RESUMO
To understand the variable response to pain, researchers have examined the change in cardiovascular measures to a uniform painful stimulation. Pain catastrophizing is the tendency to magnify or exaggerate pain sensations, and it affects the outcome of rehabilitation in a clinical setting. Its effect on cardiovascular changes during a painful stimulus is unclear. Twenty-four healthy human participants completed the study. All participants completed a cold pressor test while subjective pain intensity was measured with a numeric pain scale from 0-10. Continuous cardiac output measurements were obtained with finger-pulse plethysmograph waveform analysis. The measurements included systolic and diastolic blood pressure, heart rate averaged over 30 s intervals. Pain catastrophizing and anxiety were assessed using the pain catastrophizing scale (PCS), and Spielberger's State-Trait Anxiety Inventories, respectively. Peak pain was correlated to pain catastrophizing (r = 0.628, p < 0.01). There was a strong correlation between change in heart rate (HR) and subjective peak pain (r = 0.805, p < 0.01), total PCS (r = 0.474, p < 0.05), and the helplessness subscale of the PCS (r = 0.457, p < 0.05). Peak pain and catastrophizing explained a significant amount of the variance for the change in HR during the cold pressor test (R2 of 0.649 and 0.224 respectively, p = 0.019). These novel findings demonstrate a psycho-physiological relationship between cardiovascular changes and pain catastrophizing. Further research should include participants with subacute or persistent pain.
Assuntos
Catastrofização , Limiar da Dor , Temperatura Baixa , Frequência Cardíaca/fisiologia , Humanos , Dor , Medição da Dor , Limiar da Dor/fisiologiaRESUMO
During inflammatory processes of the central nervous system, helper T cells have the capacity to cross the blood-brain barrier and injure or kill neural cells through cytotoxic mechanisms. Glial fibrillary acidic protein (GFAP) is an intermediate filament protein that is part of the astrocyte cytoskeleton that can become fragmented in neuroinflammatory conditions. The mechanism of action by which helper T cells with cytotoxic properties injure astrocytes is not completely understood. Primary human astrocytes were obtained from fetal brain tissue. Human helper (CD4+ ) T cells were isolated from peripheral blood mononuclear cells and activated with the superantigen staphylococcal enterotoxin E (SEE). Granzyme B was detected by enzyme linked immunosorbent assay and intracellular flow cytometry. GFAP fragmentation was monitored by western blotting. Cell death was monitored by lactic acid dehydrogenase release and terminal biotin-dUTP nick labeling (TUNEL). Astrocyte migration was monitored by scratch assay. Adult human oligodendrocytes were cultured with sublethally injured astrocytes to determine support function. Helper T cells activated with SEE expressed granzyme B but not perforin. Helper T cells released granzyme B upon contact with astrocytes and caused GFAP fragmentation in a caspase-dependent, MHCII-independent manner. Sublethally injured astrocytes were not apoptotic; however, their processes were thin and elongated, their migration was attenuated, and their ability to support oligodendrocytes was reduced in vitro. Helper T cells can release granzyme B causing sublethal injury to astrocytes, which compromises the supportive functions of astrocytes. Blocking these pathways may lead to improved resolution of neuroinflammatory lesions.
Assuntos
Astrócitos/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Granzimas/metabolismo , Antígenos de Histocompatibilidade Classe II/fisiologia , Adulto , Anticorpos/farmacologia , Astrócitos/efeitos dos fármacos , Complexo CD3/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Células Cultivadas , Enterotoxinas/farmacologia , Inibidores Enzimáticos/farmacologia , Feto , Citometria de Fluxo , Humanos , Marcação In Situ das Extremidades Cortadas , Leucócitos Mononucleares , Oligodendroglia , Oligopeptídeos/farmacologia , Ferimentos e Lesões/patologiaRESUMO
Blood-brain barrier function is driven by the influence of astrocyte-secreted factors. During neuroinflammatory responses the blood-brain barrier is compromised resulting in central nervous system damage and exacerbated pathology. Here, we identified endothelial netrin 1 induction as a vascular response to astrocyte-derived sonic hedgehog that promotes autocrine barrier properties during homeostasis and increases with inflammation. Netrin 1 supports blood-brain barrier integrity by upregulating endothelial junctional protein expression, while netrin 1 knockout mice display disorganized tight junction protein expression and barrier breakdown. Upon inflammatory conditions, blood-brain barrier endothelial cells significantly upregulated netrin 1 levels in vitro and in situ, which prevented junctional breach and endothelial cell activation. Finally, netrin 1 treatment during experimental autoimmune encephalomyelitis significantly reduced blood-brain barrier disruption and decreased clinical and pathological indices of disease severity. Our results demonstrate that netrin 1 is an important regulator of blood-brain barrier maintenance that protects the central nervous system against inflammatory conditions such as multiple sclerosis and experimental autoimmune encephalomyelitis.
