RESUMO
BACKGROUND: Sepsis is one of the leading causes of mortality in intensive care units, and sedation in the intensive care unit during sepsis is usually performed intravenously. The inhalative anesthetic sevoflurane has been shown to elicit protective effects in various inflammatory studies, but its role in peritonitis-induced sepsis remains elusive. The hypothesis was that sevoflurane controls the neutrophil infiltration by stabilization of hypoxia-inducible factor 1α and elevated adenosine A2B receptor expression. METHODS: In mouse models of zymosan- and fecal-induced peritonitis, male mice were anesthetized with sevoflurane (2 volume percent, 30 min) after the onset of inflammation. Control animals received the solvent saline. The neutrophil counts and adhesion molecules on neutrophils in the peritoneal lavage of wild-type, adenosine A2B receptor -/-, and chimeric animals were determined by flow cytometry 4 h after stimulation. Cytokines and protein release were determined in the lavage. Further, the adenosine A2B receptor and its transcription factor hypoxia-inducible factor 1α were evaluated by real-time polymerase chain reaction and Western blot analysis 4 h after stimulation. RESULTS: Sevoflurane reduced the neutrophil counts in the peritoneal lavage (mean ± SD, 25 ± 17 × 105vs. 12 ± 7 × 105 neutrophils; P = 0.004; n = 19/17) by lower expression of various adhesion molecules on neutrophils of wild-type animals but not of adenosine A2B receptor -/- animals. The cytokines concentration (means ± SD, tumor necrosis factor α [pg/ml], 523 ± 227 vs. 281 ± 101; P = 0.002; n = 9/9) and protein extravasation (mean ± SD [mg/ml], 1.4 ± 0.3 vs. 0.8 ± 0.4; P = 0.002; n = 12/11) were also lower after sevoflurane only in the wild-type mice. Chimeric mice showed the required expression of the adenosine A2B receptor on the hematopoietic and nonhematopoietic compartments for the protective effects of the anesthetic. Sevoflurane induced the expression of hypoxia-inducible factor 1α and adenosine A2B receptor in the intestine, liver, and lung. CONCLUSIONS: Sevoflurane exerts various protective effects in two murine peritonitis-induced sepsis models. These protective effects were linked with a functional adenosine A2B receptor.
Assuntos
Fator 1 Induzível por Hipóxia/efeitos dos fármacos , Peritonite/complicações , Receptor A2B de Adenosina/efeitos dos fármacos , Sepse/etiologia , Sepse/prevenção & controle , Sevoflurano/farmacologia , Transdução de Sinais/efeitos dos fármacos , Anestésicos Inalatórios/farmacologia , Animais , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Peritonitis and peritonitis-associated sepsis are characterized by an increased formation of platelet-neutrophil complexes (PNCs), which contribute to an excessive migration of polymorphonuclear neutrophils (PMN) into the inflamed tissue. An important neutrophilic mechanism to capture and kill invading pathogens is the formation of neutrophil extracellular traps (NETs). Formation of PNCs and NETs are essential to eliminate pathogens, but also lead to aggravated tissue damage. The chemokine receptors CXCR4 and CXCR7 on platelets and PMNs have been shown to play a pivotal role in inflammation. Thereby, CXCR4 and CXCR7 were linked with functional adenosine A2B receptor (Adora2b) signaling. We evaluated the effects of selective CXCR4 and CXCR7 inhibition on PNCs and NETs in zymosan- and fecal-induced sepsis. We determined the formation of PNCs in the blood and, in addition, their infiltration into various organs in wild-type and Adora2b-/- mice by flow cytometry and histological methods. Further, we evaluated NET formation in both mouse lines and the impact of Adora2b signaling on it. We hypothesized that the protective effects of CXCR4 and CXCR7 antagonism on PNC and NET formation are linked with Adora2b signaling. We observed an elevated CXCR4 and CXCR7 expression in circulating platelets and PMNs during acute inflammation. Specific CXCR4 and CXCR7 inhibition reduced PNC formation in the blood, respectively, in the peritoneal, lung, and liver tissue in wild-type mice, while no protective anti-inflammatory effects were observed in Adora2b-/- animals. In vitro, CXCR4 and CXCR7 antagonism dampened PNC and NET formation with human platelets and PMNs, confirming our in vivo data. In conclusion, our study reveals new protective aspects of the pharmacological modulation of CXCR4 and CXCR7 on PNC and NET formation during acute inflammation.
