RESUMO
Purinergic signaling plays a key role in a variety of physiological functions, including regulation of immune responses. Conventional αß T cells release ATP upon TCR cross-linking; ATP binds to purinergic receptors expressed by these cells and triggers T cell activation in an autocrine and paracrine manner. Here, we studied whether similar purinergic signaling pathways also operate in the "unconventional" γδ T lymphocytes. We observed that γδ T cells purified from peripheral human blood rapidly release ATP upon in vitro stimulation with anti-CD3/CD28-coated beads or IPP. Pretreatment of γδ T cells with (10)panx-1, CBX, or Bf A reversed the stimulation-induced increase in extracellular ATP concentration, indicating that panx-1, connexin hemichannels, and vesicular exocytosis contribute to the controlled release of cellular ATP. Blockade of ATP release with (10)panx-1 inhibited Ca(2+) signaling in response to TCR stimulation. qPCR revealed that γδ T cells predominantly express purinergic receptor subtypes A2a, P2X1, P2X4, P2X7, and P2Y11. We found that pharmacological inhibition of P2X4 receptors with TNP-ATP inhibited transcriptional up-regulation of TNF-α and IFN-γ in γδ T cells stimulated with anti-CD3/CD28-coated beads or IPP. Our data thus indicate that purinergic signaling via P2X4 receptors plays an important role in orchestrating the functional response of circulating human γδ T cells.
Assuntos
Trifosfato de Adenosina/metabolismo , Comunicação Autócrina/fisiologia , Ativação Linfocitária , Receptores de Antígenos de Linfócitos T gama-delta/fisiologia , Receptores Purinérgicos P2X4/fisiologia , Linfócitos T/imunologia , Antígenos CD28/fisiologia , Complexo CD3/fisiologia , Sinalização do Cálcio , Exocitose , Humanos , Receptores Purinérgicos P2X4/análise , Receptores Purinérgicos P2X7/fisiologia , Linfócitos T/metabolismoRESUMO
We reported previously that hypertonic saline (HS) treatment can prevent or upregulate the function of polymorphonuclear neutrophils (PMNs) via A2a-type adenosine receptors or A3-type adenosine receptors (A3R), respectively. A3R translocate to the cell surface upon PMN stimulation, and thus, HS promotes PMN responses under conditions of delayed HS treatment. Here we investigated if inhibition of A3R improves the protective effects of HS resuscitation in a mouse sepsis model. We found that HS nearly triples extracellular adenosine concentrations in whole blood and that inhibition of A3R with the selective antagonist MRS-1191 dose dependently improves the inhibitory effect of HS. MRS-1191 at a concentration of 1 nM enhanced the inhibitory effect of HS and reduced stimulatory effects of delayed HS treatment. Using a mouse model of cecal ligation and puncture (CLP)-induced sepsis, we found that MRS-1191 reduces acute lung injury and PMN accumulation in lung tissue. Whereas delayed HS treatment (4 mL/kg of 7.5% NaCl) of mice 1 h after CLP aggravated PMN accumulation, lung tissue damage, and mortality 24 h after CLP, infusion of MRS-1191 (2 ng/kg body weight) combined with HS reduced these detrimental effects of delayed HS treatment. Our data thus show that A3 receptor antagonists can strengthen the beneficial effects of HS resuscitation by avoiding stimulatory adverse effects that result from delayed HS administration.