Assuntos
Barreira Hematoencefálica/metabolismo , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/metabolismo , Inflamação/metabolismo , Esclerose Múltipla/metabolismo , Fatores de Crescimento Neural/fisiologia , Fatores de Crescimento Neural/uso terapêutico , Proteínas Supressoras de Tumor/fisiologia , Proteínas Supressoras de Tumor/uso terapêutico , Animais , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Proteínas Sanguíneas/metabolismo , Barreira Hematoencefálica/efeitos dos fármacos , Células Endoteliais/metabolismo , Humanos , Inflamação/tratamento farmacológico , Mediadores da Inflamação/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Fatores de Crescimento Neural/farmacologia , Netrina-1 , Permeabilidade , Cultura Primária de Células , Junções Íntimas/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Proteínas Supressoras de Tumor/farmacologia , Regulação para CimaRESUMO
OBJECTIVE: To define changes in phenotype and functional responses of reconstituting T cells in patients with aggressive multiple sclerosis (MS) treated with ablative chemotherapy and autologous hematopoietic stem cell transplantation (HSCT). METHODS: Clinical and brain magnetic resonance imaging measures of disease activity were monitored serially in patients participating in the Canadian MS HSCT Study. Reconstitution kinetics of immune-cell subsets were determined by flow cytometry, whereas thymic function was assessed using T-cell receptor excision circle analyses as well as flow cytometry measurements of CD31+ recent thymic emigrants (RTEs). Functional assays were performed to track central nervous system-autoreactive antigen-specific T-cell responses, and the relative capacity to generate Th1, Th17, or Th1/17 T-cell responses. RESULTS: Complete abrogation of new clinical relapses and new focal inflammatory brain lesions throughout the 2 years of immune monitoring following treatment was associated with sustained decrease in naive T cells, in spite of restoration of both thymic function and release of RTEs during reconstitution. Re-emergence as well as in vivo expansion of autoreactive T cells to multiple myelin targets was evident in all patients studied. The reconstituted myelin-specific T cells exhibited the same Th1 and Th2 responses as preablation myelin-reactive T cells. In contrast, the post-therapy T-cell repertoire exhibited a significantly diminished capacity for Th17 responses. INTERPRETATION: Our results indicate that diminished Th17 and Th1/17 responses, rather than Th1 responses, are particularly relevant to the abrogation of new relapsing disease activity observed in this cohort of patients with aggressive MS following chemoablation and HSCT.
Assuntos
Transplante de Células-Tronco Hematopoéticas/métodos , Ativação Linfocitária/imunologia , Esclerose Múltipla/patologia , Esclerose Múltipla/cirurgia , Células Th17/imunologia , Células Th17/patologia , Adulto , Antígenos CD/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Citocinas/metabolismo , Feminino , Citometria de Fluxo , Seguimentos , Acetato de Glatiramer , Humanos , Imunossupressores/farmacologia , Imunossupressores/uso terapêutico , Ativação Linfocitária/efeitos dos fármacos , Contagem de Linfócitos , Linfocinas/farmacologia , Masculino , Proteína Básica da Mielina/metabolismo , Glicoproteína Mielina-Oligodendrócito/metabolismo , Peptídeos/farmacologia , Peptídeos/uso terapêutico , Células Th1/efeitos dos fármacos , Células Th1/patologia , Células Th17/efeitos dos fármacosRESUMO
Naturally occurring CD4(+)CD25(+)FOXP3(+) regulatory T cells suppress the activity of pathogenic T cells and prevent development of autoimmune responses. There is growing evidence that TLRs are involved in modulating regulatory T cell (Treg) functions both directly and indirectly. Specifically, TLR2 stimulation has been shown to reduce the suppressive function of Tregs by mechanisms that are incompletely understood. The developmental pathways of Tregs and Th17 cells are considered divergent and mutually inhibitory, and IL-17 secretion has been reported to be associated with reduced Treg function. We hypothesized that TLR2 stimulation may reduce the suppressive function of Tregs by regulating the balance between Treg and Th17 phenotype and function. We examined the effect of different TLR2 ligands on the suppressive functions of Tregs and found that activation of TLR1/2 heterodimers reduces the suppressive activity of CD4(+)CD25(hi)FOXP3(low)CD45RA(+) (naive) and CD4(+)CD25(hi)FOXP3(hi)CD45RA(-) (memory or effector) Treg subpopulations on CD4(+)CD25(-)FOXP3(-)CD45RA(+) responder T cell proliferation while at the same time enhancing the secretion of IL-6 and IL-17, increasing RORC, and decreasing FOXP3 expression. Neutralization of IL-6 or IL-17 abrogated Pam3Cys-mediated reduction of Treg suppressive function. We also found that, in agreement with recent observations in mouse T cells, TLR2 stimulation can promote Th17 differentiation of human T helper precursors. We conclude that TLR2 stimulation, in combination with TCR activation and costimulation, promotes the differentiation of distinct subsets of human naive and memory/effector Tregs into a Th17-like phenotype and their expansion. Such TLR-induced mechanism of regulation of Treg function could enhance microbial clearance and increase the risk of autoimmune reactions.