Assuntos
Armadilhas Extracelulares/efeitos dos fármacos , Receptor A2B de Adenosina/metabolismo , Receptores CXCR4/antagonistas & inibidores , Receptores CXCR/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Células Cultivadas , Armadilhas Extracelulares/metabolismo , Humanos , Masculino , Camundongos Endogâmicos C57BL , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Receptores CXCR/metabolismo , Receptores CXCR4/metabolismoRESUMO
Acute pulmonary inflammation is still a frightening complication in intensive care units and has a high mortality. Specific treatment is not available, and many details of the pathomechanism remain unclear. The recently discovered chemokine receptor CXCR7 and its ligand stromal cell-derived factor (SDF)-1 are known to be involved in inflammation. We chose to investigate the detailed role of CXCR7 in a murine model of LPS inhalation. Inflammation increased pulmonary expression of CXCR7, and the receptor was predominantly expressed on pulmonary epithelium and on polymorphonuclear neutrophil (PMNs) after transepithelial migration into the alveolar space. Specific inhibition of CXCR7 reduced transepithelial PMN migration by affecting the expression of adhesion molecules. CXCR7 antagonism reduced the most potent PMN chemoattractants CXCL1 and CXCL2/3. After inhibiting CXCR7, NF-κB phosphorylation was reduced in lungs of mice, tight junction formation increased, and protein concentration in the bronchoalveolar lavage diminished, showing the impact of CXCR7 on stabilizing microvascular permeability. In vitro studies with human cells confirmed the pivotal role of CXCR7 in pulmonary epithelium. Immunofluorescence of human lungs confirmed our in vivo data and showed an increase of the expression of CXCR7 in pulmonary epithelium. Highlighting the clinical potential of CXCR7 antagonism, nebulization of the agent before and after the inflammation showed impressive anti-inflammatory effects. Additional CXCR7 inhibition potentiated the effect of SDF-1 antagonism, most probably by downregulating SDF-1 and the second receptor of the chemokine (CXCR4) expression. In conclusion, our data identified the pivotal role of the receptor CXCR7 in pulmonary inflammation with a predominant effect on the pulmonary epithelium and PMNs.
Assuntos
Permeabilidade Capilar , Neutrófilos/imunologia , Receptores CXCR/metabolismo , Mucosa Respiratória/imunologia , Doença Aguda , Animais , Moléculas de Adesão Celular/metabolismo , Movimento Celular , Células Cultivadas , Quimiocina CXCL1/metabolismo , Quimiocina CXCL12/metabolismo , Quimiocina CXCL2/metabolismo , Humanos , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Pneumonia , Mucosa Respiratória/patologia , Migração Transendotelial e TransepitelialRESUMO
In acute pulmonary inflammation, polymorphonuclear cells (PMNs) pass a transendothelial barrier from the circulation into the lung interstitium followed by a transepithelial migration into the alveolar space. These migration steps are regulated differentially by a concept of adhesion molecules and remain-despite decades of research-incompletely understood. Current knowledge of changes in the expression pattern of adhesion molecules mainly derives from in vitro studies or from studies in extrapulmonary organ systems, where regulation of adhesion molecules differs significantly. In a murine model of lung inflammation, we determined the expression pattern of nine relevant neutrophilic adhesion molecules on their way through the different compartments of the lung. We used a flow cytometry-based technique that allowed describing spatial distribution of the adhesion molecules expressed on PMNs during their migration through the lung in detail. For example, the highest expression of CD29 was found in the intravascular compartment, highlighting its impact on the initial adhesion to the endothelium. CD47 showed its peak of expression on the later phase of transendothelial migration, whereas CD11b and CD54 expression peaked interstitial. A pivotal role for transepithelial migration was found for the adhesion molecule CD172a. Thereby, expression may correlate with functional impact for specific migration steps. In vitro studies further confirmed our in vivo findings. In conclusion, we are the first to determine the changes in expression patterns of relevant adhesion molecules on their migration through the different compartments of the lung. These findings may help to further understand the regulation of neutrophil trafficking in the lung.
Assuntos
Moléculas de Adesão Celular/metabolismo , Pulmão/imunologia , Pulmão/metabolismo , Neutrófilos/metabolismo , Pneumonia/metabolismo , Lesão Pulmonar Aguda/metabolismo , Animais , Antígeno CD11b/metabolismo , Antígeno CD47/metabolismo , Adesão Celular/efeitos dos fármacos , Citometria de Fluxo , Inflamação/imunologia , Inflamação/metabolismo , Integrina beta1/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Lipopolissacarídeos/toxicidade , Pulmão/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/efeitos dos fármacos , Pneumonia/induzido quimicamente , Pneumonia/imunologia , Receptores Imunológicos/metabolismoRESUMO
BACKGROUND: Liver ischemia/reperfusion (IR) injury is characterized by hepatic tissue damage and an inflammatory response. This is accompanied by the formation and vascular sequestration of platelet-neutrophil conjugates (PNCs). Signaling through Adora2b adenosine receptors can provide liver protection. Volatile anesthetics may interact with adenosine receptors. This study investigates potential antiinflammatory effects of the volatile anesthetic sevoflurane during liver IR. METHODS: Experiments were performed ex vivo with human blood and in a liver IR model with wild-type, Adora2a, and Adora2b mice. The effect of sevoflurane on platelet activation, PNC formation and sequestration, cytokine release, and liver damage (alanine aminotransferase release) was analyzed using flow cytometry, luminometry, and immunofluorescence. Adenosine receptor expression in liver tissue was analyzed using immunohistochemistry and real-time polymerase chain reaction. RESULTS: Ex vivo experiments indicate that sevoflurane inhibits platelet and leukocyte activation (n = 5). During liver IR, sevoflurane (2 Vol%) decreased PNC formation 2.4-fold in wild-type (P < 0.05) but not in Adora2b mice (n ≥ 5). Sevoflurane reduced PNC sequestration 1.9-fold (P < 0.05) and alanine aminotransferase release 3.5-fold (P < 0.05) in wild-type but not in Adora2b mice (n = 5). In Adora2a mice, sevoflurane also inhibited PNC formation and cytokine release. Sevoflurane diminished cytokine release (n ≥ 3) and increased Adora2b transcription and expression in liver tissue of wild-types (n = 4). CONCLUSIONS: Our experiments highlight antiinflammatory and tissue-protective properties of sevoflurane during liver IR and reveal a mechanistic role of Adora2b in sevoflurane-associated effects. The targeted use of sevoflurane not only as an anesthetic but also to prevent IR damage is a promising approach in the treatment of critically ill patients.