Assuntos
Antagonistas do Receptor A3 de Adenosina/farmacologia , Di-Hidropiridinas/farmacologia , Neutrófilos/metabolismo , Receptor A3 de Adenosina/metabolismo , Ressuscitação , Solução Salina Hipertônica/farmacologia , Sepse/terapia , Animais , Membrana Celular/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Humanos , Pulmão/metabolismo , Masculino , Camundongos , Infiltração de Neutrófilos/efeitos dos fármacos , Sepse/metabolismo , Fatores de TempoRESUMO
OBJECTIVE: Hypertonic saline fluids used to resuscitate trauma patients can prevent neutrophil-mediated lung tissue damage, making them attractive alternatives to conventional resuscitation fluids. We have previously shown that gammadeltaT cells, a small T lymphocyte subset, reduce acute inflammatory lung damage by eliminating activated neutrophils that express heat shock protein 72 on the cell surface. Here, we studied whether these protective effects of hypertonic saline are related to improved gammadeltaT cell-mediated neutrophil killing. DESIGN: Laboratory investigation. SETTING: University research laboratory. SUBJECTS: Human peripheral blood from healthy subjects--isolated gammadeltaT lymphocytes and neutrophils. INTERVENTIONS: Isolated blood cells were treated with different concentrations of hypertonic saline and endotoxin of Escherichia coli O111:B4 (lipopolysaccharide). In some experiments, gammadeltaT cells were activated by CD3 cross-linking or by phorbol-myristate acetate and ionomycin, or by phytohemagglutinin. MEASUREMENTS AND MAIN RESULTS: Clinically relevant concentrations of hypertonic saline (20 mM) significantly augmented CD69 expression of gammadeltaT cells that were stimulated with 100 ng/mL lipopolysaccharide. Additionally, lipopolysaccharide induced a three- to five-fold increase in tumor necrosis factor-alpha and interleukin-10 expression by gammadeltaT cells. This response was completely abrogated by hypertonic saline. These data indicate that hypertonic saline can modulate gammadeltaT cell functions. Stimulation of neutrophils with 1-1,000 ng/mL lipopolysaccharide caused a greater than 3-fold increase in heat shock protein-72 expression on the cell surface, which was significantly augmented by hypertonic saline. In cocultures of gammadeltaT cells with autologous neutrophils, 15.6 +/- 3.4% of all neutrophils were killed within 120 min. In the presence of lipopolysaccharide (1 microg/mL), this percentage increased to 23.7 +/- 2.1%, and it was further increased to 31.8 +/- 3.1% when 20 mM hypertonic saline was added with lipopolysaccharide. CONCLUSIONS: Our findings suggest that hypertonic saline enhances the elimination of inflammatory neutrophils by gammadeltaT cells by augmenting heat shock protein-72 expression on the cell surface of neutrophils. Hypertonic saline resuscitation may therefore protect host tissues by enhancing neutrophil clearance from the lungs.
Assuntos
Neutrófilos/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Solução Salina Hipertônica/farmacologia , Subpopulações de Linfócitos T/imunologia , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Relação Dose-Resposta a Droga , Humanos , Técnicas In Vitro , Interleucina-10/biossíntese , Lectinas Tipo C , Lipopolissacarídeos/farmacologia , Ativação de Neutrófilo , Neutrófilos/citologia , Subpopulações de Linfócitos T/metabolismo , Fator de Necrose Tumoral alfa/biossínteseRESUMO
OBJECTIVE: Hypertonic saline resuscitation reduces tissue damage by inhibiting polymorphonuclear neutrophils. Hypertonic saline triggers polymorphonuclear neutrophils to release adenosine triphosphate that is converted to adenosine, inhibiting polymorphonuclear neutrophils through A2a adenosine receptors. Polymorphonuclear neutrophils also express A3 adenosine receptors that enhance polymorphonuclear neutrophil functions. Here we investigated whether A3 receptors may diminish the efficacy of hypertonic saline in a mouse model of acute lung injury. DESIGN: Randomized animal study and laboratory investigation. SETTING: University research laboratory. INTERVENTIONS: The effect of A3 receptors on the efficacy of hypertonic saline resuscitation was assessed in A3 receptor knockout and wild-type mice. Animals were treated with hypertonic saline (7.5% NaCl, 4 mL/kg) before or after cecal ligation and puncture, and acute lung injury and mortality were determined. The effect of timing of hypertonic saline exposure on A3 receptor expression and degranulation was studied in vitro with isolated human polymorphonuclear neutrophils. MEASUREMENTS AND MAIN RESULTS: Treatment of human polymorphonuclear neutrophils with hypertonic saline before stimulation with formyl methionyl-leucyl-phenylalanine inhibited A3 receptor expression and degranulation, whereas hypertonic saline-treatment after formyl methionyl-leucyl-phenylalanine-stimulation augmented A3 receptor expression and degranulation. Acute lung injury in wild-type mice treated with hypertonic saline after cecal ligation and puncture was significantly greater than in wild-type mice pretreated with hypertonic saline. This aggravating effect of delayed hypertonic saline-treatment was absent in A3 receptor knockout mice. Similarly, mortality in wild-type mice with delayed hypertonic saline-treatment was significantly higher (88%) than in animals treated with hypertonic saline before cecal ligation and puncture (50%). Mortality in A3 receptor knockout mice remained only 50% regardless of timing of hypertonic saline administration. CONCLUSIONS: Polymorphonuclear neutrophil A3 receptors expression determines whether hypertonic saline resuscitation inhibits or aggravates polymorphonuclear neutrophil-induced acute lung injury. These findings suggest that A3 antagonists could improve the efficacy of hypertonic saline resuscitation by reducing side effects in patients whose polymorphonuclear neutrophils are activated before hypertonic saline treatment.