Assuntos
Diferenciação Celular/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Receptor 2 Toll-Like/imunologia , Separação Celular , Citometria de Fluxo , Humanos , Ativação Linfocitária/imunologia , Fenótipo , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/metabolismo , Células Th17/citologia , Células Th17/metabolismo , Receptor 2 Toll-Like/metabolismoRESUMO
Axonal damage can occur in the central nervous system following trauma, during the course of autoimmune and neurodegenerative disease and during viral and bacterial infections. The degree of axonal damage and absence of spontaneous repair are major determinants of long-term clinical outcome. While inflammation is a common feature of these conditions, the impact of particular immune cell subsets and their products on injured axons is not fully known. To investigate the impact of immune cells on neuronal viability and axonal repair, we developed an in vitro culture system in which neurons are exposed to mixed or distinct immune cell subsets. We find that total peripheral blood mononuclear cells (PBMCs) have a significant inhibitory effect on neurite outgrowth that is independent of apoptosis. Using isolated immune cells subsets, we demonstrate that activated CD4+ T cells enhance neurite outgrowth while activated NK cells and CD8+ T cells inhibit neurite outgrowth. We find that NK cell inhibition of neuronal outgrowth is dependent on MAPK activity. Our findings describe heterogeneous effects of individual immune cell subsets on neuronal growth and offer important insights into the cellular and molecular mechanisms that may impact axonal repair in inflammatory CNS conditions.
Assuntos
Células Matadoras Naturais/imunologia , Neuritos/fisiologia , Linfócitos T/imunologia , Adulto , Animais , Axônios/fisiologia , Células Cultivadas , Sistema Nervoso Central/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Células Matadoras Naturais/fisiologia , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/imunologia , Neurônios/citologia , Ratos , Linfócitos T/fisiologiaRESUMO
BACKGROUND: T helper 17 (Th17) cells produce IL-17A cytokine and can exacerbate autoimmune diseases and asthma. The ß2 adrenergic receptor is a g protein-coupled receptor that induces cAMP second messenger pathways. We tested the hypothesis that terbutaline, a ß2-adrenergic receptor-specific agonist, promotes IL-17 secretion by memory Th17 cells in a cAMP and PKA-dependent manner. METHODS: Venous peripheral blood mononuclear cells (PBMC) from healthy human participants were activated with anti-CD3 and anti-CD28 antibodies. Secreted IL-17A was measured by enzyme linked immunosorbent assay, intracellular IL-17A, and RORγ were measured using flow cytometry, and RORC by qPCR. Memory CD3+CD4+CD45RA-CD45RO+ T cells were obtained by immunomagnetic negative selection and activated with tri-antibody complex CD3/CD28/CD2. Secreted IL-17A, intracellular IL-17A, RORC were measured, and phosphorylated-serine133-CREB was measured by western blotting memory Th cells. RESULTS: Terbutaline increased IL-17A (p < 0.001), IL-17A+ cells (p < 0.05), and RORC in activated PBMC and memory Th cells. The PKA inhibitors H89 (p < 0.001) and Rp-cAMP (p < 0.01) abrogated the effects of terbutaline on IL-17A secretion in PBMC and memory T cells. Rolipram increased IL-17A (p < 0.01) to a similar extent as terbutaline. P-Ser133-CREB was increased by terbutaline (p < 0.05) in memory T cells. CONCLUSION: Terbutaline augments memory Th17 cells in lymphocytes from healthy participants. This could exacerbate autoimmune diseases or asthma, in cases where Th17 cells are considered to be pro-inflammatory.
Assuntos
Asma , Doenças Autoimunes , Humanos , Agonistas Adrenérgicos/metabolismo , Agonistas Adrenérgicos/farmacologia , Doenças Autoimunes/metabolismo , Antígenos CD28/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Interleucina-17/metabolismo , Leucócitos Mononucleares/metabolismo , Receptores Adrenérgicos/metabolismo , Terbutalina/farmacologia , Terbutalina/metabolismo , Células Th17RESUMO
Remyelination, which occurs subsequent to demyelination, contributes to functional recovery and is mediated by oligodendrocyte progenitor cells (OPCs) that have differentiated into myelinating cells. Therapeutics that impact remyelination in the CNS could be critical determinants of long-term functional outcome in multiple sclerosis (MS). Fingolimod is a S1P receptor modulator in MS clinical trials due to systemic anti-inflammatory properties, yet may impact cells within the CNS by crossing the blood-brain barrier. Previous studies using isolated dissociated cultures indicate that neural cells express S1P receptors and respond to receptor engagement. Our objective was to assess the effects of fingolimod on myelin-related processes within a multicellular environment that maintains physiological cell-cell interactions, using organotypic cerebellar slice cultures. Fingolimod treatment had no impact on myelin under basal conditions. Fingolimod treatment subsequent to lysolecithin-induced demyelination enhanced remyelination and process extension by OPCs and mature oligodendrocytes, while increasing microglia numbers and immunoreactivity for the astrocytic marker glial fibrillary acidic protein. The number of phagocytosing microglia was not increased by fingolimod. Using S1P receptor specific agonists and antagonists, we determined that fingolimod-induced effects on remyelination and astrogliosis were mediated primarily through S1P3 and S1P5, whereas enhanced microgliosis was mediated through S1P1 and S1P5. Taken together, these data demonstrate that fingolimod modulates multiple neuroglial cell responses, resulting in enhanced remyelination in organotypic slice cultures that maintain the complex cellular interactions of the mammalian brain.