Assuntos
Anestésicos Inalatórios/farmacologia , Hepatopatias/prevenção & controle , Fígado/efeitos dos fármacos , Éteres Metílicos/farmacologia , Receptor A2B de Adenosina/metabolismo , Traumatismo por Reperfusão/prevenção & controle , Adulto , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo Real , Receptor A2B de Adenosina/genética , Sevoflurano , Transdução de SinaisRESUMO
Heterotrimeric G proteins of the Gα(i) family have been implicated in signaling pathways regulating cell migration in immune diseases. The Gα(i)-protein-coupled C5a receptor is a critical regulator of IgG FcR function in experimental models of immune complex (IC)-induced inflammation. By using mice deficient for Gα(i2) or Gα(i3), we show that Gα(i2) is necessary for neutrophil influx in skin and lung Arthus reactions and agonist-induced neutrophilia in the peritoneum, whereas Gα(i3) plays a less critical but variable role. Detailed analyses of the pulmonary IC-induced inflammatory response revealed several shared functions of Gα(i2) and Gα(i3), including mediating C5a anaphylatoxin receptor-induced activation of macrophages, involvement in alveolar production of chemokines, transition of neutrophils from bone marrow into blood, and modulation of CD11b and CD62L expression that account for neutrophil adhesion to endothelial cells. Interestingly, C5a-stimulated endothelial polymorphonuclear neutrophil transmigration, but not chemotaxis, is enhanced versus reduced in the absence of neutrophil Gα(i3) or Gα(i2), respectively, and knockdown of endothelial Gα(i2) caused decreased transmigration of wild-type neutrophils. These data demonstrate that Gα(i2) and Gα(i3) contribute to inflammation by redundant, overlapping, and Gα(i)-isoform-specific mechanisms, with Gα(i2) exhibiting unique functions in both neutrophils and endothelial cells that appear essential for polymorphonuclear neutrophil recruitment in IC disease.
Assuntos
Lesão Pulmonar Aguda/imunologia , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/fisiologia , Lesão Pulmonar Aguda/genética , Lesão Pulmonar Aguda/patologia , Animais , Reação de Arthus/genética , Reação de Arthus/imunologia , Reação de Arthus/patologia , Adesão Celular/genética , Adesão Celular/imunologia , Quimiotaxia de Leucócito/genética , Quimiotaxia de Leucócito/imunologia , Modelos Animais de Doenças , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Endotélio Vascular/citologia , Endotélio Vascular/imunologia , Endotélio Vascular/patologia , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/deficiência , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/fisiologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/citologia , Neutrófilos/imunologia , Neutrófilos/patologia , Regulação para Cima/genética , Regulação para Cima/imunologiaRESUMO
Recruiting polymorphonuclear neutrophil granulocytes (PMNs) from circulation and bone marrow to the site of inflammation is one of the pivotal mechanisms of the innate immune system. During inflammation, the enzyme heme oxygenase 1 (HO-1) has been shown to reduce PMN migration. Although these effects have been described in various models, underlying mechanisms remain elusive. Recent studies revealed an influence of HO-1 on different cells of the bone marrow. We investigated the particular role of the bone marrow in terms of HO-1-dependent pulmonary inflammation. In a murine model of LPS inhalation, stimulation of HO-1 by cobalt (III) protoporphyrin-IX-chloride (CoPP) resulted in reduced segmented PMN migration into the alveolar space. In the CoPP group, segmented PMNs were also decreased intravascularly, and concordantly, mature and immature PMN populations were higher in the bone marrow. Inhibition of the enzyme by tin protoporphyrin-IX increased segmented and banded PMN migration into the bronchoalveolar lavage fluid with enhanced PMN release from the bone marrow and aggravated parameters of tissue inflammation. Oxidative burst activity was significantly higher in immature compared with mature PMNs. The chemokine stromal-derived factor-1 (SDF-1), which mediates homing of leukocytes into the bone marrow and is decreased in inflammation, was increased by CoPP. When SDF-1 was blocked by the specific antagonist AMD3100, HO-1 activation was no longer effective in curbing PMN trafficking to the inflamed lungs. In conclusion, we show evidence that the anti-inflammatory effects of HO-1 are largely mediated by inhibiting the release of segmented PMNs from the bone marrow rather than direct effects within the lung.
