RNAi screens have identified STIM1 as the putative ER Ca(2+) sensor and CRACM1 (Orai1; ) as the putative store-operated Ca(2+) channel. Overexpression of both proteins is required to reconstitute CRAC currents (I(CRAC); ). We show here that CRACM1 forms multimeric assemblies that bind STIM1 and that acidic residues in the transmembrane (TM) and extracellular domains of CRACM1 contribute to the ionic selectivity of the CRAC-channel pore. Replacement of the conserved glutamate in position 106 of the first TM domain of CRACM1 with glutamine (E106Q) acts as a dominant-negative protein, and substitution with aspartate (E106D) enhances Na(+), Ba(2+), and Sr(2+) permeation relative to Ca(2+). Mutating E190Q in TM3 also affects channel selectivity, suggesting that glutamate residues in both TM1 and TM3 face the lumen of the pore. Furthermore, mutating a putative Ca(2+) binding site in the first extracellular loop of CRACM1 (D110/112A) enhances monovalent cation permeation, suggesting that these residues too contribute to the coordination of Ca(2+) ions to the pore. Our data provide unequivocal evidence that CRACM1 multimers form the Ca(2+)-selective CRAC-channel pore.
Assuntos
Cálcio/metabolismo , Transporte de Íons/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Proteínas de Neoplasias/metabolismo , Sequência de Aminoácidos , Western Blotting , Canais de Cálcio , Linhagem Celular , Clonagem Molecular , Ácido Glutâmico/genética , Glutamina/genética , Humanos , Imunoprecipitação , Proteínas de Membrana/genética , Dados de Sequência Molecular , Mutação de Sentido Incorreto/genética , Proteína ORAI1 , Técnicas de Patch-Clamp , Estrutura Terciária de Proteína , Alinhamento de Sequência , Molécula 1 de Interação EstromalRESUMO
Depletion of intracellular calcium stores activates store-operated calcium entry across the plasma membrane in many cells. STIM1, the putative calcium sensor in the endoplasmic reticulum, and the calcium release-activated calcium (CRAC) modulator CRACM1 (also known as Orai1) in the plasma membrane have recently been shown to be essential for controlling the store-operated CRAC current (I(CRAC)). However, individual overexpression of either protein fails to significantly amplify I(CRAC). Here, we show that STIM1 and CRACM1 interact functionally. Overexpression of both proteins greatly potentiates I(CRAC), suggesting that STIM1 and CRACM1 mutually limit store-operated currents and that CRACM1 may be the long-sought CRAC channel.
Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Cálcio/deficiência , Cálcio/metabolismo , Canais de Cálcio/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Quelantes/farmacologia , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Retículo Endoplasmático/efeitos dos fármacos , Expressão Gênica/fisiologia , Humanos , Inositol 1,4,5-Trifosfato/farmacologia , Líquido Intracelular/efeitos dos fármacos , Líquido Intracelular/metabolismo , Células Jurkat , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Proteínas de Membrana/genética , Proteínas de Neoplasias/genética , Proteína ORAI1 , Molécula 1 de Interação Estromal , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
The progeny of T lymphocytes responding to immunization mostly die rapidly, leaving a few long-lived survivors functioning as immune memory. Thus, control of this choice of death versus survival is critical for immune memory. There are indications that reactive radicals may be involved in this death pathway. We now show that, in mice lacking inducible nitric oxide synthase (iNOS), higher frequencies of both CD4 and CD8 memory T cells persist in response to immunization, even when iNOS(+/+) APCs are used for immunization. Postactivation T cell death by neglect is reduced in iNOS(-/-) T cells, and levels of the antiapoptotic proteins Bcl-2 and Bcl-xL are increased. Inhibitors of the iNOS-peroxynitrite pathway also enhance memory responses and block postactivation death by neglect in both mouse and human T cells. However, early primary immune responses are not enhanced, which suggests that altered survival, rather than enhanced activation, is responsible for the persistent immunity observed. Thus, in primary immune responses, iNOS in activated T cells autocrinely controls their susceptibility to death by neglect to determine the level of persisting CD4 and CD8 T cell memory, and modulation of this pathway can enhance the persistence of immune memory in response to vaccination.