Assuntos
Cerebelo/citologia , Cerebelo/efeitos dos fármacos , Doenças Desmielinizantes/tratamento farmacológico , Imunossupressores/farmacologia , Bainha de Mielina/fisiologia , Propilenoglicóis , Esfingosina/análogos & derivados , Animais , Animais Recém-Nascidos , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Cerebelo/patologia , Cerebelo/fisiologia , Doenças Desmielinizantes/induzido quimicamente , Cloridrato de Fingolimode , Humanos , Imunossupressores/uso terapêutico , Lisofosfatidilcolinas/toxicidade , Camundongos , Microglia/citologia , Microglia/efeitos dos fármacos , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/fisiopatologia , Oligodendroglia/citologia , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/metabolismo , Propilenoglicóis/farmacologia , Propilenoglicóis/uso terapêutico , Receptores de Lisoesfingolipídeo/metabolismo , Esfingosina/farmacologia , Esfingosina/uso terapêutico , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Técnicas de Cultura de TecidosRESUMO
Human mesenchymal stem cells (hMSCs) are being considered for clinical trials of multiple sclerosis (MS). We examined the effects of adult bone marrow-derived hMSCs on responses of primary human Th1, Th17, and Th1/17 double-expressing T-cell subsets, all implicated in MS. As expected, soluble products from hMSCs inhibited Th1 responses; however, Th17 responses were increased. Secretion of interleukin (IL)-10, considered anti-inflammatory, was decreased. Pretreating hMSCs with the proinflammatory cytokine IL-1ß accentuated these effects, and caused decreases in the Th1/17 subset. These findings underscore the importance of further preclinical work and immune-monitoring to define hMSC effects on disease-relevant immune responses under variable conditions.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Células-Tronco Mesenquimais/imunologia , Células Th1/citologia , Antígenos CD/metabolismo , Linfócitos T CD4-Positivos/classificação , Linfócitos T CD4-Positivos/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Citocinas/imunologia , Citocinas/farmacologia , Ensaio de Imunoadsorção Enzimática/métodos , Citometria de Fluxo/métodos , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Ativação Linfocitária/imunologia , Células-Tronco Mesenquimais/química , Linfócitos T Auxiliares-Indutores/classificação , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Linfócitos T Auxiliares-Indutores/imunologia , Células Th1/imunologiaRESUMO
OBJECTIVE: To study antibody-independent contributions of B cells to inflammatory disease activity, and the immune consequences of B-cell depletion with rituximab, in patients with multiple sclerosis (MS). METHODS: B-Cell effector-cytokine responses were compared between MS patients and matched controls using a 3-signal model of activation. The effects of B-cell depletion on Th1/Th17 CD4 and CD8 T-cell responses in MS patients were assessed both ex vivo and in vivo, together with pharmacokinetic/pharmacodynamic studies as part of 2 rituximab clinical trials in relapsing-remitting MS. RESULTS: B Cells of MS patients exhibited aberrant proinflammatory cytokine responses, including increased lymphotoxin (LT):interleukin-10 ratios and exaggerated LT and tumor necrosis factor (TNF)-alpha secretion, when activated in the context of the pathogen-associated TLR9-ligand CpG-DNA, or the Th1 cytokine interferon-gamma, respectively. B-Cell depletion, both ex vivo and in vivo, resulted in significantly diminished proinflammatory (Th1 and Th17) responses of both CD4 and CD8 T cells. Soluble products from activated B cells of untreated MS patients reconstituted the diminished T-cell responses observed following in vivo B-cell depletion in the same patients, and this effect appeared to be largely mediated by B-cell LT and TNFalpha. INTERPRETATION: We propose that episodic triggering of abnormal B-cell cytokine responses mediates 'bystander activation' of disease-relevant proinflammatory T cells, resulting in new relapsing MS disease activity. Our findings point to a plausible mechanism for the long-recognized association between infections and new MS relapses, and provide novel insights into B-cell roles in both health and disease, and into mechanisms contributing to therapeutic effects of B-cell depletion in human autoimmune diseases, including MS.