Assuntos
Heme Oxigenase-1/metabolismo , Proteínas de Membrana/metabolismo , Neutrófilos/imunologia , Pneumonia/enzimologia , Doença Aguda , Administração por Inalação , Animais , Benzilaminas , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/imunologia , Células da Medula Óssea/patologia , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/imunologia , Quimiocina CXCL12/antagonistas & inibidores , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Ciclamos , Regulação da Expressão Gênica , Heme Oxigenase-1/genética , Compostos Heterocíclicos/farmacologia , Imunidade Inata/efeitos dos fármacos , Lipopolissacarídeos , Masculino , Proteínas de Membrana/genética , Metaloporfirinas/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Infiltração de Neutrófilos/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Neutrófilos/patologia , Pneumonia/induzido quimicamente , Pneumonia/imunologia , Pneumonia/patologia , Protoporfirinas/farmacologia , Explosão Respiratória/efeitos dos fármacosRESUMO
Pentoxifylline (PTX) has been shown to exert anti-inflammatory effects in experimental acute lung injury. However, results in humans were controversial. Recent in vitro studies suggested that the adenosine receptor A2A may be required for PTX to be effective. Therefore, we studied the association between A2A and PTX in a murine model of LPS-induced pulmonary inflammation. PTX treatment (10 mg/kg) reduced cellular influx (by 40%), microvascular permeability (30%), and the release of chemotactic cytokines into the alveolar space (TNF-α 60%, IL-6 60%, and CXCL2/3 53%, respectively). These protective effects were abolished completely in A2A(-/-) mice and in wild-type mice that had been treated with the selective A2A antagonist (1 mg/kg), but effects were not different in mice with altered adenosine levels. In vitro transmigration assays revealed a pivotal role of the endothelium in PTX-mediated PMN migration, with a reduction of 50% (2 mM PTX). This effect was also A2A dependent. Further, oxidative burst of human PMNs was A2A-dependently reduced by 53% after PTX treatment. In summary, PTX exhibits its anti-inflammatory effects in LPS-induced lung injury through an A2A-dependent pathway. These results will help to better understand previous conflicting data on PTX in inflammation and will direct further studies to consider the predominant role of A2A.
Assuntos
Anti-Inflamatórios/uso terapêutico , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pentoxifilina/uso terapêutico , Pneumonia/tratamento farmacológico , Receptor A2A de Adenosina/metabolismo , Animais , Western Blotting , Células Cultivadas , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Mutantes/genética , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Pneumonia/imunologia , Receptor A2A de Adenosina/genéticaRESUMO
ABSTRACT: Pulmonary defense mechanisms are critical for host integrity during pneumonia and sepsis. This defense is fundamentally dependent on the activation of neutrophils during the innate immune response. Recent work has shown that semaphorin 7A (Sema7A) holds significant impact on platelet function, yet its role on neutrophil function within the lung is not well understood. This study aimed to identify the role of Sema7A during pulmonary inflammation and sepsis. In patients with acute respiratory distress syndrome (ARDS), we were able to show a correlation between Sema7A and oxygenation levels. During subsequent workup, we found that Sema7A binds to the neutrophil PlexinC1 receptor, increasing integrins, and L-selectin on neutrophils. Sema7A prompted neutrophil chemotaxis in vitro and the formation of platelet-neutrophil complexes in vivo. We also observed altered adhesion and transmigration of neutrophils in Sema7A-/-animals in the lung during pulmonary inflammation. This effect resulted in increased number of neutrophils in the interstitial space of Sema7A-/- animals but reduced numbers of neutrophils in the alveolar space during pulmonary sepsis. This finding was associated with significantly worse outcome of Sema7A-/- animals in a model of pulmonary sepsis. Sema7A has an immunomodulatory effect in the lung, affecting pulmonary sepsis and ARDS. This effect influences the response of neutrophils to external aggression and might influence patient outcome. This trial was registered at www.ClinicalTrials.gov as #NCT02692118.
Assuntos
Antígenos CD , Neutrófilos , Pneumonia , Semaforinas , Sepse , Semaforinas/metabolismo , Sepse/imunologia , Sepse/metabolismo , Neutrófilos/metabolismo , Neutrófilos/imunologia , Humanos , Animais , Camundongos , Antígenos CD/metabolismo , Pneumonia/metabolismo , Pneumonia/imunologia , Proteínas Ligadas por GPI/metabolismo , Masculino , Modelos Animais de Doenças , Camundongos Knockout , Síndrome do Desconforto Respiratório/imunologia , Síndrome do Desconforto Respiratório/metabolismo , FemininoRESUMO
BACKGROUND: The authors investigated the impact of acute normovolemic hemodilution (ANH) on intrarenal oxygenation and its functional short-term consequences in pigs. METHODS: Renal microvascular oxygenation (µPO2) was measured in cortex, outer and inner medulla via three implanted optical fibers by oxygen-dependent quenching of phosphorescence. Besides systemic hemodynamics, renal function, histopathology, and hypoxia-inducible factor-1α expression were determined. ANH was performed in n = 18 pigs with either colloids (hydroxyethyl starch 6% 130/0.4) or crystalloids (full electrolyte solution), in three steps from a hematocrit of 30% at baseline to a hematocrit of 15% (H3). RESULTS: ANH with crystalloids decreased µPO2 in cortex and outer medulla approximately by 65% (P < 0.05) and in inner medulla by 30% (P < 0.05) from baseline to H3. In contrast, µPO2 remained unaltered during ANH with colloids. Furthermore, renal function decreased by approximately 45% from baseline to H3 (P < 0.05) only in the crystalloid group. Three times more volume of crystalloids was administered compared with the colloid group. Alterations in systemic and renal regional hemodynamics, oxygen delivery and oxygen consumption during ANH, gave no obvious explanation for the deterioration of µPO2 in the crystalloid group. However, ANH with crystalloids was associated with the highest formation of renal tissue edema and the highest expression of hypoxia-inducible factor-1α, which was mainly localized in distal convoluted tubules. CONCLUSIONS: ANH to a hematocrit of 15% statistically significantly impaired µPO2 and renal function in the crystalloid group. Less tissue edema formation and an unimpaired renal µPO2 in the colloid group might account for a preserved renal function.