Assuntos
Neutrófilos/fisiologia , Receptor A3 de Adenosina/biossíntese , Síndrome do Desconforto Respiratório/metabolismo , Solução Salina Hipertônica/efeitos adversos , Sepse/complicações , Animais , Degranulação Celular , Humanos , Pulmão/patologia , Camundongos , Camundongos Knockout , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/metabolismo , Elastase Pancreática/metabolismo , Cavidade Peritoneal , Peroxidase/metabolismo , Distribuição Aleatória , Síndrome do Desconforto Respiratório/etiologia , Fatores de TempoRESUMO
Elimination of activated inflammatory cells that infiltrate and damage host organs can reduce morbidity and mortality. A better understanding of the mechanisms by which these processes occur may lead to new approaches to prevent tissue damage. The lungs, gastrointestinal tract, and skin are particularly prone to infection and collateral damage by inflammatory cells. Specialized lymphocytes protect these organs from collateral tissue damage by eliminating neutrophils and macrophages from inflamed tissues. These lymphocytes recognize signals produced by inflammatory cells. One such signal is heat shock protein (Hsp) expressed on the cell surface of inflamed phagocytes. Mammalian Hsp molecules closely resemble their microbial equivalents, and therefore phagocytes decorated with these molecules are recognized as target cells. T lymphocytes bearing the gammadelta T cell receptor (TCR) elicit cytotoxic activity toward macrophages and neutrophils that express Hsp60 and Hsp70, respectively, protecting host organs from collateral tissue damage by phagocytes.
Assuntos
Proteínas de Choque Térmico/metabolismo , Inflamação/imunologia , Inflamação/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Doença Aguda , Animais , HumanosRESUMO
We have recently shown that A3 adenosine receptors and P2Y2 purinergic receptors play an important role in neutrophil chemotaxis. Chemotaxis of neutrophils to sites of infections is critical for immune defense. However, excessive accumulation of neutrophils in the lungs can cause acute lung tissue damage. Here we assessed the role of A3 and P2Y2 receptors in neutrophil sequestration to the lungs in a mouse model of sepsis. Sepsis was induced by cecal ligation and puncture (CLP) using adult male C57BL/6J mice (wild type [WT]), homozygous A3 receptor knockout (A3KO) mice, and P2Y2 receptor knockout (P2Y2KO) mice. Animals were killed 2, 4, 6, or 8 h after CLP, and peritoneal lavage fluid and blood were collected. Lungs were removed, and neutrophil infiltration was evaluated using elastase as a marker. Leukocyte and bacterial counts in peritoneal lavage fluid and blood samples were determined. Survival after sepsis was determined in a separate group. Leukocyte counts in the peritoneum were lower in A3KO and P2Y2KO mice than in WT mice. Conversely, initial leukocyte counts in the peripheral blood were higher in KO mice than in WT mice. Neutrophil sequestration to the lungs reached a maximum 2 h after CLP and remained significantly higher in WT mice compared with A3KO and P2Y2KO mice (P < 0.001). Survival after 24 h was significantly lower in WT mice (37.5%) than in A3KO or P2Y2KO mice (82.5%; P < 0.05). These data suggest that A3 and P2Y2 receptors are involved in the influx of neutrophils into the lungs after sepsis. Thus, pharmaceutical approaches that target these receptors might be useful to control acute lung tissue injury in sepsis.
Assuntos
Modelos Animais de Doenças , Pulmão/patologia , Infiltração de Neutrófilos/fisiologia , Neutrófilos/metabolismo , Neutrófilos/patologia , Receptor A3 de Adenosina/fisiologia , Receptores Purinérgicos P2/fisiologia , Sepse/patologia , Animais , Ceco , Ligadura , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Punções , Receptor A3 de Adenosina/deficiência , Receptor A3 de Adenosina/genética , Receptores Purinérgicos P2/deficiência , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2Y2 , Sepse/metabolismo , Sepse/mortalidadeRESUMO
A central event of systemic inflammation and septic organ injury is infiltration of tissues with polymorphonuclear neutrophils, likely modulated by the integrity of the extracellular matrix underlying the vascular endothelium. In the present study, the effect of matrix-modifying endoglycosidase (heparanase) on endotoxin (LPS)-induced inflammatory lung injury was investigated in rats. Animals were treated with heparanase or LPS or pretreated with heparanase before LPS injection, and acute lung injury was verified histologically and characterized by analysis of bronchoalveolar lavage fluids. Pretreatment with heparanase attenuated the mortality of animals and preserved the histological structure of the lungs. Furthermore, polymorphonuclear neutrophil accumulation and activation, analyzed by myeloperoxidase release and reactive oxygen species production associated with lung injury, were significantly reduced upon heparanase pretreatment. In addition, heparanase pretreatment elevated the IL-10 levels in the pulmonary compartment. Moreover, results from in vitro experiments have identified monocyte-derived IL-10 as an important mediator used by heparanase to suppress inflammatory reactions. The protective effect of heparanase may indicate a novel therapeutic strategy for sepsis.