Assuntos
Linfócitos B/fisiologia , Linfócitos T CD4-Positivos/fisiologia , Linfócitos T CD8-Positivos/fisiologia , Citocinas/metabolismo , Esclerose Múltipla/patologia , Linfócitos T/fisiologia , Adulto , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Murinos , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Proliferação de Células/efeitos dos fármacos , Método Duplo-Cego , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Citometria de Fluxo/métodos , Acetato de Glatiramer , Humanos , Imunossupressores/farmacologia , Interferon gama/metabolismo , Interleucina-10/metabolismo , Linfotoxina-alfa , Masculino , Pessoa de Meia-Idade , Mitógenos/farmacologia , Esclerose Múltipla/sangue , Esclerose Múltipla/imunologia , Esclerose Múltipla/terapia , Muromonab-CD3/farmacologia , Peptídeos/farmacologia , Fito-Hemaglutininas/farmacologia , Rituximab , Linfócitos T/efeitos dos fármacos , Fatores de Tempo , Fator de Necrose Tumoral alfaRESUMO
CONTEXT: Athletes are often exposed to pain due to injury and competition. Using preliminary evidence, researchers have shown that cardiovascular measures could be an objective measure of pain, but the cardiovascular response can be influenced by psychological factors, such as catastrophizing. OBJECTIVE: To use a painful cold-pressor test (CPT) to measure the relationship among catastrophizing, pain, and cardiovascular variables in athletes. DESIGN: Cohort study. SETTING: Laboratory. PATIENTS OR OTHER PARTICIPANTS: A total of 36 male rugby athletes (age = 24.0 ± 4.6 years, height = 180.0 ± 6.1 cm, mass = 90.5 ± 13.8 kg). MAIN OUTCOME MEASURE(S): We measured catastrophizing using the Pain Catastrophizing Scale and pain using a numeric pain rating scale. Cardiovascular measures were heart rate, systolic and diastolic blood pressure, and heart rate variability. RESULTS: During the CPT, participants experienced increases in pain (from 0 to 4.1 ± 2.2), systolic blood pressure (from 126.7 ± 16.5 to 149.7 ± 23.4 mm Hg), diastolic blood pressure (from 76.9 ± 8.3 to 91.9 ± 11.5 mm Hg), and heart rate variability (from 0.0164 ± 0.0121 to 0.0400 ± 0.0323 milliseconds; all P values < .001). In addition, we observed a decrease in heart rate after the CPT (P = .04). We found a correlation between athletes' pain catastrophizing and both pain intensity and change in heart rate during the CPT (P = .02 and P = .003, respectively). Linear regression indicated that pain and catastrophizing explained 29% of the variance in the change in heart rate (P = .003). CONCLUSIONS: Athletes who had catastrophizing thoughts were more likely to experience higher levels of pain and a greater cardiovascular response during a painful stimulus. The change in cardiovascular variables may be a good objective measure of pain in athletes in the future.
Assuntos
Atletas/psicologia , Pressão Sanguínea/fisiologia , Catastrofização , Frequência Cardíaca/fisiologia , Medição da Dor/métodos , Dor , Adulto , Catastrofização/fisiopatologia , Catastrofização/psicologia , Estudos de Coortes , Resposta ao Choque Frio , Feminino , Humanos , Masculino , Dor/fisiopatologia , Dor/psicologiaRESUMO
T cell activation through the T cell receptor (TCR) involves partitioning of receptors into discrete membrane compartments known as lipid rafts, and the formation of an immunological synapse (IS) between the T cell and antigen-presenting cell (APC). Compartmentalization of negative regulators of T cell activation such as cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is unknown. Recent crystal structures of B7-ligated CTLA-4 suggest that it may form lattices within the IS which could explain the mechanism of action of this molecule. Here, we show that after T cell stimulation, CTLA-4 coclusters with the TCR and the lipid raft ganglioside GM1 within the IS. Using subcellular fractionation, we show that most lipid raft-associated CTLA-4 is on the T cell surface. Such compartmentalization is dependent on the cytoplasmic tail of CTLA-4 and can be forced with a glycosylphosphatidylinositol-anchor in CTLA-4. The level of CTLA-4 within lipid rafts increases under conditions of APC-dependent TCR-CTLA-4 coligation and T cell inactivation. However, raft localization, although necessary for inhibition of T cell activation, is not sufficient for CTLA-4-mediated negative signaling. These data demonstrate that CTLA-4 within lipid rafts migrates to the IS where it can potentially form lattice structures and inhibit T cell activation.