Assuntos
Edema/etiologia , Hemodiluição/efeitos adversos , Nefropatias/etiologia , Rim/fisiopatologia , Microvasos/fisiopatologia , Oxigênio/metabolismo , Animais , Soluções Cristaloides , Modelos Animais de Doenças , Edema/fisiopatologia , Feminino , Hemodinâmica , Derivados de Hidroxietil Amido/administração & dosagem , Soluções Isotônicas/administração & dosagem , Rim/metabolismo , Nefropatias/fisiopatologia , Microvasos/metabolismo , Consumo de Oxigênio , Substitutos do Plasma/administração & dosagem , SuínosRESUMO
Extracorporeal circulation (ECC) is frequently used in intensive care patients with impaired lung or cardiac function. Despite being a life-saving therapeutic option, ECC is associated with increased risk for both bleeding and thrombosis. The management of bleeding and thromboembolic events in ECC patients is still challenging partly due to the lack of information on the pathophysiological changes in hemostasis and platelet function during the procedure. Using a combination of an ex vivo model for shear stress and a sensitive and easy-to-use laboratory method, we analyzed platelet responsiveness during ECC. After shear stress simulation in an ex vivo closed-loop ECC model, we found a significantly decreased response of α-granules after activation with adenosine diphosphate and thrombin receptor activating peptide (TRAP-6) and CD63 expression after activation with TRAP-6. Mepacrine uptake was also significantly reduced in the ex vivo shear stress model.In the same line, platelets from patients under ECC with venovenous systems and venoarterial systems showed impaired CD62P degranulation after stimulation with ADP and TRAP-6 compared with healthy control on day 1, 6, and 10 after implantation of ECC. However, no correlation between platelet degranulation and the occurrence of bleeding or thromboembolic events was observed.The used whole blood flow cytometry with immediate fixation after drawing introduces a sensitive and easy-to-use method to determine platelet activation status and our data confirm that increased shear stress conditions under ECC can cause impaired degranulation of platelet.
Assuntos
Transtornos Plaquetários , Plaquetas , Humanos , Estudos Prospectivos , Plaquetas/metabolismo , Ativação Plaquetária , Transtornos Plaquetários/etiologia , Circulação Extracorpórea/efeitos adversos , Circulação Extracorpórea/métodos , Difosfato de Adenosina/metabolismoRESUMO
Chronic wounds affect more than 2% of the population worldwide, with a significant burden on affected individuals, healthcare systems, and societies. A key regulator of the entire wound healing cascade is transforming growth factor beta (TGF-ß), which regulates not only inflammation and extracellular matrix formation but also revascularization. This present work aimed at characterizing wound tissues obtained from acute and chronic wounds regarding angiogenesis, inflammation, as well as ECM formation and degradation, to identify common disturbances in the healing process. Serum and wound tissues from 38 patients (N = 20 acute and N = 18 chronic wounds) were analyzed. The patients' sera suggested a shift from VEGF/VEGFR to ANGPT/TIE2 signaling in the chronic wounds. However, this shift was not confirmed in the wound tissues. Instead, the chronic wound tissues showed increased levels of MMP9, a known activator of TGF-ß. However, regulation of TGF-ß target genes, such as CTGF, COL1A1, or IL-6, was absent in the chronic wounds. In wound tissues, all three TGF-ß isoforms were expressed with increased levels of TGF-ß1 and TGF-ß3 and a reporter assay confirmed that the expressed TGF-ß was activated. However, Western blots and immunostaining showed decreased canonical TGF-ß signaling in the respective chronic wound tissues, suggesting the presence of a TGF-ß inhibitor. As a potential regulatory mechanism, the TGF-ß proteome profiler array suggested elevated levels of the TGF-ß pseudo-receptor BAMBI. Also, tissue expression of BAMBI was significantly increased not only in chronic wounds (10.6-fold) but also in acute wounds that had become chronic (9.5-fold). In summary, our data indicate a possible regulatory role of BAMBI in the development of chronic wounds. The available few in vivo studies support our findings by postulating a therapeutic potential of BAMBI for controlling scar formation.