Assuntos
Endotoxinas/toxicidade , Glucuronidase/uso terapêutico , Pulmão/patologia , Síndrome do Desconforto Respiratório/induzido quimicamente , Síndrome do Desconforto Respiratório/tratamento farmacológico , Animais , Linhagem Celular , Sinergismo Farmacológico , Humanos , Lipopolissacarídeos/toxicidade , Pulmão/efeitos dos fármacos , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Antibacterial therapy does not fully protect against anthrax because of severe systemic intoxication. Lysosomal processing of anthrax lethal toxin (LTX) is a key event in the disease pathogenesis, and agents interfering with this process, like chloroquine (CQ), may have practical applications. Although LTX is known to induce T-cell suppression, precise mechanisms of this phenomenon are not completely characterized. In the present study, we investigated alterations of lymphocyte ultrastructure caused by LTX and associated with favorable effect of CQ on the LTX-related dysfunction. Peripheral blood lymphocytes were activated via CD3 crosslinking in the presence or absence of LTX and CQ, and examined by transmission electron microscopy, flow cytometry and immunoblotting. Crosslinking of CD3 induced ultrastructural signs of lymphocyte activation, mostly disappeared after LTX treatment. The cell ultrastructure was well preserved in LTX-treated cells, despite dose- and time-dependent inhibition of T-cell function associated with impaired activation of mitogen-activated protein kinase. Regardless of intracellular signaling abnormalities, LTX did not decrease T-cell viability. CQ restored expression of CD69 (P<0.001) and improved phosphorylation of p38 (P=0.022) in LTX-exposed T lymphocytes. The exposure of cells to CQ, with or without LTX, led to appearance of many phagolysosomes with heterogeneous content, possibly representing unprocessed internalized material. In conclusion, LTX suppressed T-cell functions, but did not affect the viability and caused no ultrastructural damage. Ultrastructural observations indicated that CQ reduced harmful effects of LTX, possibly by interfering with lysosomal activity.
Assuntos
Antígenos de Bactérias/toxicidade , Toxinas Bacterianas/toxicidade , Cloroquina/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/ultraestrutura , Análise de Variância , Citometria de Fluxo , Humanos , Immunoblotting , Microscopia Eletrônica de TransmissãoRESUMO
Lysosomal processing of lethal toxin (LTX) is a key event in the pathogenesis of anthrax. This study investigated the ability of chloroquine (CQ) to interfere with this processing and thereby to reduce suppression of T lymphocytes. T lymphocytes isolated from blood were activated, by cross-linking of CD3, in both the absence and presence of LTX and CQ and then were assayed by flow cytometry and immunoblotting. LTX was found to disrupt intracellular signaling, and it down-regulated T lymphocyte function. CQ significantly reduced the harmful effects of LTX and protected the activation and cytokine production of T lymphocytes. This effect may indicate a promising strategy in the treatment of anthrax.
Assuntos
Antígenos de Bactérias/farmacologia , Antimaláricos/farmacologia , Toxinas Bacterianas/farmacologia , Cloroquina/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Adulto , Regulação para Baixo , Citometria de Fluxo , Humanos , Interleucina-10/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
During inflammation and sepsis, accumulation of activated neutrophils causes lung tissue damage and organ failure. Effective clearance of neutrophils reduces the risk of organ failure; however, its mechanisms are poorly understood. Because lungs are rich in gammadeltaT cells, we investigated the physiological role of these cells in the protection of lung tissue from infiltrating neutrophils. In a mouse model of sepsis, we found that the lungs of survivors contained significantly higher numbers of gammadeltaT cells than those of mice that died from sepsis. The number of gammadeltaT cells correlated inversely with the number of neutrophils in the lungs and with the degree of lung tissue damage. LPS rapidly elicited the expression of heat shock protein (HSP) 72 on the surface of human neutrophils. Inhibitors of transcription, protein synthesis, and intracellular protein transport blocked HSP72 expression, indicating that de novo synthesis is required. gammadeltaT cells targeted and rapidly killed LPS-treated neutrophils through direct cell-to-cell contact. Pre-treatment with neutralizing antibodies to HSP72 diminished neutrophil killing. Our data indicate that HSP72 expression on the cell surface predisposes inflamed neutrophils to killing by gammadeltaT cells. This intercellular exchange may allow gammadeltaT cells to resolve inflammation and limit host tissue damage during sepsis.