Assuntos
Antígenos de Diferenciação/metabolismo , Imunoconjugados , Ativação Linfocitária , Microdomínios da Membrana/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Abatacepte , Células Apresentadoras de Antígenos/imunologia , Antígenos CD , Antígenos de Diferenciação/genética , Antígeno CTLA-4 , Citometria de Fluxo , Glicosilfosfatidilinositóis/metabolismo , Humanos , Interleucina-2/antagonistas & inibidores , Células Jurkat , Microdomínios da Membrana/química , Microscopia Confocal , Dados de Sequência Molecular , Transporte Proteico , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais , Linfócitos T/citologiaRESUMO
T cell activation through the Ag receptor (TCR) requires sustained signaling from signalosomes within lipid raft microdomains in the plasma membrane. In a proteomic analysis of lipid rafts from human T cells, we identified stomatin-like protein (SLP)-2 as a candidate molecule involved in T cell activation through the Ag receptor. In this study, we show that SLP-2 expression in human primary lymphocytes is up-regulated following in vivo and ex vivo activation. In activated T cells, SLP-2 interacts with components of TCR signalosomes and with polymerized actin. More importantly, up-regulation of SLP-2 expression in human T cell lines and primary peripheral blood T cells increases effector responses, whereas down-regulation of SLP-2 expression correlates with loss of sustained TCR signaling and decreased T cell activation. Our data suggest that SLP-2 is an important player in T cell activation by ensuring sustained TCR signaling, which is required for full effector T cell differentiation, and point to SLP-2 as a potential target for immunomodulation.
Assuntos
Proteínas Sanguíneas/metabolismo , Ativação Linfocitária/imunologia , Proteínas de Membrana/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Actinas/metabolismo , Linfócitos B/imunologia , Linfócitos B/metabolismo , Proteínas Sanguíneas/genética , Células Cultivadas , Humanos , Interleucina-2/biossíntese , Tecido Linfoide/metabolismo , Proteínas de Membrana/genética , Ligação Proteica , Transporte Proteico , RNA Interferente Pequeno/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Regulação para CimaRESUMO
CONTEXT: Athletes are often exposed to pain due to injury and competition. There is preliminary evidence that cardiovascular measures could be an objective measure of pain, but the cardiovascular response can be influenced by psychological factors such as catastrophizing. OBJECTIVES: The purpose of our study was to use a painful cold pressor test to measure the relationship between catastrophizing, pain, and cardiovascular variables in athletes. DESIGN: Pre-post test. SETTING: We completed all measures in a laboratory setting. PARTICIPANTS: Thirty-six male rugby athletes participated in the study. MAIN OUTCOME MEASURES: We measured catastrophizing with the Pain Catastrophizing Scale and pain with a Numeric Pain Rating Scale. Cardiovascular measures included heart rate, systolic, and diastolic blood pressure, and heart rate variability. RESULTS: During the cold pressor test, participants experienced a significant increase in pain (0 to 4.1±2.2), systolic blood pressure (126.7±16.5mm Hg to 149.7±23.4mm Hg), diastolic blood pressure (76.9±8.3mm Hg to 91.9±11.5mm Hg) and heart rate variability (from 0.0164ms±0.0121 to 0.0400ms±0.0323) (all p<.001). In addition, there was a significant decrease in heart rate after the cold pressor test (p=0.04). There was a significant correlation between athlete's pain catastrophizing to both pain intensity and change in heart rate during the cold pressor test (p=.017 and p=.003 respectively). A significant linear regression indicated pain and catastrophizing explained 29% of the variance of the change in heart rate (p=.003). CONCLUSION: Athletes who have catastrophizing thoughts are more likely to experience higher levels of pain and a greater cardiovascular response during a painful stimulus. The change in cardiovascular variables may be a good alternative for an objective measure of pain in athletes in the future.
RESUMO
PURPOSE OF REVIEW: We focus on emerging therapies for the treatment of multiple sclerosis (MS) whose progress through late-phase clinical trials has furthered our understanding of MS pathophysiology. RECENT FINDINGS: A promising array of potential new therapies for MS is targeting a broadening range of pathophysiologic mechanisms. Essentially all emerging therapies in late-phase clinical trials continue to focus on peripheral immune mechanisms that predominate early in the illness. The success of some of these, with varying mechanisms of action, has contributed to an evolution in our conceptual framework of MS. SUMMARY: Several of the emerging therapies focusing on immune-mediated disease mechanisms seem to offer stronger efficacy than the currently approved immune modulators for MS, although with potential for serious adverse effects. These therapies have also broadened our understanding of MS pathophysiology by demonstrating that significant decreases in new disease activity can be achieved through targeting of distinct cell types and mechanisms.