Assuntos
Fator de Crescimento Transformador beta3 , Fator de Crescimento Transformador beta , Humanos , Bioensaio , Western Blotting , Inflamação , Proteínas de MembranaRESUMO
Uncontrolled transmigration of polymorphonuclear leukocytes (PMNs) into the different compartments of the lungs (intravascular, interstitial, alveolar) is a critical event in the early stage of acute lung injury and acute respiratory distress syndrome. Adenosine receptor A(2b) is highly expressed in the inflamed lungs and has been suggested to mediate cell trafficking. In a murine model of LPS-induced lung inflammation, we investigated the role of A(2b) on migration of PMNs into the different compartments of the lung. In A(2b)(-/-) mice, LPS-induced accumulation of PMNs was significantly higher in the interstitium, but not in the alveolar space. In addition, pulmonary clearance of PMNs was delayed in A(2b)(-/-) mice. Using chimeric mice, we identified A(2b) on hematopoietic cells as crucial for PMN migration. A(2b) did not affect the release of relevant chemokines into the alveolar space. LPS-induced microvascular permeability was under the control of A(2b) on both hematopoietic and nonhematopoietic cells. Activation of A(2b) on endothelial cells also reduced formation of LPS-induced stress fibers, highlighting its role for endothelial integrity. A specific A(2b) agonist (BAY 60-6583) was effective in decreasing PMN migration into the lung interstitium and microvascular permeability. In addition, in vitro transmigration of human PMNs through a layer of human endothelial or epithelial cells was A(2b) dependent. Activation of A(2b) on human PMNs reduced oxidative burst activity. Together, our results demonstrate anti-inflammatory effects of A(2b) on two major characteristics of acute lung injury, with a distinct role of hematopoietic A(2b) for cell trafficking and endothelial A(2b) for microvascular permeability.
Assuntos
Células da Medula Óssea/metabolismo , Lipopolissacarídeos/farmacologia , Pulmão/patologia , Neutrófilos/fisiologia , Pneumonia/metabolismo , Receptor A2B de Adenosina/metabolismo , Migração Transendotelial e Transepitelial , Lesão Pulmonar Aguda/imunologia , Lesão Pulmonar Aguda/metabolismo , Lesão Pulmonar Aguda/patologia , Agonistas do Receptor A2 de Adenosina/farmacologia , Aminopiridinas/farmacologia , Animais , Transplante de Medula Óssea , Líquido da Lavagem Broncoalveolar , Permeabilidade Capilar , Contagem de Células , Células Cultivadas , Quimiocinas/metabolismo , Citoesqueleto/metabolismo , Expressão Gênica , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/efeitos dos fármacos , Pneumonia/imunologia , Pneumonia/patologia , Receptor A2B de Adenosina/genética , Receptor A2B de Adenosina/fisiologia , Explosão RespiratóriaRESUMO
Acute respiratory distress syndrome is a life-threatening disease associated with high mortality. The adenosine receptor A2B (Adora2b) provides anti-inflammatory effects, which are also associated with the intracellular enzyme heme oxygenase-1 (HO-1). Our study determined the mechanism of sevoflurane's protective properties and investigated the link between sevoflurane and the impact of a functional Adora2b via HO-1 modulation during lipopolysaccharide (LPS)-induced lung injury. We examined the LPS-induced infiltration of polymorphonuclear neutrophils (PMNs) into the lung tissue and protein extravasation in wild-type and Adora2b-/- animals. We generated chimeric animals, to identify the impact of sevoflurane on Adora2b of hematopoietic and non-hematopoietic cells. Sevoflurane decreased the LPS-induced PMN-infiltration and diminished the edema formation in wild-type mice. Reduced PMN counts after sevoflurane treatment were detected only in chimeric mice, which expressed Adora2b exclusively on leukocytes. The Adora2b on hematopoietic and non-hematopoietic cells was required to improve the permeability after sevoflurane inhalation. Further, sevoflurane increased the protective effects of HO-1 modulation on PMN migration and microvascular permeability. These protective effects were abrogated by specific HO-1 inhibition. In conclusion, our data revealed new insights into the protective mechanisms of sevoflurane application during acute pulmonary inflammation and the link between sevoflurane and Adora2b, and HO-1 signaling, respectively.
Assuntos
Heme Oxigenase-1 , Pneumonia , Receptor A2B de Adenosina , Animais , Heme Oxigenase-1/metabolismo , Lipopolissacarídeos , Proteínas de Membrana , Camundongos , Neutrófilos/metabolismo , Pneumonia/tratamento farmacológico , Receptor A2B de Adenosina/metabolismo , Sevoflurano/farmacologiaRESUMO
BACKGROUND: At our institution, patients undergoing neurosurgical procedures in the posterior cranial fossa are placed either in the semisitting or in the supine position. The major risk of the semisitting positioning is a venous air embolism (VAE), which may, however, also occur in the supine position. METHODS: In a prospective single-center study with 137 patients, we evaluated the occurrence of VAEs in patients in the supine and in the semisitting position during the period from January 2014 until April 2015. All patients were monitored for VAE by the use of a transesophageal echocardiography (TEE). RESULTS: In total, 50% of the patients experienced a VAE (56% of these patients underwent surgery in the semisitting and 11% in the supine position). In total, 86% of the VAEs were detected by the use of a TEE and did not lead to any changes in the end-expiratory CO2. We only observed VAEs with a decrease in end-expiratory CO2 in the semisitting position. However, none of the patients had any hemodynamic changes due to the VAE. CONCLUSIONS: The semisitting position with TEE monitoring and a standardized protocol is a safe and advantageous technique, taking account of a significant rate of VAEs. VAEs also occur in the supine position, but less frequently.