Assuntos
Linfócitos T CD4-Positivos/enzimologia , Linfócitos T CD8-Positivos/enzimologia , Morte Celular/fisiologia , Memória Imunológica/fisiologia , Óxido Nítrico Sintase/metabolismo , Clorometilcetonas de Aminoácidos/metabolismo , Animais , Antígenos CD/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Humanos , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-2/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo II , Ácido Peroxinitroso/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Baço/citologia , Subpopulações de Linfócitos T , Proteína bcl-XRESUMO
A population of CD25(+)CD4(+) regulatory T cells (T regs) functions to maintain immunological self tolerance by inhibiting autoreactive T cell responses. CD25(+)CD4(+) T regs are present in low, but steady, numbers in the peripheral lymphoid tissues of healthy mice. Recent studies have shown that IL-2 is an essential growth factor for these cells. How this cytokine functions to regulate CD25(+)CD4(+) T reg homeostasis and prevent autoimmune disease remains unknown. In conventional CD4(+) T cells, IL-2 triggers signaling pathways that promote proliferation and survival by activating the STAT5 transcription factor and by increasing the expression of the antiapoptotic protein, Bcl-2. We show here that bcl-2 deficiency does not affect CD25(+)CD4(+) T reg homeostasis, and that ectopic expression of this molecule fails to rescue CD25(+)CD4(+) T reg numbers or to prevent the development of autoimmunity in IL-2-deficient mice. Furthermore, transient activation of STAT5 is sufficient to increase CD25(+)CD4(+) T reg numbers in IL-2-deficient mice. Our study uncovers an essential role for STAT5 in maintaining CD25(+)CD4(+) T reg homeostasis and self-tolerance.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Proteínas de Ligação a DNA/fisiologia , Homeostase/imunologia , Proteínas do Leite , Receptores de Interleucina-2/biossíntese , Tolerância a Antígenos Próprios/imunologia , Transdução de Sinais/imunologia , Transativadores/fisiologia , Animais , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Homeostase/genética , Interleucina-2/deficiência , Interleucina-2/genética , Interleucina-2/fisiologia , Contagem de Linfócitos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-bcl-2/deficiência , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Fator de Transcrição STAT5 , Tolerância a Antígenos Próprios/genética , Transdução de Sinais/genética , Transativadores/deficiência , Transativadores/genética , Transativadores/metabolismo , Regulação para Cima/genética , Regulação para Cima/imunologiaRESUMO
Signaling pathways involved in regulating T cell proliferation and survival are not well understood. Here we have investigated a possible role of the nuclear factor (NF)-kappaB pathway in regulating mature T cell function by using CD4+ T cells from p50-/- cRel-/- mice, which exhibit virtually no inducible kappaB site binding activity. Studies with these mice indicate an essential role of T cell receptor (TCR)-induced NF-kappaB in regulating interleukin (IL)-2 expression, cell cycle entry, and survival of T cells. Our results further indicate that NF-kappaB regulates TCR-induced expression of antiapoptotic Bcl-2 family members. Strikingly, retroviral transduction of CD4+ T cells with the NF-kappaB-inducing IkappaB kinase beta showed that NF-kappaB activation is not only necessary but also sufficient for T cell survival. In contrast, our results indicate a lack of involvement of NF-kappaB in both IL-2 and Akt-induced survival pathways. In vivo, p50-/- cRel-/- mice showed impaired superantigen-induced T cell responses as well as decreased numbers of effector/memory and regulatory CD4+ T cells. These findings provide the first demonstration of a role for NF-kappaB proteins in regulating T cell function in vivo and establish a critically important function of NF-kappaB in TCR-induced regulation of survival.
Assuntos
Linfócitos T CD4-Positivos/fisiologia , NF-kappa B/fisiologia , Proteínas Proto-Oncogênicas c-rel/fisiologia , Animais , Antígenos CD28/fisiologia , Ciclo Celular , Morte Celular , Sobrevivência Celular , Quinase I-kappa B , Camundongos , Subunidade p50 de NF-kappa B , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-akt , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Receptores de Antígenos de Linfócitos T/fisiologia , Receptores de Antígenos de Linfócitos T alfa-beta/análiseRESUMO
Modalities that induce specific differentiation to T cell memory in immune responses are important for vaccine design, but there is a paucity of well characterized molecular pathways useful to target for this purpose. We have shown previously that pentoxifylline (PF), a phosphodiesterase (PDE) inhibitor in common clinical use, enhances the commitment of in vitro allo-primed human T cells to secondary responsiveness, a characteristic crucial for memory T cells, which are key determinants of the longevity of the immune response. We now show that this effect can also be mediated by activation of adenylate cyclase (AC) and involves PDE4, but not PDE3 or PDE7. PF-mediated enhancement of T-cell priming is inhibited by blocking AC, is specifically signaled via cAMP-dependent protein kinase A (PKA) isoform I, and is probably independent of both nuclear factor-kappaB and the mitogen-activated protein kinase cascade. Furthermore, known pharmacological inhibitors of AC or PKA by themselves cannot block T-cell priming in the absence of PF or rolipram (Rm), and enhancement of priming requires the presence of PF only relatively late during a 4-day priming in vitro (at 48-96 h), suggesting that pharmacological extension of cAMP-mediated signaling can bring about an event critical for T cell commitment to memory. Furthermore, PF and Rm prevent induction of caspase activation and apoptosis in anti-CD3-activated human T cells. Together, our data suggest that PKA-I-mediated signals triggered by prolonging the half-life of cAMP induced during T-cell priming increase survival of activated T cells and enhance memory T cell commitment.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Isoenzimas/fisiologia , Ativação Linfocitária/fisiologia , Linfócitos T/fisiologia , 3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Adenilil Ciclases/metabolismo , Apoptose , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3 , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Humanos , Técnicas In Vitro , Pentoxifilina/farmacologia , Linfócitos T/citologia , Linfócitos T/enzimologiaRESUMO
Modalities for inducing long-lasting immune responses are essential components of vaccine design. Most currently available immunological adjuvants empirically used for this purpose cause some inflammation, limiting clinical acceptability. We show that pentoxifylline (PF), a phosphodiesterase (PDE) inhibitor in common clinical use, enhances long-term persistence of T cell responses, including protective responses to a bacterial immunogen, Salmonella typhimurium, via a cAMP-dependent protein kinase A-mediated effect on T cells if given to mice for a brief period during immunization. PF inhibits activation-mediated loss of superantigen-reactive CD4 as well as CD8 T cells in vivo without significantly affecting their activation, and inhibits activation-induced death and caspase induction in stimulated CD4 as well as CD8 T cells in vitro without preventing the induction of activation markers. Consistent with this ability to prevent activation-induced death in not only CD4 but also CD8 T cells, PF also enhances the persistence of CD8 T cell responses in vivo. Thus, specific inhibition of activation-induced T cell apoptosis transiently during immune priming is likely to enhance the persistence of CD4 and CD8 T cell responses to vaccination, and pharmacological modulators of the cAMP pathway already in clinical use can be used for this purpose as immunological adjuvants.