Assuntos
Ensaios Clínicos Fase II como Assunto , Ensaios Clínicos Fase III como Assunto , Fatores Imunológicos/uso terapêutico , Imunossupressores/uso terapêutico , Esclerose Múltipla/tratamento farmacológico , Animais , Humanos , Tolerância Imunológica , Inflamação/metabolismo , Esclerose Múltipla/imunologia , Esclerose Múltipla/patologia , Proteínas do Tecido Nervoso/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologiaRESUMO
Neuronal injury and loss are recognized features of neuroinflammatory disorders, including acute and chronic encephalitides and multiple sclerosis; destruction of astrocytes has been demonstrated in cases of Rasmussen encephalitis. Here, we show that innate immune cells (i.e. natural killer [NK] and gammadelta T cells) cause loss of neurons from primary human neuron-enriched cultures by destroying the supporting astrocytes. Interleukin 2-activated NK cells caused loss of astrocytes within 1 hour, whereas neurons were lost at 4 hours. Time-lapse imaging indicated that delayed neuron loss was due to early destruction of supporting astrocytes. Selective blocking of astrocyte death with anti-NKG2D antibodies reduced neuron loss, as did blocking of CD54 on astrocytes. gammadelta T cells also induced astrocyte cytotoxicity, leading to subsequent neuronal displacement. In astrocytes, NK cells caused caspase-dependent fragmentation of the intermediate filament proteins glial fibrillary acidic protein and vimentin, whereas anti-CD3-activated T cells produced fragmentation to a lesser extent and without measurable cytotoxicity. Glial fibrillary acidic protein fragmentation was also demonstrated in lysates from chronic multiple sclerosis plaques but not from normal control white matter. These data suggest that non-major histocompatibility complex-restricted immune effector cells may contribute to neuron loss in neuroinflammatory disorders indirectly through injury of glia.
Assuntos
Astrócitos/patologia , Citotoxicidade Imunológica , Imunidade Inata , Células Matadoras Naturais/imunologia , Neurônios/patologia , Linfócitos T/imunologia , Western Blotting , Citometria de Fluxo , Proteína Glial Fibrilar Ácida/metabolismo , Antígenos de Histocompatibilidade Classe I , Humanos , Esclerose Múltipla/imunologia , Esclerose Múltipla/metabolismo , Esclerose Múltipla/patologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , VimentinaRESUMO
The monoclonal antibody (mAb) neuronal nuclei (NeuN) labels the nuclei of mature neurons in vivo in vertebrates. NeuN has also been used to define post-mitotic neurons or differentiating neuronal precursors in vitro. In this study, we demonstrate that the NeuN mAb labels the nuclei of astrocytes cultured from fetal and adult human, newborn rat, and embryonic mouse brain tissue. A non-neuronal fibroblast cell line (3T3) also displayed NeuN immunoreactivity. We confirmed that NeuN labels neurons but not astrocytes in sections of P10 rat brain. Western blot analysis of NeuN immunoreactive species revealed a distribution of bands in nucleus-enriched fractions derived from the different cell lines that was similar, but not identical to adult rat brain homogenates. We then examined the hypothesis that the glial fibrillary acidic protein/NeuN-double positive population of cells might correspond to neuronal precursors. Although the NeuN-positive astrocytes were proliferating, no evidence of neurogenesis was detected. Furthermore, expression of additional neuronal precursor markers was not detected. Our results indicate that primary astrocytes derived from mouse, rat, and human brain express NeuN. Our findings are consistent with NeuN being a selective marker of neurons in vivo, but indicate that studies utilizing NeuN-immunoreactivity as a definitive marker of post-mitotic neurons in vitro should be interpreted with caution.
Assuntos
Antígenos Nucleares/imunologia , Astrócitos/imunologia , Núcleo Celular/imunologia , Neurônios/imunologia , Adulto , Animais , Animais Recém-Nascidos , Antígenos Nucleares/análise , Astrócitos/química , Núcleo Celular/química , Células Cultivadas , Feto , Humanos , Imuno-Histoquímica , Camundongos , Neurônios/química , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: The cold pressor test (CPT) involves acute hand or foot exposure to cold water. CPT hyper-responders have unique traits, including risk of hypertension and a greater vasoconstrictor reserve and g force tolerance compared to hypo-responders. The purpose of this study was to uncover differences in cardiovascular and sympathetic biomarkers between responder types. METHODS: Healthy volunteers (N = 30) submerged one hand into cold water (3.3 ± 0.8°C) for 5 min. Blood pressure, heart rate, cardiac output, and cardiac parameters were recorded using an automated monitor, impedance cardiography, and a beat-to-beat monitoring system. We analyzed for salivary α-amylase (SαA), which is a convenient biomarker of the sympathetic nervous system. Subjects were stratified post hoc into hyper-responders (≥ 22 mmHg) and hypo-responders (< 22 mmHg) based on change in systolic blood pressure during CPT. RESULTS: Hyper-responders had a significantly lower baseline heart rate (64 ± 7 bpm), cardiac output (5.6 ± 0.9 L · min-1), and SαA (60 ± 37 U · mL-1) compared to hypo-responders (73 ± 9 bpm, 6.9 ± 1.3 L · min-1, 165 ± 122 U · mL-1). During the cold immersion, hyper-responders had significantly higher systolic blood pressure (150 ± 14 mmHg), diastolic blood pressure (91 ± 10 mmHg), mean arterial pressure (129 ± 17 mmHg), and systemic vascular resistance (1780 ± 640 dyn · s-1 · cm-5) than hypo-responders (130 ± 14 mmHg, 81 ± 10 mmHg, 110 ± 9 mmHg, 1290 ± 220 dyn · s-1 · cm-5). The change in systolic blood pressure correlated with baseline SαA (r = -0.455, P = 0.011) and baseline heart rate (r = -0.374, P = 0.042). DISCUSSION: Baseline characteristics influenced by sympathetic tone such as SαA, heart rate, and cardiac output are indicative of responses to CPT. Our data supports the use of baseline values to predict blood pressure response to acute cold exposure and indicates an intrinsic difference between CPT responder phenotypes.Youssef M, Ghassemi A, Carvajal Gonczi CM, Kugathasan TA, Kilgour RD, Darlington PJ. Low baseline sympathetic tone correlates to a greater blood pressure change in the cold pressor test. Aerosp Med Hum Perform. 2018; 89(6):503-509.