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Embolia Aérea , Dióxido de Carbono , Embolia Aérea/diagnóstico por imagem , Embolia Aérea/epidemiologia , Embolia Aérea/etiologia , Humanos , Procedimentos Neurocirúrgicos/efeitos adversos , Procedimentos Neurocirúrgicos/métodos , Estudos Prospectivos , Decúbito DorsalRESUMO
Acute pulmonary inflammation affects over 10% of intensive care unit (ICU) patients and is associated with high mortality. Fractalkine (CX3CL1) and its receptor, CX3CR1, have been shown to affect pulmonary inflammation, but previous studies have focused on macrophages. In a murine model of acute pulmonary inflammation, we identified inflammatory hallmarks in C57BL/6J and CX3CR1-/- mice. Pulmonary inflammation was significantly enhanced in the CX3CR1-/- animals compared to the C57BL/6J animals, as assessed by microvascular permeability, polymorphonuclear neutrophil (PMN) migration into lung tissue and alveolar space. The CX3CR1-/- mice showed increased levels of apoptotic PMNs in the lungs, and further investigations revealed an increased activation of necrosome-related receptor-interacting serine/threonine-protein kinases 1 (RIPK1), 3 (RIPK3), and mixed-lineage kinase domain-like pseudokinase (MLKL). Phosphorylated MLKL leads to membrane rupture and damage-associated molecular pattern (DAMP) release, which further enhance inflammation. The release of DAMPs was significantly higher in the CX3CR1-/- mice and led to the activation of various cascades, explaining the increased inflammation. RIPK3 and MLKL inhibition improved the inflammatory response in human PMNs in vitro and confirmed our in vivo findings. In conclusion, we linked CX3CL1 to the necrosome complex in pulmonary inflammation and demonstrated a pivotal role of the necrosome complex in human PMNs.
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BACKGROUND: Peritonitis is a life-threatening condition on intensive care units. Inflammatory cytokines and their receptors drive inflammation, cause the formation of platelet-neutrophil complexes (PNCs) and therefore the migration of polymorphonuclear neutrophils (PMNs) into the inflamed tissue. CX3CL1 and its receptor CX3CR1 are expressed in various cells, and promote inflammation. The shedding of CX3CL1 is mediated by a disintegrin and metalloprotease (ADAM) 17. The role of the CX3CL1-CX3CR1 axis in acute peritonitis remains elusive. METHODS: In zymosan-induced peritonitis, we determined the formation of PNCs in the blood and the expression of PNC-related molecules on PNCs. PMN migration into the peritoneal lavage was evaluated in wild-type (WT) and CX3CR1-/- animals by flow cytometry. CX3CL1, ADAM17, and the expression of various inflammatory cytokines were detected. Further, we determined the inflammation-associated activation of the intracellular transcription factor extracellular signal-regulated kinase 1/2â(ERK1/2) by Western blot. RESULTS: The PMN accumulation in the peritoneal lavage and the PNC formation in the circulation were significantly raised in CX3CR1-/- compared with WT animals. The expression of PNC-related selectins on PNCs was significantly increased in the blood of CX3CR1-/- animals, as well as cytokine levels. Further, we observed an increased activation of ERK1/2 and elevated ADAM17 expression in CX3CR1-/- during acute inflammation. Selective ERK1/2 inhibition ameliorated inflammation-related increased ADAM17 expression. CONCLUSIONS: A CX3CR1 deficiency raised the release of inflammatory cytokines and increased the PNC formation respectively PMN migration via an elevated ERK1/2 activation during acute peritonitis. Further, we observed a link between the ERK1/2 activation and an elevated ADAM17 expression on PNC-related platelets and PMNs during inflammation. Our data thus illustrate a crucial role of CX3CR1 on the formation of PNCs and regulating inflammation in acute peritonitis.
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Plaquetas/fisiologia , Receptor 1 de Quimiocina CX3C/fisiologia , Neutrófilos/fisiologia , Peritonite/etiologia , Doença Aguda , Animais , Progressão da Doença , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Neutrophils are the most abundant immune cells in the blood. Besides common immune defense mechanisms, releasing their DNA covered with antimicrobial proteases and histones represent another strong defense mechanism: neutrophil extracellular traps. In vitro the two most common inducers of these, so called, NETs are calcium ionophores (CI) and PMA (Phorbol 12-myristate 13-acetate). Following stimulation monitoring of NET release is necessary. For now, the methods of choice are quantification of free DNA by fluorescent dyes or analysis of immunofluorescence images. As a new method we tested bio-impedance monitoring of neutrophils after stimulation with the two inducers PMA and CI in gold-electrode coated plates. Bio-impedance (cell index) was measured over time. Results were compared to the monitoring of NETs by the fluorescent DNA-binding dye Sytox Green and immunofluorescence analysis. Cell index peaked about 25 min faster following CI stimulation than following PMA stimulation. The activation in Sytox Green Assay was significantly later detectable for PMA (+ approx. 90 min) but not for CI stimulation. The earlier and faster activation by CI was also confirmed by immunofluorescence staining. Our data suggest that bio-impedance measurement allows an easy online tracking of early neutrophil activation. This offers new opportunities to monitor early phases and stimuli-dependent dynamics of NETosis.
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Our previous studies revealed a pivotal role of the chemokine stromal cell-derived factor (SDF)-1 and its receptors CXCR4 and CXCR7 on migratory behavior of polymorphonuclear granulocytes (PMNs) in pulmonary inflammation. Thereby, the SDF-1-CXCR4/CXCR7-axis was linked with adenosine signaling. However, the role of the SDF-1 receptors CXCR4 and CXCR7 in acute inflammatory peritonitis and peritonitis-related sepsis still remained unknown. The presented study provides new insight on the mechanism of a selective inhibition of CXCR4 (AMD3100) and CXCR7 (CCX771) in two models of peritonitis and peritonitis-related sepsis by injection of zymosan and fecal solution. We observed an increased expression of SDF-1, CXCR4, and CXCR7 in peritoneal tissue and various organs during acute inflammatory peritonitis. Selective inhibition of CXCR4 and CXCR7 reduced PMN accumulation in the peritoneal fluid and infiltration of neutrophils in lung and liver tissue in both models. Both inhibitors had no anti-inflammatory effects in A2B knockout animals (A2B-/-). AMD3100 and CCX771 treatment reduced capillary leakage and increased formation of tight junctions as a marker for microvascular permeability in wild type animals. In contrast, both inhibitors failed to improve capillary leakage in A2B-/- animals, highlighting the impact of the A2B-receptor in SDF-1 mediated signaling. After inflammation, the CXCR4 and CXCR7 antagonist induced an enhanced expression of the protective A2B adenosine receptor and an increased activation of cAMP (cyclic adenosine mono phosphate) response element-binding protein (CREB), as downstream signaling pathway of A2B. The CXCR4- and CXCR7-inhibitor reduced the release of cytokines in wild type animals via decreased intracellular phosphorylation of ERK and NFκB p65. In vitro, CXCR4 and CXCR7 antagonism diminished the chemokine release of human cells and increased cellular integrity by enhancing the expression of tight junctions. These protective effects were linked with functional A2B-receptor signaling, confirming our in vivo data. In conclusion, our study revealed new protective aspects of the pharmacological modulation of the SDF-1-CXCR4/CXCR7-axis during acute peritoneal inflammation in terms of the two hallmarks PMN migration and barrier integrity. Both anti-inflammatory effects were linked with functional adenosine A2B-receptor signaling.
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Benzilaminas/uso terapêutico , Ciclamos/uso terapêutico , Neutrófilos/imunologia , Peritonite/tratamento farmacológico , Receptor A2B de Adenosina/metabolismo , Receptores CXCR4/metabolismo , Receptores CXCR/metabolismo , Sepse/tratamento farmacológico , Doença Aguda , Animais , Benzilaminas/farmacologia , Permeabilidade Capilar , Quimiocina CXCL12/metabolismo , Ciclamos/farmacologia , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor A2B de Adenosina/genética , Receptores CXCR/antagonistas & inibidores , Receptores CXCR4/antagonistas & inibidores , Transdução de SinaisRESUMO
Background: Treatment of bronchopulmonary dysplasia in preterm infants is challenging due to its multifactorial origin. In rodent models of neonatal lung injury, selective inhibition of phosphodiesterase 4 (PDE4) has been shown to exert anti-inflammatory properties in the lung. We hypothesized that GSK256066, a highly selective, inhalable PDE4 inhibitor, would have beneficial effects on lung injury and inflammation in a triple hit lamb model of Ureaplasma parvum (UP)-induced chorioamnionitis, prematurity, and mechanical ventilation. Methods: Twenty-one preterm lambs were surgically delivered preterm at 129 days after 7 days intrauterine exposure to UP. Sixteen animals were subsequently ventilated for 24 hours and received endotracheal surfactant and intravenous caffeine citrate. Ten animals were randomized to receive twice a high (10 µg/kg) or low dose (1 µg/kg) of nebulized PDE4 inhibitor. Results: Nebulization of high, but not low, doses of PDE4 inhibitor led to a significant decrease in pulmonary PDE activity, and was associated with lung injury and vasculitis, influx of neutrophils, and increased proinflammatory cytokine messenger RNA levels. Conclusion: Contrary to our hypothesis, we found in our model a dose-dependent proinflammatory effect of an inhaled highly selective PDE4 inhibitor in the lung. Our findings indicate the narrow therapeutic range of inhaled PDE4 inhibitors in the preterm population.