Assuntos
Pressão Sanguínea/fisiologia , Temperatura Baixa , Imersão , Sistema Nervoso Simpático/fisiologia , Adolescente , Adulto , Débito Cardíaco/fisiologia , Feminino , Frequência Cardíaca/fisiologia , Humanos , Masculino , Saliva/enzimologia , Resistência Vascular/fisiologia , Vasoconstrição/fisiologia , Adulto Jovem , alfa-Amilases/análiseRESUMO
In autoimmunity, the balance of different helper T (Th) cell subsets can influence the tissue damage caused by autoreactive T cells. Pro-inflammatory Th1 and Th17 T cells are implicated as mediators of several human autoimmune conditions such as multiple sclerosis (MS). Autologous hematopoietic stem cell transplantation (aHSCT) has been tested in phase 2 clinical trials for MS patients with aggressive disease. Abrogation of new clinical relapses and brain lesions can be seen after ablative aHSCT, accompanied by significant reductions in Th17, but not Th1, cell populations and activity. The cause of this selective decrease in Th17 cell responses following ablative aHSCT is not completely understood. We identified an increase in the kinetics of natural killer (NK) cell reconstitution, relative to CD4+ T cells, in MS patients post-aHSCT, resulting in an increased NK cell:CD4+ T cell ratio that correlated with the degree of decrease in Th17 responses. Ex vivo removal of NK cells from post-aHSCT peripheral blood mononuclear cells resulted in higher Th17 cell responses, indicating that NK cells can regulate Th17 activity. NK cells were also found to be cytotoxic to memory Th17 cells, and this toxicity is mediated through NKG2D-dependent necrosis. Surprisingly, NK cells induced memory T cells to secrete more IL-17A. This was preceded by an early rise in T cell expression of RORC and IL17A mRNA, and could be blocked with neutralizing antibodies against CD58, a costimulatory receptor expressed on NK cells. Thus, NK cells provide initial co-stimulation that supports the induction of a Th17 response, followed by NKG2D-dependent cytotoxicity that limits these cells. Together these data suggest that rapid reconstitution of NK cells following aHSCT contribute to the suppression of the re-emergence of Th17 cells. This highlights the importance of NK cells in shaping the reconstituting immune system following aHSCT in MS patients.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Células Matadoras Naturais/imunologia , Esclerose Múltipla Recidivante-Remitente/terapia , Células Th17/imunologia , Autoimunidade , Antígenos CD58/imunologia , Citocinas/imunologia , Proteínas Ligadas por GPI/imunologia , Regulação da Expressão Gênica , Humanos , Memória Imunológica , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Interleucina-17/imunologia , Esclerose Múltipla Recidivante-Remitente/imunologia , Transplante AutólogoRESUMO
Catecholamine hormones are powerful regulators of the immune system produced by the sympathetic nervous system (SNS). They regulate the adaptive immune system by altering T-cell differentiation into T helper (Th) 1 and Th2 cell subsets, but the effect on Th17 cells is not known. Th17 cells, defined, in part, by chemokine receptor CCR6 and cytokine interleukin (IL)-17A, are crucial for mediating certain pathogen-specific responses and are linked with several autoimmune diseases. We demonstrated that a proportion of human Th17 cells express beta 2-adrenergic receptor (ß2AR), a G protein-coupled receptor that responds to catecholamines. Activation of peripheral blood mononuclear cells, which were obtained from venous blood drawn from healthy volunteers, with anti-cluster of differentiation 3 (CD3) and anti-CD28 and with a ß2-agonist drug, terbutaline (TERB), augmented IL-17A levels (P < 0.01) in the majority of samples. TERB reduced interferon gamma (IFNγ) indicating that IL-17A and IFNγ are reciprocally regulated. Similar reciprocal regulation was observed with dbcAMP. Proliferation of Th cells was monitored by carboxyfluorescein diacetate N-succinimidyl ester labeling and flow cytometry with antibody staining for CD3 and CD4. TERB increased proliferation by a small but significant margin (P < 0.001). Next, Th17 cells (CD4+ CXCR3- CCR6+ ) were purified using an immunomagnetic positive selection kit, which removes all other mononuclear cells. TERB increased IL-17A from purified Th17 cells, which argues that TERB acts directly on Th17 cells. Thus, hormone signals from the SNS maintain a balance of Th cells subtypes through the ß2AR.